11401507>Molecular dissection of the <n>importin beta1</n>-recognized nuclear targeting signal of <n>parathyroid hormone-related protein</n>. Produced by various types of solid tumors, <n>parathyroid hormone-related protein</n> (<n>PTHrP</n>) is the causative agent of humoral hypercalcemia of malignancy. The similarity of <n>PTHrP's</n> amino-terminus to that of <n>parathyroid hormone</n> enables it to share some of the latter's signalling properties, but its carboxy-terminus confers distinct functions including a role in the nucleus/nucleolus in reducing apoptosis and enhancing cell proliferation. <n>PTHrP</n> nuclear import occurs via a novel <n>importin beta1</n>-mediated pathway. The present study uses several different direct binding assays to map the interaction of <n>PTHrP</n> with <n>importin beta</n> using a series of alanine mutated <n>PTHrP</n> peptides and truncated human <n>importin beta1</n> derivatives. Our results indicate that <n>PTHrP</n> amino acids 83-93 (KTPGKKKKGK) are absolutely essential for <n>importin beta1</n> recognition with residues 71-82 (TNKVETYKEQPL) additionally required for high affinity binding; residues 380-643 of <n>importin beta1</n> are required for the interaction. Binding of <n>importin beta1</n> to <n>PTHrP</n> is reduced in the presence of the GTP-bound but not GDP-bound form of the guanine nucleotide binding protein <n>Ran</n>, consistent with the idea that <n>Ran</n>GTP binding to <n>importin beta</n> is involved in the release of <n>PTHrP</n> into the nucleus following translocation across the nuclear envelope. This study represents the first detailed examination of a modular, non-arginine-rich <n>importin beta1</n>-recognized nuclear targeting signal. Copyright 2001 Academic Press.
11327761>Functional mutagenesis of AML1/RUNX1 and PEBP2 beta/CBF beta define distinct, non-overlapping sites for DNA recognition and heterodimerization by the Runt domain. The Runt domain family of transcription factors play key roles in transcriptional regulation of definitive hematopoiesis and osteogenesis. This transcription factor family is characterized by a DNA-binding alpha-subunit harboring the Runt domain and a secondary subunit, beta, which binds to the Runt domain and enhances its interaction with DNA. Missense mutations in the Runt domain from either the blood or bone-related gene product are associated with the onset of acute human leukemia as well as a disease of skeletal patterning known as cleidocranial dysplasia. NMR "footprinting" analysis of Runt domain/beta/DNA ternary complexes in solution previously identified the likely residues that form the heterodimerization and DNA-binding surfaces of the Runt domain. Functional mutagenesis at 37 positions in the Runt domain or beta confirms the original identification of these interaction surfaces and reveals that the heterodimerization and DNA-binding surfaces of the Runt domain occur at distinct, non-overlapping sites within the domain. The analysis of an additional 21 disease-related missense mutations identified from patients with either blood or bone disease demonstrates that the primary defect in these patients is a failure in DNA-recognition by the Runt domain. The molecular basis for the DNA-binding defect is analyzed in the context of the three-dimensional structure of the Runt domain in binary and ternary protein/DNA complexes. Copyright 2001 Academic Press.
11279204>Inhibitory effect of bovine milk <n>lactoferrin</n> on the interaction between a streptococcal surface protein antigen and human <n>salivary agglutinin</n>. Human whole saliva induces aggregation of Streptococcus mutans cells via an interaction between a surface protein antigen (<n>PAc</n>) of the organism and <n>salivary agglutinin</n>. Bovine milk inhibits the saliva-induced aggregation of S. mutans. In this study, the milk component that possesses inhibitory activity against this aggregation was isolated and found to be <n>lactoferrin</n>. Surface plasmon resonance analysis indicated that bovine <n>lactoferrin</n> binds more strongly to <n>salivary agglutinin</n>, especially to high molecular mass glycoproteins, which is a component of the <n>agglutinin</n>, than to recombinant <n>PAc</n>. The binding of bovine <n>lactoferrin</n> to salivary <n>agglutinin</n> was thermostable, and the optimal pH for binding was 4.0. To identify the saliva-binding region of bovine <n>lactoferrin</n>, 11 truncated bovine <n>lactoferrin</n> fragments were constructed. A fragment corresponding to the C-terminal half of the <n>lactoferrin</n> molecule had a strong inhibitory effect on the saliva-induced aggregation of S. mutans, whereas a fragment corresponding to the N-terminal half had a weak inhibitory effect. Seven shorter fragments corresponding to <n>lactoferrin</n> residues 473-538 also showed a high ability to inhibit the aggregation of S. mutans. These results suggest that residues 473-538 of bovine <n>lactoferrin</n> are important in the inhibition of saliva-induced aggregation of S. mutans.
11259620>Direct and differential interaction of beta-arrestins with the intracellular domains of different opioid receptors. Beta-arrestins have been shown to play important roles in regulation of signaling and desensitization of <n>opioid receptors</n> in many in vivo studies. The current study was carried out to measure the direct interaction of beta-arrestins with two functional intracellular domains, the third intracellular loop (I3L) and the carboxyl terminus (CT), of <n>delta-</n>, <n>mu-</n>, and <n>kappa-opioid receptors</n> (<n>DOR</n>, <n>MOR</n>, and <n>KOR</n>, respectively). Results from the pull-down assay using <n>glutathione S-transferase</n> fusion proteins demonstrated that beta-arrestins (1 and 2) were able to bind to the I3L of <n>DOR</n> and to the CT of <n>DOR</n> and <n>KOR</n>. Surface plasmon resonance measurement gave similar results with typical dissociation equilibrium constant (K(D)) values in the micromolar range. The site-directed mutagenesis experiment further revealed that certain specific serine/threonine residues in these receptor domains play a critical role in their interaction with beta-arrestins. Taken together, our data clearly indicated that beta- arrestins interact differentially with the functional domains of different opioid receptors; this may provide a possible molecular basis for differential regulation of opioid receptors by beta-arrestins.
11233172>Wavelength-dependent spectral changes accompany <n>CN-hemin</n> binding to human <n>apohemoglobin</n>. The interaction of <n>apohemoglobin</n> with two heme derivatives, <n>CN-protohemin</n> and <n>CN-deutero-hemin</n>, was monitored at multiple Soret wavelengths (417-423 and 406-412 nm, respectively) in 0.05 M potassium phosphate buffer, pH 7.0, at 10 degrees C and revealed, as previously reported, a multiphasic kinetic reaction. Wavelength-dependent reactions were observed for both <n>CN-protohemin</n> and <n>CN-deuterohemin</n> derivatives with the alpha chain (bathochromic entity) displaying faster (4- to 7-fold) rates throughout the courses of both heme-binding reactions. The basis of this spectrally heterogeneous kinetic phenomenon could be deduced from molecular modeling studies of alpha- and beta-chain structures. Key differences in the number of stabilizing contacts of the two chains with the peripheral alpha propionyl 45(CE3); 58(E7); 61(E10) as well as the beta vinyl 38(C4); 71(E15); 106(G8) groups were found. Furthermore, RMS plots comparing apo- and heme-containing subunits reveal substantial structural disparities in the C-CD-F-FG helical regions of the alphabeta dimer interface.
11181178>Mitotic checkpoint proteins <n>HsMAD1</n> and <n>HsMAD2</n> are associated with nuclear pore complexes in interphase. <n>Mad1</n> was first identified in budding yeast as an essential component of the checkpoint system that monitors spindle assembly in mitosis and prevents premature anaphase onset. Using antibodies to the human homologue of <n>Mad1</n> (<n>HsMAD1</n>), we have begun to characterize this protein in mammalian cells. <n>HsMad1</n> is found localized at kinetochores in mitosis. The labeling is brightest in prometaphase and is absent from kinetochores at metaphase and anaphase. In cells where most chromosomes have reached the metaphase plate, those aligned at the plate show no labeling while remaining, unaligned chromosomes are still brightly labeled. We find <n>HsMad1</n> associated with <n>HsMad2</n>. Association with <n>p55CDC</n>, a protein previously shown to bind <n>HsMad2</n>, was not detected. Surprisingly, unlike any other known mitotic checkpoint proteins, <n>HsMad1</n> and <n>HsMAD2</n> were found localized at nuclear pores throughout interphase. This was confirmed by co-labeling with an antibody to known nuclear pore complex proteins and by their co-purification with enriched nuclear envelope fractions. <n>HsMad1</n> was identified serendipitously by its binding to a viral protein, HTLV-1 <n>Tax</n>, which affects transcription of viral and human proteins. The localization of <n>HsMad1</n> to nuclear pore complexes suggests an alternate, non-mitotic role for the <n>Mad1</n>/<n>Tax</n> interaction in the viral transformation of cells.
11168528><n>p35</n>/<n>cdk5</n> binds and phosphorylates <n>beta-catenin</n> and regulates <n>beta-catenin</n>/<n>presenilin-1</n> interaction. The neuronal cyclin-dependent kinase <n>p35</n>/<n>cdk5</n> comprises a catalytic subunit (<n>cdk5</n>) and an activator subunit (<n>p35</n>). To identify novel <n>p35</n>/<n>cdk5</n> substrates, we utilized the yeast two-hybrid system to screen for human <n>p35</n> binding partners. From one such screen, we identified <n>beta-catenin</n> as an interacting protein. Confirmation that <n>p35</n> binds to <n>beta-catenin</n> was obtained by using <n>glutathione S-transferase</n> (<n>GST</n>)-<n>beta-catenin</n> fusion proteins that interacted with both endogenous and transfected <n>p35</n>, and by showing that <n>beta-catenin</n> was present in <n>p35</n> immunoprecipitates. <n>p35</n> and <n>beta-catenin</n> also displayed overlapping subcellular distribution patterns in cells including neurons. Finally, we demonstrated that <n>p35</n>/<n>cdk5</n> phosphorylates <n>beta-catenin</n>. <n>beta-catenin</n> also binds to <n>presenilin-1</n> and altered <n>beta-catenin</n>/<n>presenilin-1</n> interactions may be mechanistic in Alzheimer's disease (AD). Abnormal <n>p35</n>/<n>cdk5</n> activity has also been suggested to contribute to AD. We therefore investigated how modulation of <n>p35</n>/<n>cdk5</n> activity influenced <n>beta-catenin</n>/<n>presenilin-1</n> interactions. Inhibition of <n>p35</n>/<n>cdk5</n> with roscovitine did not alter the steady state levels of either <n>beta-catenin</n> or <n>presenilin-1</n> but reduced the amount of <n>presenilin-1</n> bound to <n>beta-catenin</n>. Thus, <n>p35</n>/<n>cdk5</n> binds and phosphorylates <n>beta-catenin</n> and regulates its binding to <n>presenilin-1</n>. The findings reported here therefore provide a novel molecular framework to connect <n>p35</n>/<n>cdk5</n> with <n>beta-catenin</n> and <n>presenilin-1</n> in AD.
11157096>Association of Trk <n>neurotrophin</n> receptors with components of the cytoplasmic <n>dynein</n> motor. <n>nerve growth factor</n> (<n>NGF</n>) initiates its trophic effects by long-range signaling through binding, internalization, and transport of a ligand-receptor complex from the axon terminal to the cell body. However, the mechanism by which retrograde transport of <n>NGF</n> takes place has not been elucidated. Here we describe an interaction between the Trk receptor tyrosine kinase and a 14 kDa light chain of cytoplasmic <n>dynein</n>. After transfection in human embryonic kidney 293 cells, this 14 kDa <n>dynein</n> light chain was found to bind to <n>TrkA</n>, <n>TrkB</n>, and <n>TrkC receptors</n>. Mapping experiments indicated that the 14 kDa <n>dynein</n> light chain binds to the distal region of the <n>TrkA</n> juxtamembrane domain. Coimmunoprecipitation experiments in vivo indicate that Trk receptors are in a complex with the 14 kDa light chain and 74 kDa intermediate chain of <n>dynein</n>. Confirming the physiological relevance of this association, a marked accumulation of Trk with the 14 kDa and the 74 kDa <n>dynein</n> components was observed after ligation of the sciatic nerve. The association of Trk receptors with components of cytoplasmic <n>dynein</n> suggests that transport of <n>neurotrophins</n> during vesicular trafficking may occur through a direct interaction of the Trk receptor with the <n>dynein</n> motor machinery.
11135361>Characterization of <n>Cre</n>-loxP interaction in the major groove: hint for structural distortion of mutant <n>Cre</n> and possible strategy for HIV-1 therapy. Although the crystal structure of <n>Cre recombinase</n> complexed with DNA, named loxA, was elucidated a couple of years ago, it has not yet been determined which amino acids of the protein are involved in the specific <n>Cre</n>-loxP interaction. Arg259 and Gln90 interact with DNA substrate in the major groove from which the specificity of protein-DNA interaction comes. In this study, we substituted these residues for other amino acids. Also, two mutated DNA substrates were constructed. In each mutant, one of the bases that interact with Arg259 or Gln90 was changed into another base. In vitro binding assays and recombination assays of variant lox sites with wild-type and mutant-type <n>Cre</n> revealed that Arg259 plays a key role in <n>Cre</n>-loxP binding but Gln90 does not. However, the recombination activity still remained intact, although the binding between <n>Cre</n> and DNA substrate was not ensured. Copyright 2001 Wiley-Liss, Inc.
11111045>Identification of upstream stimulatory factor as an activator of the human <n>dipeptidyl peptidase IV</n> gene in Caco-2 cells. The 5' upstream region (-448/-443) of the human <n>dipeptidyl peptidase IV</n> gene promoter containing a consensus E-box (CACGTG) was shown to bind upstream stimulatory factor using nuclear extracts from mouse (3T3) fibroblasts and the human intestinal and hepatic epithelial cell lines Caco-2 and HepG2. Supershift analysis with specific antibodies to <n>USF-1</n> and <n>USF-2</n> indicates that <n>USF-1</n> is the primary isoform binding to the E-box in nuclear extracts from these cells. Using cell culture, transient cotransfection of USF expression vectors with <n>dipeptidyl peptidase IV</n> promoter constructs revealed that both <n>USF-1</n> and <n>USF-2</n> caused an approximately tenfold increase in reporter gene expression in Caco-2 cells. Mutant forms of <n>USF-1</n> and <n>USF-2</n> lacking the DNA binding or transcriptional activation domains were unable to stimulate reporter gene expression. Mutation of the E-box prevented binding of USF, although stimulation of reporter gene expression by cotransfection with USF was reduced by only 50%. By using a series of deletion constructs in cotransfection experiments, a second possible site of USF interaction with the <n>dipeptidyl peptidase IV</n> promoter was localized to the -119/-88 region.
11090287>Search for intermediate structures in <n>transthyretin</n> fibrillogenesis: soluble tetrameric Tyr78Phe <n>TTR</n> expresses a specific epitope present only in amyloid fibrils. Familial Amyloidotic Polyneuropathy (FAP) is caused by the assembly of <n>TTR</n> into an insoluble beta-sheet. The <n>TTR</n> tetramer is thought to dissociate into monomeric intermediates and subsequently polymerise into the pathogenic amyloid form. The biochemical mechanism behind this transformation is unknown. We characterised intermediate <n>TTR</n> structures in the in vitro amyloidogenesis pathway by destabilising the AB loop through substitution of residue 78. Changes at this residue, should destabilise the <n>TTR</n> tetrameric fold, based on the known crystallographic structure of a Leu55Pro <n>transthyretin</n> variant. We generated a soluble tetrameric form of <n>TTR</n> that is recognised by a monoclonal antibody, previously reported to react only with highly amyloidogenic mutant proteins lacking the tetrameric native fold and with amyloid fibrils. BIAcore system analysis showed that Tyr78Phe had similar binding properties as synthetic fibrils. The affinity of this interaction was 10(7) M(-1). We suggest that the tetrameric structure of Tyr78Phe is altered due to the loosening of the AB loops of the tetramer, leading to a structure that might represent an early intermediate in the fibrillogenesis pathway.
11073994>Novel <n>Upf2p</n> orthologues suggest a functional link between translation initiation and nonsense surveillance complexes. Transcripts harboring premature signals for translation termination are recognized and rapidly degraded by eukaryotic cells through a pathway known as nonsense-mediated mRNA decay (NMD). In addition to protecting cells by preventing the translation of potentially deleterious truncated peptides, studies have suggested that NMD plays a broader role in the regulation of the steady-state levels of physiologic transcripts. In Saccharomyces cerevisiae, three trans-acting factors (<n>Upf1p</n> to <n>Upf3p</n>) are required for NMD. Orthologues of <n>Upf1p</n> have been identified in numerous species, showing that the NMD machinery, at least in part, is conserved through evolution. In this study, we demonstrate additional functional conservation of the NMD pathway through the identification of <n>Upf2p</n> homologues in Schizosaccharomyces pombe and humans (<n>rent2</n>). Disruption of S. pombe UPF2 established that this gene is required for NMD in fission yeast. <n>rent2</n> was demonstrated to interact directly with <n>rent1</n>, a known trans-effector of NMD in mammalian cells. Additionally, fragments of <n>rent2</n> were shown to possess nuclear targeting activity, although the native protein localizes to the cytoplasmic compartment. Finally, novel functional domains of <n>Upf2p</n> and <n>rent2</n> with homology to <n>eukaryotic initiation factor 4G</n> (<n>eIF4G</n>) and other translational regulatory proteins were identified. Directed mutations within these so-called <n>eIF4G</n> homology (4GH) domains were sufficient to abolish the function of S. pombe <n>Upf2p</n>. Furthermore, using the two-hybrid system, we obtained evidence for direct interaction between <n>rent2</n> and human <n>eIF4AI</n> and <n>Sui1</n>, both components of the translation initiation complex. Based on these findings, a novel model in which <n>Upf2p</n> and <n>rent2</n> effects decreased translation and accelerated decay of nonsense transcripts through competitive interactions with <n>eIF4G</n>-binding partners is proposed.
11056215>An acidic amino acid cluster regulates the nucleolar localization and ribosome assembly of human <n>ribosomal protein L22</n>. The control of human <n>ribosomal protein L22</n> (<n>rpL22</n>) to enter into the nucleolus and its ability to be assembled into the ribosome is regulated by its sequence. The nuclear import of <n>rpL22</n> depends on a classical nuclear localization signal of four lysines at positions 13-16. <n>RpL22</n> normally enters the nucleolus via a compulsory sequence of KKYLKK (I-domain, positions 88-93). An acidic residue cluster at the C-terminal end (C-domain) plays a nuclear retention role. The retention is concealed by the N-domain (positions 1-9), which weakly interacts with the C-domain as demonstrated in the yeast two-hybrid system. Once it reaches the nucleolus, the question of whether <n>rpL22</n> is assembled into the ribosome depends upon the presence of the N-domain. This suggests that the N-domain, on dissociation from its interaction with the C-domain, binds to a specific region of the 28S rRNA for ribosome assembly.
11027346>Selective <n>CXCR4</n> antagonism by <n>Tat</n>: implications for in vivo expansion of coreceptor use by HIV-1. Chemokines and chemokine receptors play important roles in HIV-1 infection and tropism. <n>CCR5</n> is the major macrophage-tropic coreceptor for HIV-1 whereas <n>CXC chemokine receptor 4</n> (<n>CXCR4</n>) serves the counterpart function for T cell-tropic viruses. An outstanding biological mystery is why only R5-HIV-1 is initially detected in new seroconvertors who are exposed to R5 and X4 viruses. Indeed, X4 virus emerges in a minority of patients and only in the late stage of disease, suggesting that early negative selection against HIV-1-<n>CXCR4</n> interaction may exist. Here, we report that the HIV-1 <n>Tat</n> protein, which is secreted from virus-infected cells, is a <n>CXCR4</n>-specific antagonist. Soluble <n>Tat</n> selectively inhibited the entry and replication of X4, but not R5, virus in peripheral blood mononuclear cells (PBMCs). We propose that one functional consequence of secreted <n>Tat</n> is to select against X4 viruses, thereby influencing the early in vivo course of HIV-1 disease.
11003645>SAF-Box, a conserved protein domain that specifically recognizes scaffold attachment region DNA. SARs (scaffold attachment regions) are candidate DNA elements for partitioning eukaryotic genomes into independent chromatin loops by attaching DNA to proteins of a nuclear scaffold or matrix. The interaction of SARs with the nuclear scaffold is evolutionarily conserved and appears to be due to specific DNA binding proteins that recognize SARs by a mechanism not yet understood. We describe a novel, evolutionarily conserved protein domain that specifically binds to SARs but is not related to SAR binding motifs of other proteins. This domain was first identified in human <n>scaffold attachment factor A</n> (<n>SAF-A</n>) and was thus designated SAF-Box. The SAF-Box is present in many different proteins ranging from yeast to human in origin and appears to be structurally related to a homeodomain. We show here that SAF-Boxes from four different origins, as well as a synthetic SAF-Box peptide, bind to natural and artificial SARs with high specificity. Specific SAR binding of the novel domain is achieved by an unusual mass binding mode, is sensitive to distamycin but not to chromomycin, and displays a clear preference for long DNA fragments. This is the first characterization of a specific SAR binding domain that is conserved throughout evolution and has DNA binding properties that closely resemble that of the unfractionated nuclear scaffold.
10982864>Interaction of the <n>U3-55k</n> protein with U3 snoRNA is mediated by the box B/C motif of U3 and the WD repeats of <n>U3-55k</n>. U3 small nucleolar RNA (snoRNA) is a member of the Box C/D family of snoRNAs which functions in ribosomal RNA processing. <n>U3-55k</n> is a protein that has been found to interact with U3 but not other members of the Box C/D snoRNA family. We have found that interaction of the <n>U3-55k</n> protein with U3 RNA in vivo is mediated by the conserved Box B/C motif which is unique to U3 snoRNA. Mutation of Box B and Box C, but not of other conserved sequence elements, disrupted interaction of <n>U3-55k</n> with U3 RNA. Furthermore, a fragment of U3 containing only these two conserved elements was bound by <n>U3-55k</n> in vivo. RNA binding assays performed in vitro indicate that Box C may be the primary determinant of the interaction. We have cloned the cDNA encoding the Xenopus laevis <n>U3-55k</n> protein and find strong homology to the human sequence, including six WD repeats. Deletion of WD repeats or sequences near the C-terminus of <n>U3-55k</n> resulted in loss of association with U3 RNA and also loss of localization of <n>U3-55k</n> to the nucleolus, suggesting that protein-protein interactions contribute to the localization and RNA binding of <n>U3-55k</n> in vivo.
10967329>Convergent evolution with combinatorial peptides. Once the sequence of a genome is in hand, understanding the function of its encoded proteins becomes a task of paramount importance. Much like the biochemists who first outlined different biochemical pathways, many genomic scientists are engaged in determining which proteins interact with which proteins, thereby establishing a protein interaction network. While these interactions have evolved in regard to their specificity, affinity and cellular function over billions of years, it is possible in the laboratory to isolate peptides from combinatorial libraries that bind to the same proteins with similar specificity, affinity and primary structures, which resemble those of the natural interacting proteins. We have termed this phenomenon 'convergent evolution'. In this review, we highlight various examples of convergent evolution that have been uncovered in experiments dissecting protein-protein interactions with combinatorial peptides. Thus, a fruitful approach for mapping protein-protein interactions is to isolate peptide ligands to a target protein and identify candidate interacting proteins in a sequenced genome by computer analysis.
10956659>Interaction of the <n>factor XIII</n> activation peptide with <n>alpha-thrombin</n>. Crystal structure of its enzyme-substrate analog complex. The serine protease <n>thrombin</n> proteolytically activates <n>blood coagulation factor XIII</n> by cleavage at residue Arg(37); <n>factor XIII</n> in turn cross-links <n>fibrin</n> molecules and gives mechanical stability to the blood clot. The 2.0-A resolution x-ray crystal structure of human <n>alpha thrombin</n> bound to the factor XIII-(28-37) decapeptide has been determined. This structure reveals the detailed atomic level interactions between the <n>factor XIII</n> activation peptide and <n>thrombin</n> and provides the first high resolution view of this functionally important part of the <n>factor XIII</n> molecule. A comparison of this structure with the crystal structure of <n>fibrinopeptide A</n> complexed with <n>thrombin</n> highlights several important determinants of <n>thrombin</n> substrate interaction. First, the P1 and P2 residues must be compatible with the geometry and chemistry of the S1 and S2 specificity sites in <n>thrombin</n>. Second, a glycine in the P5 position is necessary for the conserved substrate conformation seen in both factor XIII-(28-37) and <n>fibrinopeptide A</n>. Finally, the hydrophobic residues, which occupy the aryl binding site of <n>thrombin</n> determine the substrate conformation further away from the catalytic residues. In the case of <n>factor XIII</n>-(28-37), the aryl binding site is shared by hydrophobic residues P4 (Val(34)) and P9 (Val(29)). A bulkier residue in either of these sites may alter the substrate peptide conformation.
10938104>Potentiation of <n>GATA-2</n> activity through interactions with the <n>promyelocytic leukemia protein</n> (<n>PML</n>) and the t(15;17)-generated <n>PML-retinoic acid receptor alpha oncoprotein</n>. The hematopoietically expressed GATA family of transcription factors function as key regulators of blood cell fate. Among these, <n>GATA-2</n> is implicated in the survival and growth of multipotential progenitors. Here we report that the <n>promyelocytic leukemia protein</n> (<n>PML</n>) can complex with <n>GATA-2</n> and potentiate its transactivation capacity. The binding is mediated through interaction of the zinc finger region of <n>GATA-2</n> and the B-box domain of <n>PML</n>. The B-box region of <n>PML</n> is retained in the <n>PML</n>-<n>RARalpha</n> (<n>retinoic acid receptor alpha</n>) fusion protein generated by the t(15;17) translocation characteristic of acute promyelocytic leukemia (APL). Consistent with this, we provide evidence that <n>GATA-2</n> can physically associate with <n>PML</n>-<n>RARalpha</n>. Functional experiments further demonstrated that this interaction has the capacity to render GATA-dependent transcription inducible by retinoic acid, raising the possibility that GATA target genes may be involved in the molecular pathogenesis of APL.
10921882>Negative regulation of <n>PI 3-kinase</n> by <n>Ruk</n>, a novel adaptor protein. <n>Class I(A) phosphatidylinositol 3-kinase</n> (<n>PI 3-kinase</n>) is a key component of important intracellular signalling cascades. We have identified an adaptor protein, <n>Ruk(l)</n>, which forms complexes with the <n>PI 3-kinase</n> holoenzyme in vitro and in vivo. This interaction involves the proline-rich region of <n>Ruk</n> and the SH3 domain of the p85 alpha regulatory subunit of the <n>class I(A) PI 3-kinase</n>. In contrast to many other adaptor proteins that activate <n>PI 3-kinase</n>, interaction with <n>Ruk</n>(l) substantially inhibits the lipid kinase activity of the enzyme. Overexpression of <n>Ruk</n>(l) in cultured primary neurons induces apoptosis, an effect that could be reversed by co-expression of constitutively activated forms of the p110 alpha catalytic subunit of <n>PI 3-kinase</n> or its downstream effector <n>PKB</n>/<n>Akt</n>. Our data provide evidence for the existence of a negative regulator of the <n>PI 3-kinase</n> signalling pathway that is essential for maintaining cellular homeostasis. Structural similarities between <n>Ruk</n>, <n>CIN85</n> and <n>CD2AP</n>/<n>CMS</n> suggest that these proteins form a novel family of adaptor molecules that are involved in various intracellular signalling pathways.
10908344>The human <n>RAD18 gene product</n> interacts with <n>HHR6A</n> and <n>HHR6B</n>. During DNA replication, lesion bypass is an important cellular response to unrepaired damage in the genome. In the yeast Saccharomyces cerevisiae, <n>Rad6</n> and <n>Rad18</n> are required for both the error-free and error-prone lesion bypass mechanisms. Furthermore, <n>Rad6</n>-<n>Rad18</n> interaction is thought to be critical at an early step during lesion bypass in yeast. Two closely related human homologs of yeast <n>Rad6</n> have been identified as <n>HHR6A</n> and <n>HHR6B</n>. Here, we report a full-length cDNA coding for the human homolog of yeast <n>Rad18</n>. The human RAD18 gene codes for a protein of 484 amino acid residues with a calculated molecular weight of 54 804 Da, and the gene is localized to chromosome 3 between reference intervals D3S3591 and D3S1283. Human <n>RAD18 protein</n> (<n>hRAD18</n>) was found to interact with <n>HHR6A</n> and <n>HHR6B</n>. When co-expressed in yeast cells, stable <n>hRAD18</n>-<n>HHR6A</n> and <n>hRAD18</n>-<n>HHR6B</n> protein complexes were identified and purified to near homogeneity. Thus, through interaction and complex formation with <n>HHR6A</n> and <n>HHR6B</n>, <n>RAD18 protein</n> may play an important role in lesion bypass mechanisms in humans. Consistent with its role as a fundamental lesion bypass protein, the RAD18 gene is ubiquitously expressed in various human tissues.
10884290><n>Apolipoprotein E</n>;-<n>low density lipoprotein receptor</n> interaction. Influences of basic residue and amphipathic alpha-helix organization in the ligand. Conserved lysines and arginines within amino acids 140-150 of <n>apolipoprotein (apo) E</n> are crucial for the interaction between <n>apoE</n> and the <n>low density lipoprotein receptor</n> (<n>LDLR</n>). To explore the roles of amphipathic alpha-helix and basic residue organization in the binding process, we performed site-directed mutagenesis on the 22-kDa fragment of <n>apoE</n> (amino acids 1-191). Exchange of lysine and arginine at positions 143, 146, and 147 demonstrated that a positive charge rather than a specific basic residue is required at these positions. Consistent with this finding, substitution of neutral amino acids for the lysines at positions 143 and 146 reduced the binding affinity to about 30% of the wild-type value. This reduction corresponds to a decrease in free energy of binding of approximately 600 cal/mol, consistent with the elimination of a hydrogen-bonded ion pair (salt bridge) between a lysine on <n>apoE</n> and an acidic residue on the <n>LDLR</n>. Binding activity was similarly reduced when K143 and K146 were both mutated to arginine (K143R + K146R), indicating that more than the side-chain positive charge can be important. Exchanging lysines and leucines indicated that the amphipathic alpha-helical structure of amino acids 140-150 is critical for normal binding to the <n>low density lipoprotein receptor</n>.
10862613><n>SUMO-1</n> conjugation to human <n>DNA topoisomerase II</n> isozymes. <n>Topoisomerase I</n>-mediated DNA damage induced by camptothecin has been shown to induce rapid small <n>ubiquitin</n>-related modifier <n>(SUMO)-1</n> conjugation to <n>topoisomerase I</n>. In the current study, we show that <n>topoisomerase II</n>-mediated DNA damage induced by teniposide (VM-26) results in the formation of high molecular weight conjugates of both <n>topoisomerase II alpha</n> and <n>II beta</n> isozymes in HeLa cells. Immunological characterization of these conjugates suggests that both <n>topoisomerase II alpha</n> and <n>II beta</n> isozymes are conjugated to <n>SUMO-1</n>. The involvement of <n>SUMO-1</n>/<n>UBC9</n> in the modification of <n>topoisomerase II</n> isozymes is also supported by the demonstration of physical interaction between <n>topoisomerase II</n> and <n>SUMO-1</n>/<n>UBC9</n>. Surprisingly, <n>ICRF-193</n>, which does not induce <n>topoisomerase II</n>-mediated DNA damage but traps <n>topoisomerase II</n> into a circular clamp conformation, is also shown to induce similar <n>SUMO-1</n> conjugation to <n>topoisomerase II</n> isozymes. In addition, we show that both oxidative and heat shock stresses, which can cause protein damage, rapidly increase nuclear <n>SUMO-1</n> conjugates. These studies raise the question on whether <n>SUMO-1</n> conjugation to topoisomerases is an indirect result of a DNA damage response or a direct result because of protein conformational changes.
10851237>Specific sequences of the <n>Sm</n> and <n>Sm-like</n> (<n>Lsm</n>) proteins mediate their interaction with the <n>spinal muscular atrophy disease gene product</n> (<n>SMN</n>). The <n>spinal muscular atrophy disease gene product</n> (<n>SMN</n>) is crucial for <n>small nuclear ribonuclear protein</n> (<n>snRNP</n>) biogenesis in the cytoplasm and plays a role in pre-mRNA splicing in the nucleus. <n>SMN</n> oligomers interact avidly with the <n>snRNP</n> core proteins <n>SmB</n>, <n>-D1</n>, and <n>-D3</n>. We have delineated the specific sequences in the <n>Sm</n> proteins that mediate their interaction with <n>SMN</n>. We show that unique carboxyl-terminal arginine- and glycine-rich domains comprising the last 29 amino acids of <n>SmD1</n> and the last 32 amino acids of <n>SmD3</n> are necessary and sufficient for <n>SMN</n> binding. Interestingly, <n>SMN</n> also interacts with at least two of the U6-associated <n>Sm-like</n> (<n>Lsm</n>) proteins, <n>Lsm4</n> and <n>Lsm6</n>. Furthermore, the carboxyl-terminal arginine- and glycine-rich domain of <n>Lsm4</n> directly interacts with <n>SMN</n>. This suggests that <n>SMN</n> also functions in the assembly of the U6 snRNP in the nucleus and in the assembly of other <n>Lsm</n>-containing complexes. These findings demonstrate that arginine- and glycine-rich domains are necessary and sufficient for <n>SMN</n> interaction, and they expand further the range of targets of the <n>SMN</n> protein.
10831590>Sites in the A2 subunit involved in the interfactor VIIIa interaction. <n>Factor VIIIa</n> is a trimer of the A1, A2, and A3-C1-C2 subunits. Regions in the A2 subunit that interact with the A1/A3-C1-C2 dimer were localized using synthetic peptides derived from A2 sequences showing high probability of being surface exposed. Peptides were restricted to residues 373-562 of A2 based on the earlier observation that this region of A2 reacts with A1 using a zero length cross-linker. Peptides were assessed for their capacity to inhibit the reconstitution of <n>factor VIIIa</n> from the isolated A1/A3-C1-C2 dimer and A2 subunit. Reconstitution was monitored using both regeneration of <n>factor VIIIa</n> activity and fluorescence quenching of an acrylodan-labeled A2 (Ac-A2) by fluorescein-labeled A1/A3-C1-C2. The activity assay identified four peptides as inhibitors, residues 373-395 (IC(50) = 65 micrometer), 418-428 (IC(50) = 25 micrometer), 482-493 (IC(50) = 325 micrometer), and 518-533 (IC(50) = 585 micrometer). The 373-395 and 518-533 peptides eliminated the fluorescence quenching of Ac-A2, whereas the 418-428 peptide reduced but did not eliminate Ac-A2 quenching. Peptide 482-493 had no effect on the fluorescence quenching of Ac-A2 suggesting that the peptide did not directly affect reassociation of the <n>factor VIIIa</n> subunits. These results identify three regions in the A2 subunit (373-395, 418-428, and 518-533) that interact with the A1/A3-C1-C2 dimer. Furthermore, comparison of results obtained using the two assays distinguish inhibition of the intersubunit interactions from intermolecular interactions.
10821684>Interaction of <n>charybdotoxin S10A</n> with single <n>maxi-K channels</n>: kinetics of blockade depend on the presence of the beta 1 subunit. The <n>maxi-K channel</n> from bovine aortic smooth muscle consists of a pore-forming alpha subunit and a regulatory beta1 subunit that modifies the biophysical and pharmacological properties of the alpha subunit. In the present study, we examine <n>ChTX-S10A</n> blocking kinetics of single <n>maxi-K channels</n> in planar lipid bilayers from smooth muscle or from tsA-201 cells transiently transfected with either alpha or alpha+beta 1 subunits. Under low external ionic strength conditions, <n>maxi-K channels</n> from smooth muscle showed <n>ChTX-S10A</n> block times, 48 +/- 12 s, that were similar to those expressing alpha+beta 1 subunits, 51 +/- 16 s. In contrast, with the alpha subunit alone, <n>ChTX-S10A</n> block times were much shorter, 5 +/- 0.6 s, and were qualitatively similar to previously reported values for the skeletal muscle <n>maxi-K channel</n>. Increasing the external ionic strength caused a decrease in <n>ChTX-S10A</n> block times for <n>maxi-K channel</n> complexes of alpha+beta 1 subunits but not of alpha subunits alone. These findings indicate that it may be possible to predict the association of beta 1 subunits with native <n>maxi-K channels</n> by monitoring the kinetics of <n>ChTX</n> blockade of single channels, and they suggest that <n>maxi-K channels</n> in skeletal muscle do not contain a beta 1 subunit like the one present in smooth muscle. To further test this hypothesis, we examined the binding and cross-linking properties of [(125)I]-<n>IbTX</n>-D19Y/Y36F to both bovine smooth muscle and rabbit skeletal muscle membranes. [(125)I]-<n>IbTX</n>-D19Y/Y36F binds to rabbit skeletal muscle membranes with the same affinity as it does to smooth muscle membranes. However, specific cross-linking of [(125)I]-<n>IbTX</n>-D19Y/Y36F was observed into the beta 1 subunit of smooth muscle but not in skeletal muscle. Taken together, these data suggest that studies of <n>ChTX</n> block of single <n>maxi-K channels</n> provide an approach for characterizing structural and functional features of the alpha/beta 1 interaction.
10802067>The Krüppel-like transcriptional factors <n>Zf9</n> and <n>GKLF</n> coactivate the human <n>keratin 4</n> promoter and physically interact. <n>Zf9</n>/<n>CPBP</n>/<n>KLF6</n> is a widely expressed member of the Krüppel-like family of transcriptional factors which regulates gene expression in hepatic stellate cells. Because of its ubiquitous expression including in the esophagus, we have explored its function in the esophageal squamous epithelium, a model system to study cellular proliferation and differentiation. Reverse transcription-PCR (RT-PCR) and Western blot analyses revealed that <n>Zf9</n> was highly expressed in human esophageal squamous cancer cell lines. Additionally, <n>Zf9</n> localizes to the esophageal squamous epithelium by immunohistochemistry. Using transient transfection, <n>Zf9</n> transactivates the human <n>keratin 4</n> (K4) promoter reporter gene construct in a subset of the esophageal cancer cell lines through indirect mechanisms. Co-transfection of <n>Zf9</n> and <n>GKLF</n>/<n>KLF4</n>, which is also a member of the Krüppel-like factors and expressed in the esophageal squamous epithelium, leads to coactivation in an additive fashion. Furthermore, we demonstrate that there is a physical interaction between <n>GKLF</n> and <n>Zf9</n>, a novel finding for Krüppel-like family members.
10788327>Uncoupling <n>actin</n> filament fragmentation by <n>cofilin</n> from increased subunit turnover. The <n>actin depolymerizing factor</n> (<n>ADF</n>)/<n>cofilin</n> family of proteins interact with <n>actin</n> monomers and filaments in a pH-sensitive manner. When <n>ADF</n>/<n>cofilin</n> binds <n>F-actin</n> it induces a change in the helical twist and fragmentation; it also accelerates the dissociation of subunits from the pointed ends of filaments, thereby increasing treadmilling or depolymerization. Using site-directed mutagenesis we characterized the two <n>actin</n>-binding sites on human <n>cofilin</n>. One target site was chosen because we previously showed that the <n>villin</n> head piece competes with <n>ADF</n> for binding to <n>F-actin</n>. Limited sequence homology between <n>ADF</n>/<n>cofilin</n> and the part of the <n>villin</n> headpiece essential for actin binding suggested an actin-binding site on <n>cofilin</n> involving a structural loop at the opposite end of the molecule to the alpha-helix already implicated in <n>actin</n> binding. Binding through the alpha-helix is primarily to monomeric <n>actin</n>, whereas the loop region is specifically involved in filament association. We have characterized the <n>actin</n> binding properties of each site independently of the other. Mutation of a single lysine residue in the loop region abolishes binding to filaments, but not to monomers. Using the mutation analogous to the phosphorylated form of <n>cofilin</n> (<n>S3D</n>), we show that filament binding is inhibited at physiological ionic strength but not under low salt conditions. At low ionic strength, this mutant induces both the twist change and fragmentation characteristic of wild-type <n>cofilin</n>, but does not activate subunit dissociation. The results suggest a two-site binding to filaments, initiated by association through the loop site, followed by interaction with the adjacent subunit through the "helix" site at the opposite end of the molecule. Together, these interactions induce twist and fragmentation of filaments, but the twist change itself is not responsible for the enhanced rate of <n>actin</n> subunit release from filaments.
10772640>Stability and interactions of recombinant human <n>nerve growth factor</n> in different biological matrices: in vitro and in vivo studies. The purpose of this investigation was to characterize the stability, activity, and interactions of recombinant human <n>nerve growth factor</n> (<n>rhNGF</n>) in various biological matrices in vitro and in vivo. <n>rhNGF</n> (10 microg/ml) remained stable in human plasma for up to 4 days at 37 degrees C. There was a decrease in the recovery of <n>rhNGF</n> after incubation at lower concentrations (20 ng/ml) and for longer time periods (3 and 5 days at 37 degrees C). Size exclusion HPLC analysis indicated that <n>rhNGF</n> forms high molecular weight (HMW) complexes after long incubation periods. We confirmed that <n>alpha(2)-macroglobulin</n> (<n>alpha(2)M</n>) is the major plasma component that binds to <n>rhNGF</n>. Furthermore, this interaction was considerably increased by treatment of plasma with primary amines such as CH(3)NH(2). Changes in the pH environment did not affect the interaction of <n>rhNGF</n> with <n>alpha(2)M</n>. We also determined that the binding of <n>rhNGF</n> to CH(3)NH(2)-treated pure <n>alpha(2)M</n> or <n>alpha(2)M</n> present in human plasma substantially diminished its immunoreactivity and bioactivity detection. The interaction of <n>rhNGF</n> with activated <n>alpha(2)M</n> was reversed and inhibited by coincubation with dimethyl sulfoxide. Released <n>rhNGF</n> under these conditions was fully bioactive. (125)I-<n>rhNGF</n> also binds to <n>alpha(2)M</n> by forming similar (125)I-<n>rhNGF</n>/HMW complexes in plasma after i.v. administration in rats and mice. Sixty minutes after dosing in rats, most of the labeled material was in the form of a (125)I-<n>rhNGF</n>/HMW complex. These studies have provided a better understanding of the nature of the interactions of <n>rhNGF</n> with plasma components as well as methods to enhance, reverse, and inhibit these interactions.
10759550><n>Cadherin</n> interaction probed by atomic force microscopy. Single molecule atomic force microscopy was used to characterize structure, binding strength (unbinding force), and binding kinetics of a classical <n>cadherin</n>, <n>vascular endothelial (VE)-cadherin</n>, secreted by transfected Chinese hamster ovary cells as cis-dimerized full-length external domain fused to Fc-portion of human <n>IgG</n>. In physiological buffer, the external domain of <n>VE-cadherin</n> dimers is a approximately 20-nm-long rod-shaped molecule that collapses and dissociates into monomers (V-shaped structures) in the absence of Ca(2+). Trans-interaction of dimers is a low-affinity reaction (K(D) = 10(-3)-10(-5) M, k(off) = 1.8 s(-1), k(on) = 10(3)-10(5) M(-1) x s(-1)) with relatively low unbinding force (35-55 pN at retrace velocities of 200-4,000 nm x s(-1)). Higher order unbinding forces, that increase with interaction time, indicate association of <n>cadherins</n> into complexes with cumulative binding strength. These observations favor a model by which the inherently weak unit binding strength and affinity of <n>cadherin</n> trans-interaction requires clustering and cytoskeletal immobilization for amplification. Binding is regulated by low-affinity Ca(2+) binding sites (K(D) = 1.15 mM) with high cooperativity (Hill coefficient of 5.04). Local changes of free extracellular Ca(2+) in the narrow intercellular space may be of physiological importance to facilitate rapid remodeling of intercellular adhesion and communication.
10745073>Interaction of a novel cysteine and histidine-rich cytoplasmic protein with <n>galectin-3</n> in a carbohydrate-independent manner. We have used the yeast two-hybrid system to search for cytoplasmic proteins that might assist in the intracellular trafficking of the soluble <n>beta-galactoside-binding protein</n>, <n>galectin-3</n>. We utilised as bait murine full-length <n>galectin-3</n> to screen a murine 3T3 cDNA library. Several interacting clones were found to encode a partial open reading frame and a full-length clone was obtained by rapid amplification of cDNA ends methodology. In various assays in vitro the novel protein was shown to bind <n>galectin-3</n> in a carbohydrate-independent manner. The novel protein contains an unusually high content of cysteine and histidine residues and shows significant sequence homologies with several metal ion-binding motifs present in known proteins. Confocal immunofluorescence microscopy of permeabilised 3T3 cells shows a prominent perinuclear, as well as cytoplasmic, localisation of the novel protein.
10722602>Staphylococcus aureus <n>protein A</n> recognizes platelet <n>gC1qR</n>/<n>p33</n>: a novel mechanism for staphylococcal interactions with platelets. The adhesion of Staphylococcus aureus to platelets is a major determinant of virulence in the pathogenesis of endocarditis. Molecular mechanisms mediating S. aureus interactions with platelets, however, are incompletely understood. The present study describes the interaction between S. aureus <n>protein A</n> and <n>gC1qR</n>/<n>p33</n>, a multifunctional, ubiquitously distributed cellular protein, initially described as a binding site for the globular heads of <n>C1q</n>. Suspensions of fixed S. aureus or purified <n>protein A</n>, chemically cross-linked to agarose support beads, were found to capture native <n>gC1qR</n> from whole platelets. Moreover, biotinylated <n>protein A</n> bound specifically to fixed, adherent, human platelets. This interaction was inhibited by unlabeled <n>protein A</n>, soluble recombinant <n>gC1qR</n> (<n>rgC1qR</n>), or anti-<n>gC1qR</n> antibody F(ab')(2) fragments. The interaction between <n>protein A</n> and platelet <n>gC1qR</n> was underscored by studies illustrating preferential recognition of the <n>protein A</n>-bearing S. aureus Cowan I strain by <n>gC1qR</n> compared to recognition of the <n>protein A</n>-deficient Wood 46 strain, as well as inhibition of S. aureus Cowan I strain adhesion to immobilized platelets by soluble <n>protein A</n>. Further characterization of the <n>protein A</n>-<n>gC1qR</n> interaction by solid-phase enzyme-linked immunosorbent assay techniques measuring biotinylated <n>gC1qR</n> binding to immobilized <n>protein A</n> revealed specific binding that was inhibited by soluble <n>protein A</n> with a 50% inhibitory concentration of (3.3 +/- 0.7) x 10(-7) M (mean +/- standard deviation; n = 3). Rabbit <n>immunoglobulin G</n> (<n>IgG</n>) also prevented <n>gC1qR</n>-<n>protein A</n> interactions, and inactivation of <n>protein A</n> tyrosil residues by hyperiodination, previously reported to prevent the binding of <n>IgG</n> Fc, but not Fab, domains to <n>protein A</n>, abrogated <n>gC1qR</n> binding. These results suggest similar <n>protein A</n> structural requirements for <n>gC1qR</n> and <n>IgG</n> Fc binding. Further studies of structure and function using a truncated <n>gC1qR</n> mutant lacking amino acids 74 to 95 demonstrated that the <n>protein A</n> binding domain lies outside of the <n>gC1qR</n> amino-terminal alpha helix, which contains binding sites for the globular heads of <n>C1q</n>. In conclusion, the data implicate the platelet <n>gC1qR</n> as a novel cellular binding site for staphylococcal <n>protein A</n> and suggest an additional mechanism for bacterial cell adhesion to sites of vascular injury and thrombosis.
10706722>Chemokines fail to up-regulate <n>beta 1 integrin</n>-dependent adhesion in human Th2 T lymphocytes. Th1 and Th2 cells are functionally distinct subsets of CD4+ T lymphocytes whose tissue-specific homing to sites of inflammation is regulated in part by the differential expression of <n>P-</n> and <n>E-selectin</n> ligands and selected chemokine receptors. Here we investigated the expression and function of <n>beta 1 integrins</n> in Th1 and Th2 cells polarized in vitro. Th1 lymphocytes adhere transiently to the extracellular matrix ligands <n>laminin 1</n> and <n>fibronectin</n> in response to chemokines such as <n>RANTES</n> and <n>stromal cell-derived factor-1</n>, and this process is paralleled by the activation of the <n>Rac1 GTPase</n> and by a rapid burst of <n>actin</n> polymerization. Selective inhibitors of <n>phosphoinositide-3 kinase</n> prevent efficiently all of the above processes, whereas the <n>protein kinase C</n> inhibitor bisindolylmaleimide prevents chemokine-induced adhesion without affecting <n>Rac1</n> activation and <n>actin</n> polymerization. Notably, chemokine-induced adhesion to <n>beta 1 integrin</n> ligands is markedly reduced in Th2 cells. Such a defect cannot be explained by a reduced sensitivity to chemokine stimulation in this T cell subset, nor by a defective activation of the signaling cascade involving <n>phosphoinositide-3 kinase</n>, <n>Rac1</n>, and <n>actin</n> turnover, as all these processes are activated at comparable levels by chemokines in the two subsets. We propose that reduced <n>beta 1 integrin</n>-mediated adhesion in Th2 cells may restrain their ability to invade and/or reside in sites of chronic inflammation, which are characterized by thickening of basement membranes and extensive fibrosis, requiring efficient interaction with organized extracellular matrices.
10679202>Cloning and characterization of a novel adaptor protein, <n>CIN85</n>, that interacts with <n>c-Cbl</n>. The <n>c-Cbl</n> protooncogene product is a prominent substrate of protein tyrosine kinases and is rapidly tyrosine-phosphorylated upon stimulation of a wide variety of cell-surface receptors. We have identified a novel <n>c-Cbl</n>-interacting protein termed <n>CIN85</n> with a molecular mass of 85 kDa which shows similarity to adaptor proteins, <n>CMS</n> and <n>CD2AP</n>. <n>CIN85</n> mRNA is expressed ubiquitously in normal human tissues and cancer cell lines analyzed. <n>CIN85</n> was basally associated with <n>c-Cbl</n>. For interaction of <n>CIN85</n> with <n>c-Cbl</n>, the second SH3 domain of <n>CIN85</n> was shown to serve as a central player. The <n>CIN85</n>-<n>c-Cbl</n> association was enhanced shortly after stimulation of 293 cells with <n>epidermal growth factor</n> (<n>EGF</n>) and gradually diminished to a basal level, which correlated with a tyrosine phosphorylation level of <n>c-Cbl</n>. Our results suggest that <n>CIN85</n> may play a specific role in the <n>EGF</n> receptor-mediated signaling cascade via its interaction with <n>c-Cbl</n>.
10644998><n>EB3</n>, a novel member of the <n>EB1</n> family preferentially expressed in the central nervous system, binds to a CNS-specific <n>APC</n> homologue. <n>APCL</n>, a homologue of the <n>adenomatous polyposis coli (APC) tumor suppressor</n>, can deplete cytoplasmic <n>beta-catenin</n> like <n>APC</n>. However, as its biological function remains unclear, we have been using a yeast two-hybrid system to search for proteins that associate with its carboxyl region. Among several cDNA clones we isolated from a fetal-brain cDNA library as candidates, six included an identical sequence with significant homology to <n>EB1</n>, a protein known to bind to <n>APC</n>. The full-length cDNA of this novel homologue of <n>EB1</n>, named <n>EB3</n>, encoded a protein of 282 amino acids with 54% identity to <n>EB1</n>, and it was expressed preferentially in brain tissue on Northern blots. Confocal microscopy demonstrated that exogenous <n>EB3</n>, like <n>EB1</n>, is associated with the cytoplasmic microtubule network. Moreover, in these experiments <n>EB3</n> and <n>APCL</n> appeared together in the perinucleus and the cytoplasmic microtubule network. Since <n>APCL</n> is also expressed highly and specifically in the central nervous system, <n>APCL</n>-<n>EB3</n> interaction may be specific to the CNS, possibly involving stability and/or extension of microtubules during neuritogenesis.
10617208>Inhibition of msl-2 splicing by <n>Sex-lethal</n> reveals interaction between <n>U2AF35</n> and the 3' splice site AG. The protein <n>Sex-lethal</n> (<n>SXL</n>) controls dosage compensation in Drosophila by inhibiting the splicing and translation of male-specific-lethal-2 (msl-2) transcripts. Here we report that splicing inhibition of msl-2 requires a binding site for <n>SXL</n> at the polypyrimidine (poly(Y)) tract associated with the 3' splice site, and an unusually long distance between the poly(Y) tract and the conserved AG dinucleotide at the 3' end of the intron. Only this combination allows efficient blockage of U2 small nuclear ribonucleoprotein particle binding and displacement of the large subunit of the U2 auxiliary factor (<n>U2AF65</n>) from the poly(Y) tract by <n>SXL</n>. Crosslinking experiments with ultraviolet light indicate that the small subunit of <n>U2AF</n> (<n>U2AF35</n>) contacts the AG dinucleotide only when located in proximity to the poly(Y) tract. This interaction stabilizes <n>U2AF65</n> binding such that <n>SXL</n> can no longer displace it from the poly(Y) tract. Our results reveal a novel function for <n>U2AF35</n>, a critical role for the 3' splice site AG at the earliest steps of spliceosome assembly and the need for a weakened <n>U2AF35</n>-AG interaction to regulate intron removal.
10601333><n>Gemin3</n>: A novel DEAD box protein that interacts with <n>SMN</n>, the spinal muscular atrophy gene product, and is a component of gems. The survival of motor neurons (<n>SMN</n>) gene is the disease gene of spinal muscular atrophy (SMA), a common motor neuron degenerative disease. The <n>SMN</n> protein is part of a complex containing several proteins, of which one, <n>SIP1</n> (<n>SMN interacting protein 1</n>), has been characterized so far. The <n>SMN</n> complex is found in both the cytoplasm and in the nucleus, where it is concentrated in bodies called gems. In the cytoplasm, <n>SMN</n> and <n>SIP1</n> interact with the <n>Sm</n> core proteins of spliceosomal small nuclear ribonucleoproteins (<n>snRNP</n>s), and they play a critical role in <n>snRNP</n> assembly. In the nucleus, <n>SMN</n> is required for pre-mRNA splicing, likely by serving in the regeneration of <n>snRNPs</n>. Here, we report the identification of another component of the <n>SMN</n> complex, a novel DEAD box putative RNA helicase, named <n>Gemin3</n>. <n>Gemin3</n> interacts directly with <n>SMN</n>, as well as with <n>SmB</n>, <n>SmD2</n>, and <n>SmD3</n>. Immunolocalization studies using mAbs to <n>Gemin3</n> show that it colocalizes with <n>SMN</n> in gems. <n>Gemin3</n> binds <n>SMN</n> via its unique COOH-terminal domain, and <n>SMN</n> mutations found in some SMA patients strongly reduce this interaction. The presence of a DEAD box motif in <n>Gemin3</n> suggests that it may provide the catalytic activity that plays a critical role in the function of the <n>SMN</n> complex on RNPs.
10572088>Kinetic resolution of two mechanisms for high-affinity <n>granulocyte-macrophage colony-stimulating factor</n> binding to its receptor. <n>Granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) is an important hematopoietic cytokine that exerts its effects by interaction with the <n>GM-CSF receptor</n> (<n>GMR</n>) on the surface of responsive cells. The <n>GM-CSF receptor</n> consists of two subunits: GMRalpha, which binds <n>GM-CSF</n> with low affinity, and <n>GMR</n>beta, which lacks intrinsic ligand-binding capability but complexes with GMRalpha to form a high-affinity receptor (<n>GMRalpha/beta</n>). We conducted dynamic kinetic analyses of <n>GM-CSF</n> receptors to define the role of GMRbeta in the interaction of ligand and receptor. Our data show that <n>GMRalpha/beta</n> exhibits a higher k(on) than GMRalpha, indicating that GMRbeta facilitates ligand acquisition to the binding pocket. Heterogeneity with regard to <n>GM-CSF</n> dissociation from <n>GMRalpha/beta</n> points to the presence of loose and tight ligand-receptor complexes in high-affinity binding. Although the loose complex has a k(off) similar to GMRalpha, the lower k(off) indicates that GMRbeta inhibits <n>GM-CSF</n> release from the tight receptor complex. The two rates of ligand dissociation may provide for discrete mechanisms of interaction between <n>GM-CSF</n> and its high-affinity receptor. These results show that the beta subunit functions to stabilize ligand binding as well as to facilitate ligand acquisition.
10551879><n>Cyclic AMP-dependent protein kinase</n> binding to A-kinase anchoring proteins in living cells by fluorescence resonance energy transfer of <n>green fluorescent protein</n> fusion proteins. A-kinase anchoring proteins tether <n>cAMP-dependent protein kinase</n> (<n>PKA</n>) to specific subcellular locations. The purpose of this study was to use fluorescence resonance energy transfer to monitor binding events in living cells between the type II regulatory subunit of <n>PKA</n> (RII) and the RII-binding domain of the human <n>thyroid RII anchoring protein</n> (<n>Ht31</n>), a peptide containing the PKA-binding domain of an A-kinase anchoring protein. RII was linked to <n>enhanced yellow fluorescent protein</n> (<n>EYFP</n>), <n>Ht31</n> was linked to <n>enhanced cyan fluorescent protein</n> (<n>ECFP</n>), and these constructs were coexpressed in Chinese hamster ovary cells. Upon excitation of the donor fluorophore, <n>Ht31</n>.<n>ECFP</n>, an increase in emission of the acceptor fluorophore, RII.<n>EYFP</n>, and a decrease in emission from <n>Ht31</n>.<n>ECFP</n> were observed. The emission ratio (acceptor/donor) was increased 2-fold (p &#60; 0.05) in cells expressing <n>Ht31</n>.<n>ECFP</n> and RII.<n>EYFP</n> compared with cells expressing <n>Ht31P</n>.<n>ECFP</n>, the inactive form of <n>Ht31</n>, and RII.<n>EYFP</n>. These results provide the first in vivo demonstration of RII/<n>Ht31</n> interaction in living cells and confirm previous in vitro findings of RII/<n>Ht31</n> binding. Using surface plasmon resonance, we also showed that the <n>green fluorescent protein</n> tags did not significantly alter the binding of <n>Ht31</n> to RII. Thus, fluorescence resonance energy transfer can be used to directly monitor protein-protein interactions of the <n>PKA</n> signaling pathway in living cells.
10527805>Alzheimer's disease associated <n>presenilin 1</n> interacts with HC5 and ZETA, subunits of the catalytic 20S proteasome. Proteolytic processing and degradation tightly regulate the amount of stable, functional <n>presenilin 1</n> (<n>PSEN1</n>) in the cell. The approximately 46-kDa <n>PSEN1</n> holoprotein is cleaved into an approximately 30-kDa N-terminal fragment (NTF) and an approximately 20-kDa C-terminal fragment (CTF) by an unknown protease. The fragments are stabilized in a high molecular weight complex and nonincorporated fragments and excess holoprotein are degraded by the 26S proteasome. The tight balance between, on the one hand, processing and incorporation into the stable complex and, on the other hand, proteolytic degradation of excess <n>PSEN1</n>, indicates that minor changes in one of these two processes could be pathologically relevant. Here we demonstrate the direct physical interaction between <n>PSEN1</n> and two subunits, HC5 and ZETA, of the 20S proteasome. These interactions were identified using an interaction trap screening and were further established in an in vitro binding assay. Furthermore, we were able to coimmunoprecipitate the transfected binding partners, as well as the endogenous <n>PSEN1</n> and ZETA proteins from HEK 293T cells. Finally, degradation of ubiquitinated wild-type and mutant <n>PSEN1</n> by the 26S proteasome was demonstrated. In conclusion, we report a direct interaction between <n>PSEN1</n> and subunits of the 20S catalytic particle of the 26S proteasome, further establishing the involvement of proteasomal degradation in the regulation of <n>PSEN1</n> turnover.
10506185>A peptide representing the carboxyl-terminal tail of the <n>met receptor</n> inhibits kinase activity and invasive growth. Interaction of the <n>hepatocyte growth factor</n> (<n>HGF</n>) with its receptor, the <n>Met tyrosine kinase</n>, results in invasive growth, a genetic program essential to embryonic development and implicated in tumor metastasis. Met-mediated invasive growth requires autophosphorylation of the receptor on tyrosines located in the kinase activation loop (Tyr(1234)-Tyr(1235)) and in the carboxyl-terminal tail (Tyr(1349)-Tyr(1356)). We report that peptides derived from the <n>met receptor</n> tail, but not from the activation loop, bind the receptor and inhibit the kinase activity in vitro. Cell delivery of the tail receptor peptide impairs <n>HGF</n>-dependent <n>Met</n> phosphorylation and downstream signaling. In normal and transformed epithelial cells, the tail receptor peptide inhibits <n>HGF</n>-mediated invasive growth, as measured by cell migration, invasiveness, and branched morphogenesis. The <n>Met</n> tail peptide inhibits the closely related <n>Ron receptor</n> but does not significantly affect the <n>epidermal growth factor</n>, <n>platelet-derived growth factor</n>, or <n>vascular endothelial growth factor receptor</n> activities. These experiments show that carboxyl-terminal sequences impair the catalytic properties of the <n>met receptor</n>, thus suggesting that in the resting state the nonphosphorylated tail acts as an intramolecular modulator. Furthermore, they provide a strategy to selectively target the MET proto-oncogene by using small, cell-permeable, peptide derivatives.
10487747><n>GRASP55</n>, a second mammalian <n>GRASP</n> protein involved in the stacking of Golgi cisternae in a cell-free system. We have identified a 55 kDa protein, named <n>GRASP55</n> (<n>Golgi reassembly stacking protein of 55 kDa</n>), as a component of the Golgi stacking machinery. <n>GRASP55</n> is homologous to <n>GRASP65</n>, an N-ethylmaleimide-sensitive membrane protein required for the stacking of Golgi cisternae in a cell-free system. <n>GRASP65</n> exists in a complex with the <n>vesicle docking protein receptor GM130</n> to which it binds directly, and the <n>membrane tethering protein p115</n>, which also functions in the stacking of Golgi cisternae. <n>GRASP55</n> binding to <n>GM130</n>, could not be detected using biochemical methods, although a weak interaction was detected with the yeast two-hybrid system. Cryo-electron microscopy revealed that <n>GRASP65</n>, like <n>GM130</n>, is present on the cis-Golgi, while <n>GRASP55</n> is on the medial-Golgi. Recombinant <n>GRASP55</n> and antibodies to the protein block the stacking of Golgi cisternae, which is similar to the observations made for <n>GRASP65</n>. These results demonstrate that <n>GRASP55</n> and <n>GRASP65</n> function in the stacking of Golgi cisternae.
10448035>Human <n>Rad51</n> amino acid residues required for <n>Rad52</n> binding. The <n>Rad51</n> protein, a homologue of the bacterial <n>RecA</n> protein, is an essential factor for both meiotic and mitotic recombination. The N-terminal domain of the human <n>Rad51</n> protein (<n>HsRad51</n>) directly interacts with DNA. Based on a yeast two-hybrid analysis, it has been reported that the N-terminal region of the Saccharomyces cerevisiae <n>Rad51</n> protein binds <n>Rad52</n>;S. cerevisiae <n>Rad51</n> and <n>Rad52</n> both activate the homologous pairing and strand exchange reactions. Here, we show that the <n>HsRad51</n> N-terminal region, which corresponds to the <n>Rad52</n>-binding region of <n>ScRad51</n>, does not exhibit strong binding to the human <n>Rad52</n> protein (<n>HsRad52</n>). To investigate its function, the C-terminal region of <n>HsRad51</n> was randomly mutagenized. Although this region includes the two segments corresponding to the putative DNA-binding sites of <n>RecA</n>, all seven of the mutants did not decrease, but instead slightly increased, the DNA binding. In contrast, we found that some of these <n>HsRad51</n> mutations significantly decreased the <n>HsRad52</n> binding. Therefore, we conclude that these amino acid residues are required for the <n>HsRad51</n>.<n>HsRad52</n> binding. <n>HsRad52</n>, as well as S. cerevisiae <n>Rad52</n>, promoted homologous pairing between ssDNA and dsDNA, and higher homologous pairing activity was observed in the presence of both <n>HsRad51</n> and <n>HsRad52</n> than with either <n>HsRad51</n> or <n>HsRad52</n> alone. The <n>HsRad51</n> F259V mutation, which strongly impaired the <n>HsRad52</n> binding, decreased the homologous pairing in the presence of both <n>HsRad51</n> and <n>HsRad52</n>, without affecting the homologous pairing by <n>HsRad51</n> alone. This result suggests the importance of the <n>HsRad51</n>.<n>HsRad52</n> interaction in homologous pairing.
10425523>Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat <n>insulin-like growth factor binding protein (IGFBP)-5</n> are important determinants in binding <n>IGF-I</n>. The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in <n>IGF</n> binding, but only recently have some of the critical residues in the <n>IGFBP</n> sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat <n>IGFBP-5</n> that are involved in binding <n>IGF-I</n>. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of <n>rIGFBP-5</n>. Relative to wild-type <n>rIGFBP-5</n>, an 8-fold reduction in affinity for human <n>IGF-I</n> was found for recombinant G203K protein in both <n>IGF-I</n> ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in <n>IGFBP-5</n> are important determinants in binding <n>IGF-I</n>, and due to their complete conservation in all <n>IGFBP</n> sequences, we suggest that they are likely to be involved in binding <n>IGF-I</n> in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of <n>IGFBP-5</n>, demonstrating that the C-terminus contains partially overlapping <n>IGF-I</n> and ECM binding sites. We therefore propose that <n>heparin</n> binding to basic amino acids in <n>IGFBP-5</n> between 201-218 may physically occlude subsequent interaction between <n>IGF-I</n> and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound <n>IGFBP-5</n> for <n>IGF-I</n>.
10400685>Intact LIM 3 and LIM 4 domains of <n>paxillin</n> are required for the association to a novel polyproline region (Pro 2) of <n>protein-tyrosine phosphatase-PEST</n>. The <n>focal adhesion protein p130</n>(<n>Cas</n>) was identified as a substrate for the <n>protein-tyrosine phosphatase (PTP)-PEST</n>, and the specificity of this interaction is mediated by a dual mechanism involving a Src homology 3 domain-mediated binding and PTP domain recognition. Recently, <n>paxillin</n> was also demonstrated to interact with <n>PTP-PEST</n> (Shen, Y., Schneider, G., Cloutier, J. F., Veillette, A., and Schaller, M. D. (1998) J. Biol. Chem. 273, 6474-6481). In the present study, we show that amino acids 344-397 of <n>PTP-PEST</n> are sufficient for the binding to <n>paxillin</n>. We demonstrate that a proline-rich segment of <n>PTP-PEST</n> (Pro 2), 355PPEPHPVPPILTPSPPSAFP374, is essential for this interaction in vivo. Furthermore, mutation of proline residues within the Pro 2 motif reveal that proline 362 is critical for the binding of <n>paxillin</n>. Conversely, using deletion and point mutants of <n>paxillin</n>, LIM 3 and 4 domains were both found to be necessary for binding of <n>PTP-PEST</n>. Finally, using a "substrate trapping" approach, we demonstrate that, unlike <n>p130</n>(<n>Cas</n>), <n>paxillin</n> is not a substrate for <n>PTP-PEST</n>. In conclusion, we show that a novel proline-rich motif found in <n>PTP-PEST</n> serves as a ligand for the LIM domains of <n>paxillin</n>. Interestingly, the focal adhesion targeting of <n>paxillin</n> is mediated by LIM 3. Thus, we propose that <n>PTP-PEST</n>, by a competition with the ligand of <n>paxillin</n> in the focal adhesion complex, could contribute to the removal of <n>paxillin</n> from the adhesion sites and consequently promote focal adhesion turnover.
10380882><n>MBP1</n>: a novel mutant <n>p53</n>-specific protein partner with oncogenic properties. Using a yeast two-hybrid screening strategy with a common tumour-derived <n>p53</n> mutant as bait, we identified several mutant <n>p53</n>-interacting partners including the known proteins wild-type (wt) <n>p53</n>, <n>hUBC9</n> and <n>GBP</n>/<n>PIAS1</n>. In addition, a novel protein partner was identified which we have termed <n>MBP1</n>, for <n>Mutant p53-Binding Protein 1</n>. <n>MBP1</n> is a new member of the emerging fibulin gene family, which currently comprises <n>fibulin-1</n>, <n>fibulin-2</n> and <n>S1-5</n>. Expression of <n>MBP1</n> mRNA is differentially regulated both temporally during development of the mouse embryo and in a tissue-specific manner within the adult. Specific interaction between <n>MBP1</n> and mutant <n>p53</n> was illustrated by both two-hybrid analysis in yeast and co-immunoprecipitation in mammalian cells. <n>MBP1</n> displayed the following order of binding specificity towards different <n>p53</n> forms: H175 > G281 > H273 > or = W248>wt <n>p53</n>. Thus, <n>MBP1</n> appears to bind preferentially to <n>p53</n> mutants of the 'structural' rather than 'contact' class, reflecting a potential bias towards those mutants having a significant alteration in conformation from that assumed by wt <n>p53</n>. We propose that <n>MBP1</n> is the product of a candidate oncogene as rates of both neoplastic transformation and tumour cell growth were shown to be significantly enhanced when the protein is ectopically overexpressed. Furthermore, <n>MBP1</n> may play a role in determining if a 'gain of function' effect is seen with certain <n>p53</n> mutants.
10357818><n>Latent membrane protein 1</n> of Epstein-Barr virus interacts with <n>JAK3</n> and activates STAT proteins. <n>Latent membrane protein 1</n> (<n>LMP1</n>) acts like a permanently activated receptor of the tumor necrosis factor (TNF)-receptor superfamily and is absolutely required for B cell immortalization by Epstein-Barr virus. Molecular and biochemical approaches demonstrated that <n>LMP1</n> usurps cellular signaling pathways resulting in the induction of <n>NF-kappaB</n> and <n>AP-1</n> via two C-terminal activating regions. We demonstrate here that a third region encompassing a proline rich sequence within the 33 bp repetitive stretch of <n>LMP1's</n> C-terminus is required for the activation of <n>Janus kinase 3</n> (<n>JAK3</n>). The interaction of <n>LMP1</n> and <n>JAK3</n> leads to the enhanced tyrosine auto/transphosphorylation of <n>JAK3</n> within minutes after crosslinking of a conditional <n>NGF</n>-R:<n>LMP1</n> chimera and is a prerequisite for the activation of STAT transcription factors. These results reveal a novel activating region in the <n>LMP1</n> C-terminus and identify the JAK/STAT pathway as a target of this viral integral membrane protein in B cells.
10318834>Shared and unique determinants of the <n>erythropoietin (EPO) receptor</n> are important for binding <n>EPO</n> and <n>EPO mimetic peptide</n>. We have shown previously that Phe93 in the extracellular domain of the <n>erythropoietin (EPO) receptor</n> (<n>EPOR</n>) is crucial for binding <n>EPO</n>. Substitution of Phe93 with alanine resulted in a dramatic decrease in <n>EPO</n> binding to the Escherichia coli-expressed extracellular domain of the <n>EPOR</n> (<n>EPO-binding protein</n> or <n>EBP</n>) and no detectable binding to full-length mutant receptor expressed in COS cells. Remarkably, Phe93 forms extensive contacts with a peptide ligand in the crystal structure of the <n>EBP</n> bound to an <n>EPO-mimetic peptide</n> (<n>EMP1</n>), suggesting that Phe93 is also important for <n>EMP1</n> binding. We used alanine substitution of <n>EBP</n> residues that contact <n>EMP1</n> in the crystal structure to investigate the function of these residues in both <n>EMP1</n> and <n>EPO</n> binding. The three largest hydrophobic contacts at Phe93, Met150, and Phe205 and a hydrogen bonding interaction at Thr151 were examined. Our results indicate that Phe93 and Phe205 are important for both <n>EPO</n> and <n>EMP1</n> binding, Met150 is not important for <n>EPO</n> binding but is critical for <n>EMP1</n> binding, and Thr151 is not important for binding either ligand. Thus, Phe93 and Phe205 are important binding determinants for both <n>EPO</n> and <n>EMP1</n>, even though these ligands share no sequence or structural homology, suggesting that these residues may represent a minimum epitope on the <n>EPOR</n> for productive ligand binding.
10219086>Directional binding of <n>HMG-I(Y)</n> on four-way junction DNA and the molecular basis for competitive binding with <n>HMG-1</n> and <n>histone H1</n>. <n>Histone H1</n>, <n>HMG-1</n> and <n>HMG-I(Y)</n> are mammalian nuclear proteins possessing distinctive DNA-binding domain structures that share the common property of preferentially binding to four-way junction (4H) DNA, an in vitro mimic of the in vivo genetic recombination intermediate known as the Holliday junction. Nevertheless, these three proteins bind to 4H DNA in vitro with very different affinities and in a mutually exclusive manner. To investigate the molecular basis for these distinctive binding characteristics, we employed base pair resolution hydroxyl radical footprinting to determine the precise sites of nucleotide interactions of both <n>HMG-1</n> and <n>histone H1</n> on 4H DNA and compared these contacts with those previously described for <n>HMG-I(Y)</n> on the same substrate. Each of these proteins had a unique binding pattern on 4H DNA and yet shared certain common nucleotide contacts on the arms of the 4H DNA molecule near the branch point. Both the <n>HMG-I(Y)</n> and <n>HMG-1</n> proteins made specific contacts across the 4H DNA branch point, as well as interacting at discrete sites on the arms, whereas the globular domain of <n>histone H1</n> bound exclusively to the arms of the 4H DNA substrate without contacting nucleotides at the crossover region. Experiments employing the chemical cleavage reagent 1, 10-orthophenanthroline copper(II) attached to the C-terminal end of a site-specifically mutagenized <n>HMG-I(Y)</n> protein molecule demonstrated that this protein binds to 4H DNA in a distinctly polar, direction-specific manner. Together these results provide an attractive molecular explanation for the observed mutually exclusive 4H DNA-binding characteristics of these proteins and also allow for critical assessment of proposed models for their interaction with 4H DNA substrates. The results also have important implications concerning the possible in vivo roles of <n>HMG-I(Y)</n>, <n>histone H1</n> and <n>HMG-1</n> in biological processes such as genetic recombination and retroviral integration.
10198166>Isolation and functional characterization of the human <n>90K</n> promoter. <n>90K</n> is a secreted protein thought to be involved in the body's defense against pathogens and cancer. To elucidate its transcriptional regulation, the promoter of human <n>90K</n> (HGMW-approved symbol <n>LGAL S3BP</n>) was isolated and characterized. Analysis of the 3. 3-kb 5'-flanking region revealed that it is a TATA-less promoter, but neither GC-rich nor dependent on SP1 sites. <n>RNase</n> protection assays detected one major transcription start site (+1) and several minor transcription start sites upstream and downstream. Deletion studies defined a minimal promoter (-103 --> -49) and indirectly suggested positive synergism between different elements within it. Consistent with the proposed function of <n>90K</n>, its promoter activity could be stimulated by poly(I). poly(C), mimicking viral infection. Two regions mediating induction by poly(I). poly(C) (-171 --> -112, -32 --> 46) were identified by deletion mutants. A small region around the minimal promoter (-99 --> -12) was highly homologous between human and mouse. While both human and mouse minimal promoters contained an <n>interferon</n>-responsive element (IRF-E), the human minimal promoter was not inducible by poly(I). poly(C) in contrast to that of the mouse. Point mutations 30 bp upstream of the IRF-E, however, conferred inducibility to the human minimal promoter, suggesting interaction between different promoter elements.
10089499>Biochemical and crystallographic characterization of homologous non-peptidic <n>thrombin</n> inhibitors having alternate binding modes. The X-ray crystallographic structure of [N-(3-phenylpropionyl)-N-(phenethyl)]-Gly-boroLys-OH (HPBK, Ki = 0. 42 nM, crystallographic R factor to 1.8 A resolution, 19.6%) complexed with human alpha-<n>thrombin</n> shows that the boron adopts a tetrahedral geometry and is covalently bonded to the active serine, Ser195. The HPBK phenethyl aromatic ring forms an edge-to-face interaction with the indole side chain of Trp215. Four HPBK analogs containing either electron-withdrawing or electron-donating substitutents at the 3' position of the phenethyl ring were synthesized in an attempt to modulate ligand affinity by inductive stabilization of the edge-to-face interaction. Refined crystallographic structures of the trifluoromethyl (Ki = 0.37 nM, crystallographic R factor to 2.0 A resolution = 18.7%), fluoro (Ki = 0.60; R factor to 2.3 A resolution = 18.4%), methoxy (Ki = 0.91 nM, R factor to 2.2 A resolution = 19.8%) and methyl (Ki = 0.20 nM, R factor to 2.5 A resolution = 16.9%) HPBK analogs complexed with <n>thrombin</n> revealed two binding modes for the closely related compounds. A less than 1.5-fold variation in affinity was observed for analogs (trifluoromethyl-HPBK and fluoro-HPBK) binding with the edge-to-face interaction. The slight inductive modulation is consistent with the overall weak nature of the edge-to-face interaction. Owing to an unexpected rotation of the phenethyl aromatic ring, the 3' substituent of two analogs, methoxy-HPBK and methyl-HPBK, made direct contact with the Trp215 indole side chain. Increased affinity of the 3' methyl analog is attributed to favorable interactions between the methyl group and the Trp215 indole ring. Differences in inhibitor, <n>thrombin</n> and solvent structure are discussed in detail. These results demonstrate the subtle interplay of weak forces that determine the equilibrium binding orientation of inhibitor, solvent and protein.
10050856>The <n>Rab5</n> effector <n>EEA1</n> is a core component of endosome docking. Intracellular membrane docking and fusion requires the interplay between soluble factors and SNAREs. The SNARE hypothesis postulates that pairing between a vesicular <n>v-SNARE</n> and a target membrane <n>z-SNARE</n> is the primary molecular interaction underlying the specificity of vesicle targeting as well as lipid bilayer fusion. This proposal is supported by recent studies using a minimal artificial system. However, several observations demonstrate that SNAREs function at multiple transport steps and can pair promiscuously, questioning the role of SNAREs in conveying vesicle targeting. Moreover, other proteins have been shown to be important in membrane docking or tethering. Therefore, if the minimal machinery is defined as the set of proteins sufficient to reproduce in vitro the fidelity of vesicle targeting, docking and fusion as in vivo, then SNAREs are not sufficient to specify vesicle targeting. Endosome fusion also requires cytosolic factors and is regulated by the small GTPase <n>Rab5</n>. Here we show that <n>Rab5</n>-interacting soluble proteins can completely substitute for cytosol in an in vivo endosome-fusion assay, and that the <n>Rab5</n> effector <n>EEA1</n> is the only factor necessary to confer minimal fusion activity. <n>Rab5</n> and other associated proteins seem to act upstream of <n>EEA1</n>, implying that <n>Rab5</n> effectors comprise both regulatory molecules and mechanical components of the membrane transport machinery. We further show that <n>EEA1</n> mediates endosome docking and, together with SNAREs, leads to membrane fusion.
9973489>Identification of residues in the first domain of human <n>Fc alpha receptor</n> essential for interaction with <n>IgA</n>. The FcR family contains multiple receptors for Igs, of which the most distantly related ( approximately 20%) is the <n>IgA</n> receptor (human <n>Fc alpha R</n>), being more homologous ( approximately 35%) to another family of killer-inhibitory receptor-related immunoreceptors with a 19q13.4 chromosomal location in humans. This study of the <n>Fc alpha R</n> demonstrated that, like several <n>IgG</n> receptors, <n>Fc alpha R</n> is a low affinity receptor for Ab (Ka approximately 106 M-1). Rapid dissociation of the rs<n>Fc alpha R</n>:<n>IgA</n> complex (t1/2 approximately 25 s) suggests that monomer <n>IgA</n> would bind transiently to cellular <n>Fc alpha R</n>s, while <n>IgA</n> immune complexes could bind avidly. Mutagenesis of histidyl 85 and arginyl 82, in the FG loop of domain 1, demonstrated that these residues were essential for the <n>IgA</n>-binding activity of <n>Fc alpha R</n>, while arginyl 87 makes a minor contribution to the binding activity of the receptor. This site is unusual among the Fc receptors (<n>Fc gamma RII</n>, <n>Fc gamma RIII</n>, and <n>Fc epsilon RI</n>), in which the ligand binding site is in domain 2 rather than domain 1, but like <n>Fc alpha R</n>, the FG loop comprises part of the ligand binding site. The putative F and G strands flanking the <n>Fc alpha R</n> ligand binding site are highly homologous in the other killer-inhibitory receptor-related immunoreceptors, suggesting they comprise a conserved structural element on which divergent FG loops are presented and participate in the specific ligand interactions of each of these receptors.
9890995>Definition of the sites of interaction between the protein <n>tyrosine phosphatase SHP-1</n> and <n>CD22</n>. <n>CD22</n> phosphorylation is an early event of B cell antigen receptor engagement and results in the recruitment of the negative regulatory tyrosine phosphatase, <n>SHP-1</n>. Peptides representing the potential phosphorylation sites within the cytoplasmic domain of <n>CD22</n> have been used to stimulate <n>SHP-1</n> catalytic activity and to inhibit the binding of <n>SHP-1</n> to <n>CD22</n> (Doody, G., Justement, L., Delibrias, C., Matthews, R., Lin, J., Thomas, M., and Fearon, D. (1995) Science 269, 242-244). However, the sites of phosphorylation within the cytoplasmic domain of <n>CD22</n> and the importance of each for the recruitment and activation of <n>SHP-1</n> remain unknown. Here we demonstrate that there are multiple sites within the cytoplasmic domain of <n>CD22</n> that interact with the Src homology 2 domains of <n>SHP-1</n>. Nevertheless, a minimum of two tyrosines in <n>CD22</n> is required for the association with <n>SHP-1</n>. Furthermore, both Src homology 2 domains of <n>SHP-1</n> are necessary for efficient binding to <n>CD22</n>.
9869654>Lipid binding-induced conformational changes in the N-terminal domain of human <n>apolipoprotein E</n>. The N-terminal domain of human <n>apolipoprotein E3</n> (<n>apoE3</n>) adopts an elongated, globular four helix bundle conformation in the lipid-free state. Upon lipid binding, the protein is thought to undergo a significant conformational change that is essential for manifestation of its <n>low density lipoprotein receptor</n> recognition properties. We have used fluorescence resonance energy transfer (FRET) to characterize helix repositioning which accompanies lipid interaction of this protein. ApoE3(1-183) possesses a single cysteine at position 112 and four tryptophan residues (positions 20, 26, 34, and 39). Modification of Cys112 with the chromophore, N-iodoacetyl-N'-(5-sulfo-1-naphthyl)etheylenediamine (AEDANS) was specific and did not alter the secondary structure content of the protein. The efficiency of energy transfer from donor Trp residues to the AEDANS moiety was 49% in buffer, consistent with close proximity of the chromophores. Guanidine HCl titration experiments induced characteristic changes in the efficiency of energy transfer, indicating that FRET data faithfully reports on the conformational status of the protein. Interaction of AEDANS-<n>apoE3</n>(1-183) with dimyristoylphosphatidylcholine to form disk particles, or with detergent micelles, resulted in large decreases in the efficiency of energy transfer. Distance calculations based on the FRET measurements revealed that lipid binding increases the average distance between the four Trp donors and the AEDANS acceptor from 23 A to 44 A.The results obtained demonstrate the utility of FRET to investigate conformational adaptations of exchangeable <n>apolipoproteins</n> and are consistent with the hypothesis that, upon lipid binding, <n>apoE3</n>(1-183) undergoes conformational opening, repositioning helix 1 and 3 to adopt a receptor-active conformation.
9843378>SH3 binding domains in the <n>dopamine D4 receptor</n>. The <n>dopamine D4 receptor</n> is a G protein-coupled receptor (GPCR) that belongs to the dopamine D2-like receptor family. Functionally, the D2-like receptors are characterized by their ability to inhibit <n>adenylyl cyclase</n>. The <n>dopamine D4 receptor</n> as well as many other catecholaminergic receptors contain several putative SH3 binding domains. Most of these sites in the <n>D4 receptor</n> are located in a polymorphic repeat sequence and flanking sequences in the third intracellular loop. Here we demonstrate that this region of the <n>D4 receptor</n> can interact with a large variety of SH3 domains of different origin. The strongest interactions were seen with the SH2-SH3 adapter proteins <n>Grb2</n> and <n>Nck</n>. The repeat sequence itself is not essential in this interaction. The data presented indicate that the different SH3 domains in the adapter proteins interact in a cooperative fashion with two distinct sites immediately upstream and downstream from the repeat sequence. Removal of all the putative SH3 binding domains in the third intracellular loop of the <n>dopamine D4 receptor</n> resulted in a receptor that could still bind spiperone and dopamine. Dopamine could not modulate the coupling of these mutant receptors to <n>adenylyl cyclase</n> and <n>MAPK</n>, although dopamine modulated receptor-G protein interaction appeared normal. The receptor deletion mutants show strong constitutive internalization that may account for the deficiency in functional activation of second messengers. The data indicates that the <n>D4 receptor</n> contains SH3 binding sites and that these sites fall within a region involved in the control of receptor internalization.
9820523>A site for <n>CD4</n> binding in the beta 1 domain of the MHC class II protein <n>HLA-DR1</n>. Using a lymphocyte binding assay, we have previously demonstrated that the <n>CD4</n> protein can mediate cell adhesion by direct interaction with MHC class II molecules. In this report, we have used this assay to test whether synthetic peptides, corresponding to DR beta sequences, could inhibit <n>CD4</n>-class II adhesion. A peptide derived from sequences within the <n>beta1</n> domain (DR beta 41-55), as well as two peptides derived from sequences within the beta 2 domain (DR beta 121-135 and DR beta 141-155), were shown to inhibit <n>CD4</n>-class II adhesion. Inasmuch as a site for <n>CD4</n> binding in the beta 2 domain had been previously documented, these studies were designed to investigate the role of the beta 1 domain as an additional site of interaction with <n>CD4</n>. Sixteen site-specific mutations were engineered within the <n>beta1</n> domain of DR beta 1*0101. Several mutations were shown to disrupt <n>CD4</n>-dependent T cell activation. Based on these results, we propose a model for the molecular interaction of <n>CD4</n> with MHC class II proteins in which both the beta 1 and beta 2 domains of class II interact with the two amino-terminal Ig-like domains of <n>CD4</n>.
9799109>Characterization of <n>brain PCTAIRE-1 kinase</n> immunoreactivity and its interactions with <n>p11</n> and <n>14-3-3</n> proteins. An antibody directed against the C-terminal part of <n>PCTAIRE-1</n> recognized three proteins in rodent brain. The high-molecular-mass band is most abundant in the cerebellum, hippocampus and cortex. It migrated at the same apparent molecular mass as recombinant <n>PCTAIRE-1</n> and interacted, like recombinant <n>PCTAIRE-1</n>, with <n>p11</n> and <n>14-3-3</n> proteins. Combination of <n>p11</n> or <n>14-3-3</n> affinity resins with immunoprecipitation and peptide elution allowed us to obtain a purified full-length <n>PCTAIRE-1</n> preparation having significant kinase activity. These results suggest that <n>PCTAIRE-1</n> is an active kinase in brain. The catalytic core region of <n>PCTAIRE-1</n> which is common for all cyclin-dependent kinases, does not interact with <n>p11</n> and <n>14-3-3</n> proteins in the two-hybrid assay. Full interaction with <n>p11</n> and <n>14-3-3</n> proteins requires both, the N-terminal and C-terminal ends of <n>PCTAIRE-1</n>, suggesting that complex three-dimensional arrangements are responsible for these interactions. A low-molecular-mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy-terminal part of <n>PCTAIRE-1</n>, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein yielded cDNA and genomic (a gene of about 10 kb on human chromosome 1q24-1q25 and clone 262D12) sequences, allowing us to compose a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100% identity with parts of the 26 kDa protein coding sequence. Our results allow us to attribute these widely distributed cDNA sequences to an existing 26-kDa protein and to localize a gene within two recently published genomic sequences.
9786917><n>CBP</n> alleviates the intramolecular inhibition of <n>ATF-2</n> function. The transcription factor <n>ATF-2</n> (also called <n>CRE-BP1</n>), whose DNA-binding domain consists of a basic amino acid cluster and a leucine zipper (b-ZIP) region, binds to the cAMP response element as a homodimer or as a heterodimer with <n>c-Jun</n>. The amino-terminal region of <n>ATF-2</n> containing the transcriptional activation domain is phosphorylated by stress-activated kinases, which leads to activation of <n>ATF-2</n>. We report here that <n>CBP</n>, which was originally identified as a co-activator of <n>CREB</n>, directly binds to the b-ZIP region of <n>ATF-2</n> via a Cys/His-rich region termed C/H2, and potentiates trans-activation by <n>ATF-2</n>. The b-ZIP region of <n>ATF-2</n> was previously shown to interact with the amino-terminal region intramolecularly and to inhibit trans-activating capacity. The binding of <n>CBP</n> to the b-ZIP region abrogates this intramolecular interaction. The <n>adenovirus 13S E1A protein</n> which binds to the b-ZIP region of <n>ATF-2</n> also inhibited this intramolecular interaction, suggesting that both <n>CBP</n> and <n>13S E1A</n> share a similar function as positive regulators of <n>ATF-2</n>. We found that the b-ZIP regions of <n>c-Jun</n> and <n>CREB</n> also interact with the C/H2 domain of <n>CBP</n>, suggesting that <n>CBP</n> acts as a regulator for a group of b-ZIP-containing proteins. These results shed light on a novel aspect of <n>CBP</n> function as a regulator for a group of b-ZIP-containing proteins.
9761476>Lysine-50 is a likely site for anchoring the <n>plasminogen</n> N-terminal peptide to lysine-binding kringles. Interactions between the kringle 4 (K4) domain of human <n>plasminogen</n> (<n>Pgn</n>) and segments of the N-terminal Glu1-Lys77 peptide (NTP) have been investigated via 1H-NMR at 500 MHz. NTP peptide stretches devoid of Lys residues but carrying an internal Arg residue show negligible affinity toward K4 (equilibrium association constant Ka &#60; 0.05 mM(-1)). In contrast, while most fragments containing an internal Lys residue exhibit affinities comparable to that shown by the blocked Lys derivative Nalpha-acetyl-L-lysine-methyl ester (Ka approximately 0.2 mM(-1), peptides encompassing Lys50O consistently show higher Ka values. Among the investigated linear peptides, Nalpha-acetyl-Ala-Phe-Tyr-His-Ser-Ser-Lys5O-Glu-Gln-NH2 (AcAFYHSK5OEQ-NH2) exhibits the strongest interaction with K4 (Ka approximately 1.4 mM(-1)), followed by AcYHSK50EQ-NH2 (Ka approximately 0.9 mM(-1)). Relative to the wild-type sequence, mutated hexapeptides exhibit lesser affinity for K4. When a Lys50 --> Ser mutation was introduced (==> AcYHSS50EQ-NH2), binding was abolished. The Ile27-lle56 construct (L-NTP) contains the Lys50 site within a loop constrained by two cystine bridges. The propensity of recombinant <n>Pgn</n> K1 (rK1) and K2 (rK2) modules, and of <n>Pgn</n> fragments encompassing the intact K4 and K5 domains, for binding L-NTP, was investigated. We find that L-NTP interacts with rK1, rK2, K4, and K5-all lysine-binding kringles-in a fashion that closely mimics what has been observed for the Glul-HSer57 N-terminal fragment of <n>Pgn</n> (CB-NTP). Thus, both the constellation of kringle lysine binding site (LBS) aromatic residues that are perturbed upon complexation of L-NTP and magnitudes of kringle-L-NTP binding affinities (rK1, Ka approximately 4.3 mM(-1); rK2, Ka approximately 3.7 mM(-1; K4, Ka approximately 6.4 mM(1); and K5, Ka approximately 2.1 mM(-1)) are essentially the same as for the corresponding kringle-CB-NTP pairs. Molecular modeling studies suggest that the Glu39-Lys50 stretch in NTP generates an area that complements, both topologically and electrostatically, the solvent-exposed kringle LBS surface.
9740790>The <n>V protein</n> of the paramyxovirus SV5 interacts with <n>damage-specific DNA binding protein</n>. The simian parainfluenza virus 5 (SV5) V/P gene encodes two proteins: <n>V</n> and the <n>phosphoprotein P</n>. The <n>V</n> and <n>P</n> proteins are amino coterminal for 164 residues, but they have unique carboxyl termini. The unique carboxyl terminus of <n>V</n> contains seven cysteine residues, resembles a zinc finger, and binds two atoms of zinc. In a <n>glutathione-S-transferase</n> (<n>GST</n>)-fusion protein selection of cell lysate assay, the <n>GST</n>-<n>V protein</n> was found to interact with the 127-kDa subunit (DDB1) of the <n>damage-specific DNA binding protein</n> (<n>DDB</n>) [also known as <n>UV-damaged DNA binding protein</n> (<n>UV-DDB</n>), <n>xeroderma pigmentosum group E binding factor</n> (<n>XPE-BF</n>), and the hepatitis B virus <n>X-associated protein 1</n> (<n>AP-1</n>)]. A reciprocal <n>GST</n>-DDB1 fusion protein selection assay of SV5-infected cell lysates showed that DDB1 and <n>V</n> interact, and it was found that <n>V</n> and DDB1 could be coimmunoprecipitated from SV5-infected cells or from cells expressing <n>V</n> and DDB1 using the vaccinia virus T7 expression system. The interaction of <n>V</n> and DDB1 involves the carboxyl-terminal domain of <n>V</n> in that either deletion of the <n>V</n> carboxyl-terminal domain or substitution of the cysteine residues (C189, C193, C205, C207, C210, C214, and C217) in the zinc-binding domain with alanine was able to disrupt binding to DDB1. The <n>V protein</n>s of the mumps virus, human parainfluenza virus 2 (hPIV2), and measles virus have also been found to interact with DDB1 in <n>GST</n>-fusion protein selection assays using in vitro transcribed and translated DDB1.
9716151>Effects of radiographic contrast agents on <n>thrombin</n> formation and activity. Clinical trials suggest that the risk of thrombosis during coronary angioplasty is lower with ionic contrast agents than with nonionic contrast agents. However, the molecular mechanisms underlying this effect are unknown. This study examined the effects of contrast agents on <n>thrombin</n> formation and its interaction with substrates, inhibitors, and ligands to define potential mechanisms by which contrast agents affect thrombus formation. Two ionic agents, diatrizoate and ioxaglate, and one nonionic agent, ioversol, were studied. Ionic agents inhibited <n>factor X</n> activation by the <n>tissue factor</n>-<n>factor VIIa</n> complex more potently than ioversol (53 +/- 3.7, 43.0 +/- 1.9, and 26.5 +/- 2.4% inhibition by diatrizoate, ioxaglate, and ioversol, respectively, at concentrations of 5%). Ionic contrast agents were potent inhibitors of <n>prothrombinase</n> function, inhibiting <n>thrombin</n> formation by >75% at contrast concentrations of 0.6% (p &#60;0.005). Ioversol inhibited <n>prothrombinase</n> to a significantly lesser extent than ionic agents. Clotting assays suggested that ioxaglate was the most potent inhibitor of <n>thrombin</n> generation in plasma despite having the least effect on <n>fibrin</n> polymerization. Contrast agents inhibited binding of <n>thrombin</n> to <n>fibrin</n>, with ionic agents producing a more potent effect than ioversol (p &#60;0.02). However, contrast agents did not inhibit <n>thrombin</n>-mediated platelet activation, had only a minor effect on inhibition of <n>thrombin</n> by <n>antithrombin III</n>, and did not affect <n>thrombin</n>-<n>hirudin</n> interactions. In summary, these studies identify specific mechanisms by which radiographic contrast agents inhibit <n>thrombin</n> formation and function -- i.e. inhibition of <n>tissue factor</n>-dependent <n>factor Xa</n> generation, inhibition of the <n>prothrombinase</n> complex, and inhibition of <n>thrombin</n> binding to <n>fibrin</n>. These findings may help to explain the reduced risk of thrombosis during coronary angioplasty associated with ionic contrast agents.
9683573>A novel human DnaJ protein, <n>hTid-1</n>, a homolog of the Drosophila tumor suppressor protein <n>Tid56</n>, can interact with the human papillomavirus type 16 <n>E7 oncoprotein</n>. We have cloned <n>hTid-1</n>, a human homolog of the Drosophila tumor suppressor protein <n>Tid56</n>, by virtue of its ability to form complexes with the human papillomavirus <n>E7 oncoprotein</n>. The carboxyl terminal cysteine-rich metal binding domain of <n>E7</n> is the major determinant for interaction with <n>hTid-1</n>. The carboxyl terminus of <n>E7</n> is essential for the functional and structural integrity of <n>E7</n> and has previously been shown to function as a multimerization domain. The <n>hTid-1</n> protein is a member of the DnaJ-family of chaperones. Its mRNA is widely expressed in human tissues, including the HPV-18-positive cervical carcinoma cell line HeLa and human genital keratinocytes, the normal host cells of the HPVs. The <n>hTid-1</n> gene has been mapped to the short arm of chromosome 16. The large tumor antigens of polyomaviruses encode functional J-domains that are important for viral replication as well as cellular transformation. The ability of HPV <n>E7</n> to interact with a cellular DnaJ protein suggests that these two viral oncoproteins may target common regulatory pathways through J-domains.
9657960>Chaperonins. The molecular chaperones are a diverse set of protein families required for the correct folding, transport and degradation of other proteins in vivo. There has been great progress in understanding the structure and mechanism of action of the chaperonin family, exemplified by Escherichia coli <n>GroEL</n>. The chaperonins are large, double-ring oligomeric proteins that act as containers for the folding of other protein subunits. Together with its co-protein <n>GroES</n>, <n>GroEL</n> binds non-native polypeptides and facilitates their refolding in an ATP-dependent manner. The action of the <n>ATPase</n> cycle causes the substrate-binding surface of <n>GroEL</n> to alternate in character between hydrophobic (binding/unfolding) and hydrophilic (release/folding). ATP binding initiates a series of dramatic conformational changes that bury the substrate-binding sites, lowering the affinity for non-native polypeptide. In the presence of ATP, <n>GroES</n> binds to <n>GroEL</n>, forming a large chamber that encapsulates substrate proteins for folding. For proteins whose folding is absolutely dependent on the full <n>GroE</n> system, ATP binding (but not hydrolysis) in the encapsulating ring is needed to initiate protein folding. Similarly, ATP binding, but not hydrolysis, in the opposite <n>GroEL</n> ring is needed to release <n>GroES</n>, thus opening the chamber. If the released substrate protein is still not correctly folded, it will go through another round of interaction with <n>GroEL</n>.
9575161>Characterization of human hect domain family members and their interaction with <n>UbcH5</n> and <n>UbcH7</n>. The hect domain protein family was originally identified by sequence similarity of its members to the C-terminal region of <n>E6-AP</n>, an E3 <n>ubiquitin</n>-protein ligase. Since the C terminus of <n>E6-AP</n> mediates thioester complex formation with <n>ubiquitin</n>, a necessary intermediate step in <n>E6-AP</n>-dependent <n>ubiquitination</n>, it was proposed that members of the hect domain family in general have E3 activity. The hect domain is approximately 350 amino acids in length, and we show here that the hect domain of <n>E6-AP</n> is necessary and sufficient for <n>ubiquitin</n> thioester adduct formation. Furthermore, the human genome encodes at least 20 different hect domain proteins, and in further support of the hypothesis that hect domain proteins represent a family of E3s, several of these are shown to form thioester complexes with <n>ubiquitin</n>. In addition, some hect domain proteins interact preferentially with <n>UbcH5</n>, whereas others interact with <n>UbcH7</n>, indicating that human hect domain proteins can be grouped into at least two classes based on their E2 specificity. Since E3s are thought to play a major role in substrate recognition, the presence of a large family of E3s should contribute to ensure the specificity and selectivity of <n>ubiquitin</n>-dependent proteolytic pathways.
9556563><n>Ciao 1</n> is a novel WD40 protein that interacts with the tumor suppressor protein <n>WT1</n>. The <n>Wilms tumor suppressor protein</n>, <n>WT1</n>, is a transcription factor capable of activating or repressing transcription of various cellular genes. The mechanisms involved in regulating the transcriptional activities of <n>WT1</n> are beginning to be unraveled. It appears that physical interactions of other cellular proteins (<n>p53</n> and <n>par-4</n>) with <n>WT1</n> can modulate the function of <n>WT1</n>. Here, we report the identification and cloning of a novel <n>WT1</n>-interacting protein termed <n>Ciao 1</n>, a member of the WD40 family of proteins. <n>Ciao 1</n> specifically interacts with <n>WT1</n> both in vitro and in vivo. This interaction alters the mobility of a <n>WT1</n>.DNA complex in gel shift assays, and results in a decrease in transcriptional activation mediated by <n>WT1</n>. <n>Ciao 1</n> does not inhibit binding of <n>WT1</n> to its consensus nucleotide sequence and does not affect the repression activity of <n>WT1</n>. Thus, <n>Ciao 1</n> appears to specifically modulate the transactivation activity of <n>WT1</n> and may function to regulate the physiological functions of <n>WT1</n> in cell growth and differentiation.
9571026>Structural and functional analysis of the 1:1 growth hormone:receptor complex reveals the molecular basis for receptor affinity. The designed G120R mutant of human growth hormone (<n>hGH</n>) is an antagonist and can bind only one molecule of the growth hormone receptor. We have determined the crystal structure of the 1:1 complex between this mutant and the receptor extracellular domain (hGHbp) at 2.6 A resolution, and used it to guide a detailed survey of the structural and functional basis for hormone-receptor recognition. The overall structure of the complex is very similar to the equivalent portion of the 1:2 complex, showing that formation of the active complex does not involve major conformational changes. However, a segment involved in receptor-receptor interactions in the 1:2 complex is disordered in this structure, suggesting that its productive conformation is stabilized by receptor dimerization.The hormone binding site of the receptor comprises a central hydrophobic patch dominated by Trp104 and Trp169, surrounded by a hydrophilic periphery containing several well-ordered water molecules. Previous alanine scanning showed that the hydrophobic "hot spot" confers most of the binding energy. The new structural data, coupled with binding and kinetic analysis of further mutants, indicate that the hot spot is assembled cooperatively and that many residues contribute indirectly to binding. Several hydrophobic residues serve to orient the key tryptophan residues; kinetic analysis suggests that Pro106 locks the Trp104 main-chain into a required conformation. The electrostatic contacts of Arg43 to <n>hGH</n> are less important than the intramolecular packing of its alkyl chain with Trp169. The true functional epitope that directly contributes binding energy may therefore comprise as few as six side-chains, participating mostly in alkyl-aromatic stacking interactions. Outside the functional epitope, multiple mutation of residues to alanine resulted in non-additive increases in affinity: up to tenfold for a hepta-alanine mutant. Contacts in the epitope periphery can therefore attenuate the affinity of the central hot spot, perhaps reflecting a role in conferring specificity to the interaction.
9563526>NMR analyses of the interactions of human <n>annexin I</n> with ATP, Ca2+, and Mg2+. Human <n>annexin I</n> is a member of the annexin family of calcium-dependent phospholipid binding proteins. The structure of an N-terminally truncated human <n>annexin I</n> (<n>delta-annexin I</n>) and its interactions with Ca2+, Mg2+, and ATP were studied at the atomic level using nuclear magnetic resonance (NMR) spectroscopy. Since <n>delta-annexin I</n> is a large protein, with a molecular weight of 35 kDa, a site-specific (carbonyl-13C, amide-15N) labeling technique was used to determine the interaction sites of <n>delta-annexin I</n> with Ca2+, Mg2+, and ATP. The 13C NMR study focused on the carbonyl carbon resonances of the histidine residues of <n>delta-annexin I</n>. We found that ATP binds to <n>delta-annexin I</n>, and that the ATP binding site is located in the 1-domain of <n>annexin I</n>. We also found that histidine-52 is involved in that site, and that the binding ratio of ATP to <n>delta-annexin I</n> is 1:1.
9488727>Association of <n>activating transcription factor 2</n> (<n>ATF2</n>) with the <n>ubiquitin</n>-conjugating enzyme <n>hUBC9</n>. Implication of the <n>ubiquitin</n>/proteasome pathway in regulation of <n>ATF2</n> in T cells. <n>activating transcription factor 2</n> (<n>ATF2</n>) is regulated by phosphorylation via the <n>Jun N-terminal kinase</n>, and its binding activity is markedly induced at late stages of T and B lymphocyte activation (Feuerstein, N., Firestein, R., Aiyer, N., Xiao, H., Murasko, D., and Cristofalo, V. (1996) J. Immunol. 156, 4582-4593). To identify proteins that interact specifically with <n>ATF2</n> in lymphocytes, the yeast two-hybrid interaction system was employed using <n>ATF2</n> cDNA as a "bait." In two separate screenings, a clone was identified that revealed a novel sequence with homology to several members of the <n>ubiquitin</n>-conjugating enzyme family. An identical sequence was recently reported as the human homolog of the yeast <n>UBC9</n>, <n>hUBC9</n>. Northern blot analysis revealed a 1.3-kilobase RNA transcript, which showed differential levels of expression in various human tissues and a moderate induction after a 48-h stimulation of peripheral blood T lymphocytes. An antibody that was generated against the bacterially expressed <n>glutathione S-transferase</n>-<n>hUBC9</n> detected a approximately 19-kDa protein, which localizes predominantly in the nuclei of T cells. Further quantitative assays using the yeast two-hybrid system confirmed a high and specific level of interaction of <n>hUBC9</n> with <n>ATF2</n> and lack of interaction with <n>lamin</n> or control vectors. Two other cyclic AMP-responsive element-binding transcription factors, <n>CREB</n> and <n>ATF1</n>, also showed significant levels of interaction with <n>hUBC9</n>. However, this interaction was severalfold lower as compared with <n>ATF2</n>. Far Western blot analysis confirmed the specific binding of <n>ATF2</n> and <n>hUBC9</n> also in vitro. Evidence is presented that indicates a physiological significance for the interaction of <n>hUBC9</n> with <n>ATF2</n>. (a) We show that <n>ATF2</n> is ubiquitinated in vivo and in vitro, and (b) <n>ATF2</n> ubiquitination in vitro is facilitated by addition of purified <n>hUBC9</n>. (c) <n>ATF2</n> is shown to undergo a proteolytic process, which is rapidly regulated upon T cell activation concomitant with induction of <n>ATF2</n> phosphorylation. (d) A proteasome inhibitor delays the down-regulation of <n>ATF2</n> phophorylation after T cell activation. Taken collectively, these results implicate a role for <n>hUBC9</n> and the <n>ubiquitin</n>/proteasome pathway in regulation of <n>ATF2</n> in T cells.
9514571>Binding of complement subcomponent <n>C1q</n> to Streptococcus pyogenes: evidence for interactions with the <n>M5</n> and <n>FcRA76</n> proteins. Binding of <n>C1q</n>, the first component of the complement system, to some human pathogens has been earlier reported. In the present study, direct binding of <n>C1q</n> to group A streptococci (GAS) of various serotypes as well as some other Gram-positive and Gram-negative species was demonstrated. The interaction between <n>C1q</n> and GAS was investigated more in detail. In hot neutral extracts of a number of GAS strains two components of 64 and 52 kDa, respectively, bound <n>C1q</n>; alkaline and SDS extracts yielded the 52 kDa component as the main <n>C1q</n>-binding substance. <n>Trypsin</n> treatment of the SDS extracts of two GAS strains suggested the <n>C1q</n>-binding component(s) to be of protein nature. <n>C1q</n>-binding material purified from the SDS extract of an avirulent strain, type T27, was separated in 12% SDS-PAGE and probed in Western blot with human <n>C1q</n> and <n>fibrinogen</n>, conjugated to <n>horse radish peroxidase</n> (<n>HRP</n>) as well as rabbit <n>IgG</n> antibodies complexed to <n>HRP</n> (PAP system). The 52 kDa component was non-reactive with <n>fibrinogen</n> or rabbit <n>IgG</n>. However, <n>C1q</n>-binding components purified from the alkaline extracts of two M-positive strains revealed strong binding of either <n>fibrinogen</n> (type M5) or both <n>fibrinogen</n> and rabbit <n>IgG</n> (type M76); the molecular mass of these components. 55 kDa and 43-40 kDa, respectively, was in agreement with the reported molecular mass of the <n>M5</n> and <n>FcRA76</n> proteins. Our findings suggest that <n>C1q</n> may interact with GAS through certain M-family proteins as well as by a so far unidentified surface factor of protein nature occurring in most GAS strains. The involvement of M-family proteins, regarded as virulence factors of these organisms, may suggest the interaction of GAS with <n>C1q</n> as biologically important.
9466043>DNA-binding activity of wild-type <n>p53</n> protein is mediated by the central part of the molecule and controlled by its C terminus. The DNA binding activity of wild type <n>p53</n> is central to its activity. The "central" part of the molecule, where most mutations appear in primary human tumors, is the actual DNA binding domain. The C-terminal part was shown to exert a negative effect on the DNA binding activity. In the present study we show that while anti-p53 antibodies recognizing the C terminus of the wild type <n>p53</n> facilitate DNA binding activity, blocking of the wild type specific epitope by specific anti-p53 antibodies, inhibited the DNA binding activity of the wild type <n>p53</n> protein. An alternatively spliced <n>p53</n> protein exhibits an augmented DNA binding activity. The fact that most <n>p53</n> mutants have lost the wild type <n>p53</n> conformation specific epitope, coupled with the observation that blocking of this site by binding specific antibodies, prevents the interaction of wild type <n>p53</n> with DNA, suggests that maintaining the correct structural conformation of this site is central for DNA binding activity. Still, the internal structure of the <n>p53</n> target and particularly the length of the sequence between the two tandem inverted repeats, is critical for protein-DNA interaction behavior.
9398285>Participation of the N-terminal region of <n>Cepsilon3</n> in the binding of human <n>IgE</n> to its high-affinity receptor <n>FcepsilonRI</n>. The binding of <n>immunoglobulin E</n> (<n>IgE</n>) to its high-affinity receptor (<n>FcepsilonRI</n>) expressed on mast cells and basophils is central to the development of an allergic reaction. Previous studies have implicated the third constant domain of <n>IgE</n>-Fc (<n>Cepsilon3</n>) as the site of the interaction with <n>FcepsilonRI</n>. We have prepared a series of site-directed mutants of human <n>IgE</n>-Fc, particularly focusing on the N-terminal "linker" region and AB loop of <n>Cepsilon3</n>. The kinetics of binding <n>IgE</n> and its Fc fragments to the immobilized receptor were determined by surface plasmon resonance (SPR), and two phases of binding were observed. We identified one mutation in the N-terminal linker region, R334S, that has a dramatic effect on binding. R334S lowers the affinity of <n>IgE</n>-Fc for <n>FcepsilonRI</n> by 120-fold, principally through an increase in the dissociation rate of the slower phase of the interaction. This mutation has a similar effect in <n>Fcepsilon3-4</n>, a truncated form of <n>IgE</n>-Fc which lacks the <n>Cepsilon2</n> domain pair, and thus it does not exert its effect through altering the quaternary structure of <n>IgE</n>-Fc, firmly implicating Arg334 as a contact residue in the complex. However R334S has no effect on the binding of <n>FcepsilonRII</n> (<n>CD23</n>), the low-affinity receptor for <n>IgE</n>, demonstrating the structural integrity of the mutated <n>IgE</n>-Fc. Circular dichroism spectroscopy and thermal stability studies further indicate that the R334S mutation does not disorder or destabilize the structure of <n>IgE</n>-Fc or <n>Fcepsilon3-4</n>. These results demonstrate the importance of the N-terminal linker region of <n>Cepsilon3</n> in the interaction of <n>IgE</n> with <n>FcepsilonRI</n>.
9420225>Amino-terminal substitutions in the <n>CCR5</n> coreceptor impair <n>gp120</n> binding and human immunodeficiency virus type 1 entry. The <n>CC-chemokine receptor</n> <n>CCR5</n> is required for the efficient fusion of macrophage (M)-tropic human immunodeficiency virus type 1 (HIV-1) strains with the plasma membrane of <n>CD4</n>+ cells and interacts directly with the viral surface glycoprotein <n>gp120</n>. Although receptor chimera studies have provided useful information, the domains of <n>CCR5</n> that function for HIV-1 entry, including the site of <n>gp120</n> interaction, have not been unambiguously identified. Here, we use site-directed, alanine-scanning mutagenesis of <n>CCR5</n> to show that substitutions of the negatively charged aspartic acid residues at positions 2 and 11 (D2A and D11A) and a glutamic acid residue at position 18 (E18A), individually or in combination, impair or abolish <n>CCR5</n>-mediated HIV-1 entry for the ADA and JR-FL M-tropic strains and the DH123 dual-tropic strain. These mutations also impair <n>Env</n>-mediated membrane fusion and the <n>gp120</n>-<n>CCR5</n> interaction. Of these three residues, only D11 is necessary for <n>CC-chemokine</n>-mediated inhibition of HIV-1 entry, which is, however, also dependent on other extracellular <n>CCR5</n> residues. Thus, the <n>gp120</n> and <n>CC-chemokine</n> binding sites on <n>CCR5</n> are only partially overlapping, and the former site requires negatively charged residues in the amino-terminal <n>CCR5</n> domain.
9352222>Increased expression of <n>CD44</n> on astrocytoma cells induced by binding <n>myelin basic protein</n>. An astrocytoma cell line (HTB-14), expressing high amounts of a <n>CD44</n> variant compared to other astrocytoma lines was shown to bind <n>myelin basic protein</n> to a greater extent than low expressing lines in a concentration-dependent manner. The <n>CD44</n> variant expressed by HTB-14 cells was determined to migrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular mass of 100 kDa compared to that from white matter which had a molecular mass of 80 kDa. The most cationic component of <n>myelin basic protein</n> (<n>MBP</n>), (component 1) bound more avidly than the least cationic isomer (component 8). Internalization of <n>MBP</n> was demonstrated by immunogold electron microscopy and was localized to the perinuclear area with some gold particles in the cytoplasm but not near the plasma membrane. Colocalization with <n>glial fibrillary acid protein</n> suggested an interaction between these two molecules. Binding and internalization of <n>MBP</n> was accompanied by an increase in <n>CD44</n> as determined by quantitation of gold particles and the measurement of <n>CD44</n> by sandwich enzyme-linked immunosorbent assay. The implication of these studies for the mechanism of demyelination is discussed.
9334241>The <n>Groucho</n>/<n>transducin</n>-like enhancer of split transcriptional repressors interact with the genetically defined amino-terminal silencing domain of <n>histone H3</n>. <n>Groucho</n> is a transcriptional repressor implicated in Notch signaling and involved in neural development and segmentation in Drosophila. We are investigating the molecular mechanisms underlying the functions of <n>Groucho</n> and its mammalian homologs, the <n>transducin-like Enhancer of split</n> (<n>TLE</n>) proteins. We report that <n>Groucho</n>/<n>TLEs</n> are associated with chromatin in live cells and that they co-purify with isolated histones. Affinity chromatography and far Western blotting studies show further that native <n>Groucho</n>/<n>TLE</n> proteins interact specifically with <n>histone H3</n> and not with other core histones. This interaction is mediated by the <n>H3</n> amino-terminal domain previously shown by genetic analysis in yeast to be essential for the role of <n>H3</n> in transcriptional silencing. We also demonstrate that <n>Groucho</n>/<n>TLEs</n> form oligomeric structures in vivo. These combined findings suggest that transcription complexes containing <n>Groucho</n>/<n>TLEs</n> may associate with chromatin through interactions with the amino terminus of <n>histone H3</n> and that these interactions may be propagated along the chromosome due to the ability of <n>Groucho</n>/<n>TLE</n>s to participate in higher order structures.
9311810>Herpes simplex virus 1 alpha regulatory protein <n>ICP0</n> interacts with and stabilizes the cell cycle regulator <n>cyclin D3</n>. The herpes simplex virus 1 (HSV-1) <n>infected-cell protein 0</n> (<n>ICP0</n>) has the characteristics of a promiscuous transactivator of genes introduced into cells by infection or transfection. To identify cellular proteins interacting with <n>ICP0</n>, we used a domain of exon II of <n>ICP0</n> that is known to be crucial for regulatory function of the protein as bait in the yeast two-hybrid screen. Our results were as follows. (i) A cDNA in a positive yeast colony was found to encode <n>cyclin D3</n>, a cell cycle regulator of G1 phase. (ii) A purified chimeric protein consisting of <n>glutathione S-transferase</n> (<n>GST</n>) fused to <n>cyclin D3</n> specifically formed complexes with <n>ICP0</n> contained in HSV-1-infected cell lysate. (iii) To enhance the expression of <n>cyclin D3</n>, the gene was inserted into the viral genome and overexpressed in infected cells. The overexpressed <n>cyclin D3</n> colocalized with <n>ICP0</n> in nuclear structures characteristic of ND10 and which earlier have been reported to contain <n>ICP0</n>. (iv) The accumulation of <n>cyclin D3</n> protein in Vero cells infected with an <n>alpha0</n> deletion mutant was reduced relative to that of cells infected with wild-type virus or a recombinant virus in which the deleted <n>alpha0</n> sequences were restored. (v) Lysates of Spodoptera frugiperda Sf9 cells doubly infected with baculoviruses genetically engineered to express <n>cyclin D3</n> and <n>cyclin-dependent kinase 4</n> (<n>CDK4</n>) phosphorylated <n>GST</n> fused to <n>retinoblastoma protein</n> (<n>GST</n>-<n>pRb</n>) but did not phosphorylate the <n>GST</n>-<n>alpha0</n>(20-241) or <n>GST</n>-<n>alpha0</n>(543-768) fusion protein or immunoprecipitated <n>ICP0</n> proteins. Moreover, the chimeric <n>GST</n>-<n>ICP0</n>(exon II) protein shown to bind <n>cyclin D3</n> had no effect on the activity of the kinase on <n>GST</n>-<n>pRb</n> when added to mixtures of lysates of Sf9 cells which coexpressed <n>cyclin D3</n> and <n>CDK4</n>. These results indicate that <n>ICP0</n> interacts with, colocalizes with, and stabilizes the <n>cyclin D3</n> cell cycle regulator and does not affect its interaction with the cyclin-dependent kinase.
9261175>Association of the <n>T-cell protein tyrosine phosphatase</n> with <n>nuclear import factor p97</n>. Alternative splicing of the <n>T-cell protein tyrosine phosphatase</n> (<n>TCPTP</n>) transcript generates two forms of the enzyme that differ at their extreme C termini: a 48-kDa endoplasmic reticulum-associated form and a 45-kDa nuclear form. By affinity chromatography, using <n>GST</n>-<n>TCPTP</n> fusion proteins, we have isolated three cytoplasmic proteins of 120, 116, and 97 kDa that interact with <n>TCPTP</n>. The <n>p120</n> protein associated with residues 377-415 from the C terminus of the 48-kDa form of <n>TCPTP</n>, whereas the recognition site for <n>p97</n> and <n>p116</n> was mapped to residues 350-381 encompassing the <n>TCPTP</n> nuclear localization sequence (NLS). The <n>TCPTP</n> NLS was shown to be bipartite, requiring basic residues 350-358 (basic cluster I) and 377-381 (basic cluster II), the sites of interaction with <n>p97</n> and <n>p116</n>, for efficient nuclear translocation. The interaction between <n>p97</n>, <n>p116</n>, and the <n>TCPTP</n> NLS appeared unique in that these proteins did not form a stable interaction with the classical NLS of <n>SV40 large T antigen</n> or the standard bipartite NLS of <n>nucleoplasmin</n>. Sequence analysis of <n>p97</n> identified it as the <n>nuclear import factor p97</n> (<n>importin</n>-beta), which is an essential component of the nuclear import machinery. In assays in vitro in permeabilized cells, <n>p97</n> was necessary but not sufficient for optimal nuclear import of <n>TCPTP</n>. We found that <n>TCPTP</n> co-immunoprecipitated with the <n>nuclear import factor p97</n> from cell lysates and that purified recombinant <n>p97</n> and <n>TCPTP</n> interacted directly in vitro. These results indicate selectivity in the binding of <n>p97</n> and <n>p116</n> to the <n>TCPTP</n> NLS and suggest that <n>p97</n> may mediate events that are distinct from the classical nuclear import process. Moreover, these results demonstrate that the C-terminal segment of <n>TCPTP</n> contains docking sites for interaction with proteins that may function to target the enzyme to defined intracellular locations and in the process regulate <n>TCPTP</n> function.
9202023>Characterization of the WW domain of human <n>yes-associated protein</n> and its polyproline-containing ligands. We had previously identified the WW domain as a novel globular domain that is composed of 38-40 semiconserved amino acids and is involved in mediating protein-protein interaction. The WW domain is shared by proteins of diverse functions including structural, regulatory, and signaling proteins in yeast, nematode, and mammals. Functionally it is similar to the Src homology 3 domain in that it binds polyproline ligands. By screening a 16-day mouse embryo expression library, we identified two putative ligands of the WW domain of <n>Yes kinase-associated protein</n> which we named <n>WW domain-binding proteins 1</n> and <n>2</n>. These proteins interacted with the WW domain via a short proline-rich motif with the consensus sequence of four consecutive prolines followed by a tyrosine. Herein, we report the cDNA cloning and characterization of the human orthologs of <n>WW domain-binding proteins 1</n> and <n>2</n>. The products encoded by these cDNA clones represent novel proteins with no known function. Furthermore, these proteins show no homology to each other except for a proline-rich motif. By fluorescence in situ hybridization on human metaphase chromosomes, we mapped the human genes for <n>WW domain-binding proteins 1</n> and <n>2</n> to chromosomes 2p12 and 17q25, respectively. In addition, using site-directed mutagenesis, we determined which residues in the WW domain of <n>Yes kinase-associated protein</n> are critical for binding. Finally, by synthesizing peptides in which the various positions of the four consecutive proline-tyrosine motif and the five surrounding residues were replaced by all possible amino acid residues, we further elucidated the binding requirements of this motif.
9209500>Characterization of mouse <n>ALCAM</n> (<n>CD166</n>): the <n>CD6</n>-binding domain is conserved in different homologs and mediates cross-species binding. Activated leukocyte cell adhesion molecule (<n>ALCAM</n>; <n>CD166</n>) is a member of the immunoglobulin gene superfamily (IgSF) which is expressed by activated leukocytes and thymic epithelial cells and is a ligand for the lymphocyte antigen <n>CD6</n>. Herein, we report on the isolation and characterization of cDNA clones encoding mouse <n>ALCAM</n> (<n>mALCAM</n>). Comparison of the predicted amino acid sequence of <n>mALCAM</n> and human <n>ALCAM</n> (<n>hALCAM</n>) showed an overall identity of 93%. Binding studies with truncated forms of the extracellular region of <n>mALCAM</n> showed that the <n>CD6</n> binding site is located in the N-terminal Ig-like domain and that <n>mALCAM</n> is capable of binding both human and mouse <n>CD6</n>. Mutagenesis studies on <n>hALCAM</n> suggested that residues critical for <n>CD6</n> binding map to the predicted A'GFCC'C beta-sheet of <n>ALCAM's</n> N-terminal binding domain. Residue differences in the N-terminal domains of <n>mALCAM</n> and <n>hALCAM</n> were analyzed with the aid of a molecular model of <n>ALCAM</n>. All residues critical for <n>CD6</n> binding are conserved in both <n>mALCAM</n> and <n>hALCAM</n>, whereas residue differences map to the predicted BED face which is opposite the <n>CD6</n> binding site on <n>hALCAM</n>. These findings provide a molecular rationale for the observed cross-species <n>CD6</n>/<n>ALCAM</n> interaction and the apparent inability to generate monoclonal antibodies (mAb) against the <n>CD6</n> binding site. RNA blot analysis showed that mRNA transcripts encoding <n>mALCAM</n> are expressed in the brain, lung, liver, and the kidney, as well as by activated leukocytes and a number of cell lines. A rat mAb specific for <n>mALCAM</n> was produced and by two-color immunofluorescence studies was shown to bind to both activated <n>CD4</n>+ and CD8+ T cells.
9144171>Protein binding and signaling properties of <n>RIN1</n> suggest a unique effector function. Human <n>RIN1</n> was first characterized as a <n>RAS</n> binding protein based on the properties of its carboxyl-terminal domain. We now show that full-length <n>RIN1</n> interacts with activated <n>RAS</n> in mammalian cells and defines a minimum region of 434 aa required for efficient <n>RAS</n> binding. <n>RIN1</n> interacts with the effector domain of <n>RAS</n> and employs some <n>RAS</n> determinants that are common to, and others that are distinct from, those required for the binding of <n>RAF1</n>, a known <n>RAS</n> effector. The same domain of <n>RIN1</n> that binds <n>RAS</n> also interacts with <n>14-3-3</n> proteins, extending the similarity between <n>RIN1</n> and other <n>RAS</n> effectors. When expressed in mammalian cells, the <n>RAS</n> binding domain of <n>RIN1</n> can act as a dominant negative signal transduction blocker. The amino-terminal domain of <n>RIN1</n> contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This <n>RIN1</n> sequence shows preferential binding to the ABL-SH3 domain in vitro. Moreover, the amino-terminal domain of <n>RIN1</n> directly associates with, and is tyrosine phosphorylated by, <n>c-ABL</n>. In addition, <n>RIN1</n> encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that <n>RIN1</n> is able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of <n>RIN1</n> regulation.
9103436><n>Adenosine deaminase</n> binding to human <n>CD26</n> is inhibited by <n>HIV-1 envelope glycoprotein</n> <n>gp120</n> and viral particles. <n>CD26</n>, known to be the <n>adenosine deaminase (ADA)-binding protein</n>, has been implicated in HIV infection. Several studies have revealed a correlation between depletion of <n>CD4</n>+/<n>CD26</n>+ T lymphocytes, increased serum levels of <n>ADA</n>, and the evolution of AIDS in infected individuals. We show that in human B and T cell lines, irrespective of <n>CD4</n> expression, 125I-labeled <n>ADA</n> binding to <n>CD26</n> is inhibited by recombinant soluble <n>HIV-1 envelope glycoprotein</n> <n>gp120</n> and by HIV-1 infectious particles. Accordingly, an anti-<n>CD4</n> mAb, which inhibits the binding of <n>gp120</n> to <n>CD4</n> and blocks viral infection, did not affect inhibition of 125I-labeled <n>ADA</n> binding to <n>CD26</n> by HIV particles. On the other hand, mAbs directed against the V3 loop and the C-terminal region of <n>gp120</n> abolished completely the inhibitory effect. Overlapping synthetic peptides covering the entire <n>gp120</n> sequence were tested to map the region in <n>gp120</n> responsible for <n>ADA</n> binding inhibition. Only peptides in the C3 region significantly inhibited the binding of <n>ADA</n> to <n>CD26</n>. These results provide indirect evidence for the interaction of <n>gp120</n> with <n>CD26</n> and indicate that a specific function of <n>gp120</n> is the inhibition of <n>ADA</n> binding to <n>CD26</n> in both <n>CD4</n>+ and <n>CD4</n>- cells. Because <n>ADA</n> deficiency leads to severe combined immunodefiency syndrome, it remains possible that HIV particle-mediated blockade of <n>ADA</n>-<n>CD26</n> interaction may have significant consequences in the pathogenesis of AIDS.
9054371>Native and multimeric <n>vitronectin</n> exhibit similar affinity for <n>heparin</n>. Differences in <n>heparin</n> binding properties induced upon denaturation are due to self-association into a multivalent form. For many years, the concept that the <n>heparin</n>-binding sequence is sequestered within <n>vitronectin</n> and exposed upon denaturation of the protein has guided experimental design and interpretation of related structure-function studies on the protein. To evaluate binding of <n>heparin</n> to both native and denatured/renatured <n>vitronectin</n>, methods for monitoring binding in solution have been developed. A fluorescence method based on changes in an extrinsic probe attached to <n>heparin</n> has been used to evaluate <n>heparin</n> binding to native and denatured/renatured <n>vitronectin</n>. This approach indicates that there are not major differences in intrinsic <n>heparin</n>-binding affinities between native and renatured protein and invalidate the currently accepted model for a cryptic <n>heparin</n>-binding sequence in the protein. Denaturation and renaturation of <n>vitronectin</n> under near physiological solution conditions is accompanied invariably by self-association of the protein into a multimeric form (Zhuang, P., Blackburn, M. N., and Peterson, C. B. (1996) J. Biol. Chem. 271, 14323-14332), resulting in exposure of multiple <n>heparin</n>-binding sites on the surface of the oligomer. On the basis of the binding data from solution studies and interaction of the native monomer and the denatured multimeric form of <n>vitronectin</n> with a <n>heparin</n> column, along with evaluation of the ionic strength dependence of <n>heparin</n> binding to these <n>vitronectin</n> forms in solution, an alternative model is favored to account for the altered <n>heparin</n> binding properties of <n>vitronectin</n> associated with denaturation of the protein. This model proposes that multivalent interactions between <n>heparin</n> and multimeric <n>vitronectin</n> are responsible for differences in <n>heparin</n> affinity chromatography and ionic strength dependence compared with the native protein.
9041197>Adhesion of multiple myeloma peripheral blood B cells to bone marrow fibroblasts: a requirement for <n>CD44</n> and <n>alpha4beta7</n>. We have earlier described the presence of phenotypically unusual monoclonal B cells within the peripheral blood of multiple myeloma (MM) patients. To determine the biological properties of these B cells as compared to B cells from normal donors, we investigated the potential of <n>CD19</n>+ MM blood B cells to adhere to endothelial cell and bone marrow (BM)-fibroblast monolayers. We find that 30-60% of freshly isolated <n>CD19</n>+ MM blood B cells adhere to endothelial cell monolayers, and 50-80% adhere to BM fibroblast monolayers. The adhesion of MM blood B cells to either monolayer was not increased by in vitro activation, suggesting that these cells were activated in vivo. In contrast, fewer than 10% of <n>CD19</n>+ B cells from peripheral blood of normal donors adhered. Function-blocking monoclonal antibodies (mAbs) were used to determine which adhesion receptors were involved in <n>CD19</n>+ MM blood B cell interaction with BM fibroblasts. mAbs against <n>very late antigen 4</n>, the beta7-integrin subunit, and <n>CD44</n>, but not mAbs against <n>very late antigen 5</n> and <n>beta1</n>, inhibited adhesion 61, 50, and 30%, respectively. The lack of inhibition with mAbs against <n>beta1</n> implicates <n>alpha4beta7</n> but not <n>alpha4beta1</n> in adhesion of <n>CD19</n>+ MM blood B cells. To determine the <n>alpha4beta7</n> ligand that mediated MM blood B cell adhesion, mAbs against <n>vascular cellular adhesion molecule 1</n> and <n>fibronectin</n>, as well as CS1 and RGD peptides, were used as inhibitors. These were unable to reduce the adhesion of <n>CD19</n>+ MM blood B cells to BM fibroblasts, suggesting that <n>fibronectin</n> and <n>vascular cellular adhesion molecule 1</n> are not involved in adhesion. Also, adhesion of MM blood B cells to <n>mucosal addressin cell adhesion molecule 1</n>-transfected Chinese hamster ovary cells was not enhanced compared to control-transfected Chinese hamster ovary cells, suggesting that <n>mucosal addressin cell adhesion molecule 1</n> was not promoting adhesion of these cells. These data implicate <n>CD44</n>:<n>HA</n> interactions, as well as <n>alpha4beta7</n> and an as yet unidentified ligand in the adhesion of in vivo activated MM blood B cell adhesion to BM fibroblasts. The adhesion properties of MM <n>CD19</n>+ B cells distinguishes them from normal B cells. Although the malignant status of these cells is as yet undefined, their adhesion properties implicate MM blood B cells in migratory spread of the disease.
9003062>Characterization of the binding of <n>amyloid-beta</n> peptide to cell culture-derived native <n>apolipoprotein E2</n>, <n>E3</n>, and <n>E4</n> isoforms and to isoforms from human plasma. The epsilon 4 allele of <n>apolipoprotein E</n> (<n>apoE</n>, protein; APOE, gene) is a major risk factor for Alzheimer's disease (AD). Genetically, the frequency of the epsilon 4 allele is enriched in early-onset sporadic, late-onset familial, and common late-onset sporadic AD. <n>ApoE</n> is found in the extracellular <n>amyloid-beta</n> (<n>A beta</n>) deposits that are characteristic features of AD. In this study, we examined the interaction between <n>A beta</n> and <n>apoE</n> isoforms. The <n>apoE</n> isoforms used in this study were either produced by stably transfected Chinese hamster ovary cells (CHO) or were from human plasma. We report that when similar concentrations of the <n>apoE</n> isoforms were used, native nonpurified <n>apoE3</n> from recombinant CHO-derived sources bound <n>A beta</n>, but <n>apoE4</n> did not. In fact, in our system, binding of recombinant <n>apoE4</n> to <n>A beta</n> was never detectable, even after incubation for 4 days. Furthermore, using the same assay conditions, native <n>apoE2</n>, like <n>apoE3</n>, binds <n>A beta</n> avidly. Furthermore, when human plasma <n>apoE</n> isoforms are tested in <n>A beta</n> binding experiments, <n>apoE3</n> bound <n>A beta</n> more avidly than <n>apoE4</n>, and a major <n>apoE</n>/<n>A beta</n> complex (the 40-kDa form) was observed with plasma <n>apoE3</n> but not <n>apoE4</n>. These data extend our understanding of <n>apoE</n> isoform-dependent binding of <n>A beta</n> by associating <n>apoE2</n> with efficient <n>apoE</n>/<n>A beta</n> complex formation and demonstrate that native <n>apoE3</n> (whether recombinant or derived from human plasma) forms sodium dodecyl sulfate-stable <n>apoE</n>/<n>A beta</n> complexes more readily than native <n>apoE4</n>. The different <n>A beta</n>-binding properties of native <n>apoE4</n> versus native <n>apoE3</n> provide insight into the molecular mechanisms by which the APOE epsilon 4 allele exerts its risk factor effects in AD.
8982277>A site of interaction between <n>pleckstrin's</n> PH domains and <n>G beta gamma</n>. <n>Pleckstrin</n> is a 40 kDa substrate for <n>protein kinase C</n> found in platelets and neutrophils. Based upon its sequence, <n>pleckstrin</n> contains two of the recently-described PH domains that are thought to be binding motifs for phosphatidyl 4,5-bisphosphate (PIP2) and/or <n>G protein beta gamma</n> heterodimers (<n>G beta gamma</n>). In the present studies we have examined the interaction between <n>pleckstrin</n> and <n>G beta gamma</n> by incubating <n>pleckstrin</n> fusion proteins with lysates from human platelets. In this analysis, both the N-terminal and C-terminal PH domains from <n>pleckstrin</n> bound <n>G beta gamma</n> in vitro, as did peptides containing as little as the first 30 residues of the C-terminal <n>pleckstrin</n> PH domain. Introduction of a point mutation into this region, analogous to the mutation in the <n>Btk</n> PH domain that causes X-linked immunodeficiency disease (XID) in mice, dramatically disrupted this interaction. We propose that <n>pleckstrin</n> may interact with <n>G beta gamma</n>, and that one potential site for this interaction involves the first 30 residues of <n>pleckstrin's</n> C-terminal PH domain.
8910522><n>Receptor-associated protein</n> and members of the <n>low density lipoprotein receptor</n> family share a common epitope. An extended model for the development of passive Heymann nephritis. Heymann nephritis is an experimental rat model for human membranous glomerulonephritis. Two target antigens have been identified in the proximal tubule brush border of rat kidneys. One of them is <n>megalin</n>, a 600-kDa membrane protein that belongs to the family of <n>low density lipoprotein receptor</n> (<n>LDLR</n>)-related proteins. The other one is <n>receptor-associated protein</n> (<n>RAP</n>), a polypeptide of 40 kDa that associates with members of the <n>LDLR</n> family. Here we show that antibodies produced against recombinant human <n>RAP</n> strongly cross-react with the chicken oocyte receptor for <n>very low density lipoprotein</n> and <n>vitellogenin</n> (<n>LR8</n>), and with two other members of the <n>LDLR</n> family, <n>LDLR-related protein</n> and <n>megalin</n>. The interaction of this antibody with <n>LR8</n> showed binding characteristics exactly as those demonstrated for the physiological ligands of this receptor, in that binding of the antibody: (i) is Ca2+-dependent; (ii) is abolished by unfolding of the cysteine-rich binding domain by reduction; and (iii) interferes with the binding of <n>very low density lipoprotein</n> and <n>vitellogenin</n>. Immunopurification of the <n>LR8</n>-specific subpopulation of the polyclonal antiserum yielded an <n>IgG</n> fraction strongly reacting with <n>LR8</n> as well as with <n>RAP</n>. Using recombinant fragments of <n>RAP</n> and peptide mapping, the cross-reacting epitope(s) could be narrowed down to three short sequences (5-7 residues) in the COOH-terminal part of the protein. After immunization with <n>RAP</n>, anti-<n>LR8</n> antibodies and anti-<n>RAP</n> antibodies arise simultaneously, indicating that the receptor-specific activity is not due to anti-idiotypic antibodies. These findings suggest the existence of a common epitope(s) on <n>RAP</n> and members of the LDL receptor family. Based on these results, we present an extended molecular model for the development of passive Heymann nephritis.
8900150>Transcriptional regulation of the human <n>alpha2(I) collagen</n> gene. Combined action of upstream stimulatory and inhibitory cis-acting elements. This study identifies three regions of the human <n>alpha2(I) collagen</n> promoter involved in the binding of nuclear factors. These regions include sequences from -173 to -155 (footprint I), -133 to -119 (footprint II), and -101 to -72 (footprint III). A novel positive cis-element containing a TCCTCC motif was identified within footprint II. In addition, we demonstrated that a pyrimidine-rich region within footprint I is a binding site for a transcriptional repressor, and a CCAAT motif within footprint III is a binding site for a transcriptional activator. Comparative functional analysis of the cis-acting elements within the proximal 350 base pairs of this promoter, including previously characterized Sp1 binding sites at -300, indicates that constitutive activity of this promoter is regulated equivalently by the three positive cis-acting elements at -300, -125, and -80. Mutations in the repressor site at -160 increase constitutive activity by 4-6-fold. However, simultaneous mutations of the repressor site and the cis-regulatory element at either the -300 or -125 sites result in no increase in constitutive transcription activity suggesting interaction between the activators and repressor elements. In contrast, simultaneous mutation of the CCAAT motif and the repressor site results in about a 4-fold increase, suggesting that activation via the CCAAT motif may be independent of this repressor.
8879225>The domain on the <n>Duffy blood group antigen</n> for binding Plasmodium vivax and P. knowlesi malarial parasites to erythrocytes. Plasmodium vivax and the related simian malarial parasite P. knowlesi use the <n>Duffy blood group antigen</n> as a receptor to invade human erythrocytes and region II of the parasite ligands for binding to this erythrocyte receptor. Here, we identify the peptide within the <n>Duffy blood group antigen</n> of human and rhesus erythrocytes to which the P. vivax and P. knowlesi ligands bind. Peptides from the NH2-terminal extracellular region of the <n>Duffy</n> antigen were tested for their ability to block the binding of erythrocytes to transfected Cos cells expressing on their surface region II of the <n>Duffy</n>-binding ligands. The binding site on the human <n>Duffy</n> antigen used by both the P. vivax and P. knowlesi ligands maps to a 35-amino acid region. A 34-amino acid peptide from the equivalent region of the rhesus <n>Duffy</n> antigen blocked the binding of P. vivax to human erythrocytes, although the P. vivax ligand expressed on Cos cells does not bind rhesus erythrocytes. The binding of the rhesus peptide, but not the rhesus erythrocyte, to the P. vivax ligand was explained by interference of carbohydrate with the binding process. Rhesus erythrocytes, treated with <n>N-glycanase</n>, bound specifically to P. vivax region II. Thus, the interaction of P. vivax ligand with human and rhesus erythrocytes appears to be mediated by a peptide-peptide interaction. Glycosylation of the rhesus <n>Duffy</n> antigen appears to block binding of the P. vivax ligand to rhesus erythrocytes.
8872163>The impact of <n>DR3</n> microvariation on peptide binding: the combinations of specific DR beta residues critical to binding differ for different peptides. HLA-DR molecules are a group of highly polymorphic glycoprotein heterodimers that present peptide antigens to T lymphocytes for immune surveillance. To assess the significance of limited polymorphism on the functional differentiation of DR molecules, the binding of several immunogenic peptides to the <n>DR3</n> microvariants [DR(alpha, beta 1*0302) and DR(alpha, beta 1*0301)] and to mutants of these <n>DR3</n> molecules was examined. This analysis has shown that each residue (DR beta 26, DR beta 28, DR beta 47, and DR beta 86), which differentiates these two <n>DR3</n> molecules, contributes to their functional distinction and that the relative contribution of each residue varies for different peptide/<n>DR3</n> complexes. For example, DR beta 28 and DR beta 86 controlled the mycobacterium tuberculosis <n>65-kD heat shock protein</n> peptides 3-13 and 4-15 (HSP) binding specificity to DR (alpha, beta 1*0301). [HSP does not bind to DR(alpha, beta 1*0302)], whereas DR beta 26, DR beta 28, and DR beta 86 controlled the influenza <n>hemagglutinin</n> peptide 306-318 (<n>HA</n>) binding specificity to DR(alpha, beta 1*0302). [<n>HA</n> does not bind to DR(alpha, beta 1*0301).] In comparison, DR beta 86 alone controlled the binding level difference of sperm whale <n>myoglobin</n> peptide 132-151 (<n>SWM</n>) and of <n>myelin basic protein</n> peptide 152-170 (<n>MBP</n>) [both bind to DR(alpha, beta 1*0301) at levels five times greater than to DR(alpha, beta 1*0302)] to the <n>DR3</n> molecules. Although not critical, additional DR beta residues influenced the binding level of individual peptides of each of the <n>DR3</n> molecules and, again, the combinations of these residues differed for different peptide/<n>DR3</n> complexes. These data showed that individual DR residues vary in their relative contribution to the interaction between a specific DR molecule and different peptides and that limited polymorphism can create substantial differences in the peptide binding profiles among DR molecules.
8702735>Defining a novel cis-element in the 3'-untranslated region of mammalian <n>ribonucleotide reductase</n> component R2 mRNA. cis-trans-interactions and message stability. Mammalian <n>ribonucleotide reductase</n> is a highly regulated activity essential for DNA synthesis and repair. The 3'-untranslated region (3'-UTR) of mammalian <n>ribonucleotide reductase</n> R2 mRNA has been implicated in the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate-mediated stabilization of mouse BALB/c 3T3 R2 message. We investigated the possibility that the 3'-UTR contains regulatory information for R2 mRNA turnover. Using 3'-end-labeled RNA in gel shift and UV cross-linking analyses, we detected in the 3'-UTR a novel 9-nucleotide cis-element, 5'-UCGUGUGCU-3', which interacted with a widely distributed cellular cytosolic protease-sensitive factor(s) in a sequence-specific manner to form a 45-kDa R2 binding protein complex. The binding activity was redox-sensitive and down-regulated by 12-O-tetradecanoylphorbol-13-acetate and okadaic acid in a dose-dependent manner. Insertion of a 154-base pair fragment containing the cis-element led to markedly reduced accumulation of chloramphenicol acetyltransferase hybrid mRNA relative to the same insert carrying a series of G --> A mutations within this element that eliminated binding. We suggest that the 9-nucleotide region functions as a destabilizing element. These results provide a model for <n>ribonucleotide reductase</n> gene expression through a novel and specific mRNA cis-trans-interaction involving a phosphorylation signal pathway that leads to changes in the stability of R2 message.
8764047>Interaction cloning of <n>NS1-I</n>, a human protein that binds to the nonstructural <n>NS1</n> proteins of influenza A and B viruses. The yeast interaction trap system was used to identify, <n>NS1-I</n> (for <n>NS1 interactor</n>), which is a human protein that binds to the nonstructural <n>NS1</n> protein of the influenza A virus. <n>NS1-I</n> is a human homolog of the porcine <n>17beta-estradiol dehydrogenase precursor protein</n>, to which it is 84% identical. We detected only one <n>NS1-I</n> mRNA species, of about 3.0 kb, in HeLa cells, and the <n>NS1-I</n> cDNA was found to have a coding capacity for a 79.6-kDa protein. However, immunoblot analysis detected predominantly a 55-kDa protein in human cells, suggesting that <n>NS1-I</n>, like the porcine <n>17beta-estradiol dehydrogenase</n>, is posttranslationally processed. Using an in vitro coprecipitation assay, we showed that <n>NS1-I</n> interacts with <n>NS1</n> proteins from extracts of cells infected with five different influenza A virus strains as well as with the <n>NS1</n> of an influenza B virus. The fact that influenza A and influenza B virus <n>NS1</n> proteins bind to <n>NS1-I</n> suggests that this cellular protein plays a role in the influenza virus life cycle.
8676466>Activation of transcription factor <n>NF-kappaB</n> by the <n>Tat</n> protein of human immunodeficiency virus type 1. A recombinant <n>Tat</n> protein was used to investigate the molecular mechanisms of transcriptional activation of the human immunodeficiency virus type 1 long terminal repeat (LTR). Liposome-mediated delivery of this protein to responsive cells results in dose-dependent LTR activation. As evaluated by mRNA quantitation with competitive PCR, the activation response is rapid and transient, peaking at 5 h after the beginning of <n>Tat</n> treatment. In vivo footprinting experiments at the LTR showed that transcriptional activation is concomitant with a modification of the protein-DNA interaction pattern at the downstream kappaB site of the enhancer and at the adjacent Sp1 boxes. The effects of <n>Tat</n> on the enhancer are mediated by <n>Tat</n>-induced nuclear translocation of <n>NF-kappaB</n>, which parallels the kinetics of transcriptional activation. This induction results from degradation of the inhibitor <n>IkappaB-alpha</n>, is blocked under antioxidant conditions and by a protease inhibitor, and occurs as a rapid response in different cell types. The functional response to <n>Tat</n> is impaired upon cell treatment with a kappaB site decoy or with sodium salicylate, an inhibitor of <n>NF-kappaB</n> activation. These results show that <n>NF-kappaB</n> activation by <n>Tat</n> is important for LTR transcriptional activation. Furthermore, they suggest that some of the pleiotropic effects of <n>Tat</n> on cellular functions can be mediated by induction of <n>NF-kappaB</n>.
8654375>The B cell coactivator <n>Bob1</n> shows DNA sequence-dependent complex formation with <n>Oct-1</n>/<n>Oct-2</n> factors, leading to differential promoter activation. We have shown previously that both octamer binding transcription factors, namely the ubiquitous <n>Oct-1</n> and the B cell-specific <n>Oct-2A</n> protein, can be enhanced in transcriptional activity by their association with the B cell-specific coactivator protein <n>Bob1</n>, also called <n>OBF-1</n> or <n>OCA-B</n>. Here we study the structural requirements for ternary complex formation of DNA-Oct-<n>Bob1</n> and coactivation function of <n>Bob1</n>. In analogy to DNA-bound transcription factors, <n>Bob1</n> has a modular structure that includes an interaction domain (amino acids 1-65) and a C-terminal domain (amino acids 65-256), both important for transcriptional activation. A mutational analysis has resolved a region of seven amino acids (amino acids 26-32) in the N-terminus of <n>Bob1</n> that are important for contacting the DNA binding POU domain of <n>Oct-1</n> or <n>Oct-2</n>. In contrast to the viral coactivator <n>VP16</n> (<n>vmw65</n>), which interacts with <n>Oct-1</n> via the POU homeosubdomain, <n>Bob1</n> association with Oct factors requires residues located in the POU-specific subdomain. Because the same residues are also involved in DNA recognition, we surmised that this association would affect the DNA binding specificity of the Oct-<n>Bob1</n> complex compared with free Oct factors. While <n>Oct-1</n> or <n>Oct-2</n> bind to a large variety of octamer sequences, <n>Bob1</n> ternary complex formation is indeed highly selective and occurs only in a subset of these sequences, leading to the differential coactivation of octamer-containing promoters. The results uncover a new level in selectivity that furthers our understanding in the regulation of cell type-specific gene expression.
8639776>Mouse <n>P-selectin glycoprotein ligand-1</n>: molecular cloning, chromosomal localization, and expression of a functional <n>P-selectin receptor</n>. A mouse homolog of <n>P-selectin glycoprotein ligand-1</n> (<n>PSGL-1</n>), a <n>P-selectin</n> receptor on myeloid cells, has been cloned using the human cDNA sequence to probe a cDNA library prepared from the mouse WEHI-3 monocytic cell line and a genomic DNA library prepared from 129/SvJ mouse tissue. The gene flanking the entire open reading frame of 397 amino acids is composed of a single exon. Mouse and human <n>PSGL-1</n> show an overall similarity of 67% and an identity of 50% and contain a similar domain organization. However, there are 10 threonine/serine-rich decameric repeats in mouse <n>PSGL-1</n> as compared with 15 threonine-rich repeats in human <n>PSGL-1</n>. When the mouse <n>PSGL-1</n> cDNA is coexpressed with an alpha 1,3/1,4 fucosyltransferase cDNA in COS cells, a functional protein is expressed on the COS cell surface mediating binding to human <n>P-selectin</n>. The mouse <n>PSGL-1</n> gene, Selpl, was mapped to a position on mouse chromosome 5 (Chr 5). Northern blot analyses of mouse tissues showed moderate expression of a <n>PSGL-1</n> mRNA species in most tissues including heart, kidney, liver, muscle, ovary, and stomach and high levels of expression in blood, bone marrow, brain, adipose tissue, spleen, and thymus. Whereas certain mouse myeloid cell lines including PU5-1.8, WEHI-3B, and 32DC13 express high levels of <n>PSGL-1</n> mRNA, only WEHI-3B and 32DC13 bind to <n>P-selectin</n>; this interaction is blocked by anti-<n>PSGL-1</n> antibody. WEHI-3B cells bind significantly better to <n>P-selectin</n> than to <n>E-selectin</n>. Although comparable <n>P-selectin</n> binding is observed in 32DC13 cells, these cells bind better to <n>E-selectin</n>. Binding of 32DC13 cells to <n>E-selectin</n> is not blocked by anti-<n>PSGL-1</n> antibody. Treatment of WEHI-3B cells with trypsin or neuraminidase abolished their ability to interact with <n>P-selectin</n>. These results indicate that mouse <n>PSGL-1</n> has structural and functional homology to human <n>PSGL-1</n> but is characterized by differences in the composition and number of the decameric repeats. <n>PSGL-1</n> on mouse myeloid cells is critical for high-affinity binding to <n>P-selectin</n> but not <n>E-selectin</n>.
8626553>Characterization of the interaction between <n>RhoGDI</n> and <n>Cdc42Hs</n> using fluorescence spectroscopy. The <n>GDP-dissociation-inhibitor</n> (<n>GDI</n>) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how <n>GDI</n> interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the <n>GDI</n> to the Rho-like protein, <n>Cdc42Hs</n>. We show here that when the <n>GDI</n> interacts with <n>Cdc42Hs</n> that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence. The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda-expressed <n>Cdc42Hs</n> and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed <n>Cdc42Hs</n> and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying <n>GDI</n> activity. A truncated <n>Cdc42Hs</n> mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to <n>GDI</n> regulation also does not show changes in the fluorescence of its bound Mant-GDP upon <n>GDI</n> addition. Thus, the GDI-induced quenching of Mant-GDP provides a direct read-out for the binding of the <n>GDI</n> to <n>Cdc42Hs</n>. Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for <n>Cdc42Hs</n>-GDI interactions with an apparent Kd value of 30 nM. A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to <n>Cdc42Hs</n>. These results suggest that the <n>GDI</n> can bind to the GDP-bound and GTP-bound forms of <n>Cdc42Hs</n> equally well. We also have used the fluorescence assay for <n>GDI</n> interactions to demonstrate that the differences in functional potency observed between the <n>GDI</n> molecule and a related human leukemic protein, designated <n>LD4</n>, are due to differences in their binding affinities for <n>Cdc42Hs</n>. This, together with the results from studies using <n>GDI</n>/<n>LD4</n> chimeras, allow us to conclude that a limit region within the carboxyl-terminal domain of the <n>GDI</n> molecule is responsible for its ability to bind with higher affinity (compared with <n>LD4</n>) to <n>Cdc42Hs</n>.
8807192>The type III connecting segment of <n>fibronectin</n> contains an aspartic acid residue that regulates the rate of binding to <n>integrin alpha 4 beta 1</n>. The type III connecting segment (IIICS) within <n>fibronectin</n> is the major binding site for the <n>integrin alpha 4 beta 1</n>. Most integrin ligands have an essential acidic residue within their integrin binding site, in IIICS this residue is hypothesized to be the aspartic acid at position 21. Alanine scanning mutagenesis was used to determine the amino acid residues within the intact IIICS domain required for interaction with <n>alpha 4 beta 1</n>. IIICS was cloned and expressed as a fusion protein with <n>glutathione S-transferase</n>. This recombinant form of IIICS supports the adhesion of CHO cells that express human <n>alpha 4 beta 1</n> in a cation dependent manner. Alanine scanning mutagenesis of the EILDVP sequence in recombinant IIICS demonstrated that only two of these residues are critical for adhesion of <n>alpha 4 beta 1</n> expressing cells. Mutations of leucine at position 20 and aspartic acid at position 21 to alanine significantly reduced cell adhesion. Conservative mutations of aspartic acid at position 21 to asparagine or glutamic acid also reduced the ability of the recombinant protein to support cell adhesion, although not to the same extent as the corresponding alanine replacement. Most importantly, we show that although the mutation of asp 21 impairs cell adhesion, an examination of cell adhesion as a function of time demonstrated that asp 21 is not necessary for cell adhesion through <n>alpha 4 beta 1</n>. In comparison to wild type IIICS, the asp 21 to ala mutant supported minimal adhesion at early time points (10-30 min.), but was equivalent to wild type IIICS in supporting adhesion over one hour.
8700889>Human immunodeficiency virus type 1 and 2 <n>Tat</n> proteins specifically interact with <n>RNA polymerase II</n>. The <n>Tat</n>-responsive region (TAR) element is a critical RNA regulatory element in the human immunodeficiency virus (HIV) long terminal repeat, which is required for activation of gene expression by the transactivator protein <n>Tat</n>. Recently, we demonstrated by gel-retardation analysis that <n>RNA polymerase II</n> binds to TAR RNA and that <n>Tat</n> prevents this binding even when <n>Tat</n> does not bind to TAR RNA. These results suggested that direct interactions between <n>Tat</n> and <n>RNA polymerase II</n> may prevent <n>RNA polymerase II</n> pausing and lead to <n>Tat</n>-mediated increases in transcriptional elongation. To test this possibility, we performed protein interaction studies with <n>RNA polymerase II</n> and both the HIV-1 and the closely related HIV-2 <n>Tat</n> protein. These studies indicated that both the HIV-1 and HIV-2 <n>Tat</n> proteins could specifically interact with <n>RNA polymerase II</n>. Mutagenesis of both HIV-1 and HIV-2 <n>Tat</n> demonstrated that the basic domains of both the HIV-1 and HIV-2 <n>Tat</n> proteins were required for this interaction. Furthermore, "far Western" analysis suggested that the largest subunit of <n>RNA polymerase II</n> was the site for interaction with <n>Tat</n>. The interactions between <n>Tat</n> and <n>RNA polymerase II</n> were of similar magnitude to those detected between <n>RNA polymerase II</n> and the cellular transcription factor <n>RAP30</n>, which stably associates with <n>RNA polymerase II</n> during transcriptional elongation. These studies are consistent with the model that <n>RNA polymerase II</n> is a cellular target for <n>Tat</n> resulting in <n>Tat</n>-mediated increases in transcriptional elongation from the HIV long terminal repeat.
8639676>Domain structure and conformation of <n>histidine-proline-rich glycoprotein</n>. The complete primary structure of rabbit plasma <n>histidine-proline-rich glycoprotein</n> (<n>HPRG</n>), also known as <n>histidine-rich glycoprotein</n>, was determined by a combination of cDNA and peptide sequencing. Limited proteolysis with <n>plasmin</n> yielded three disulfide-linked fragments that were further purified. Reduction of the disulfide bonds with dithiothreitol under nondenaturing conditions releases the central, histidine-proline-rich domain, which contains 15 tandem repeats of the pentapeptide [H/P]-[H/P]PHG. The N-terminal fragment (295 amino acids), consisting of two <n>cystatin</n>-like modules, is bound to the proline-rich C-terminal fragment (105 amino acids) via a buried disulfide bond whose reduction requires prior denaturation. Far-UV circular dichroism spectra revealed beta-sheet with some alpha-helix, polyproline-II helix, and random coil in the secondary structure of the N-terminal, central, and C-terminal domains, respectively. The modular architecture of <n>HPRG</n> suggests that it may have several independent binding sites and that its biological role may be to bring two or more ligands together. The histidine-proline-rich domain, which contains 34 of the 53 histidine residues of <n>HPRG</n>, binds <n>heparin</n> and has an isoelectric point of 7.15 and a relatively high apparent pKa (7.0) of its histidine residues, and thus it probably mediates the interaction between <n>HPRG</n> and <n>heparin</n>, which is strikingly sensitive to pH in the range 7.0-7.4 [Peterson et al. (1987) J. Biol. Chem. 262, 7567-7574]. Solvent perturbation and second-derivative UV spectroscopy of <n>HPRG</n> revealed changes in the environment of tryptophan residues upon lowering the pH. This transition had a midpoint at pH 6.0 and required the disulfide bond bridging the histidine-proline-rich domain to the N/C fragment. The data are consistent with the mutual repulsion of protonated histidine residues in the histidine-proline-rich region causing a conformational change transmitted to the rest of the molecule via the disulfide bond.
8550612>Transcriptional analysis of the 5'-noncoding region of the human <n>involucrin</n> gene. Human <n>involucrin</n> whose gene transcription is directed by a 2456-nucleotide (nt) 5'-noncoding region is a structural component of the epithelial cornified layer. Transient transfection assays demonstrated that this region is transcriptionally active in multiplying keratinocytes and is enhanced by 2 mM CaCl2 treatment. Calcium-independent transcriptional activity and the interaction with the <n>AP-1</n> transcriptional factor was located on the proximal part (nt -159 to -1) of the 5'-noncoding region. However, CaCl2 responsiveness was mapped to a distal 1185-nt fragment (nt -2456 to -1272). Moreover, this fragment potentiated the <n>Herpes simplex thymidine kinase</n> promoter in normal keratinocytes and is responsive to calcium treatment in a cell type-specific manner. Interestingly, the absence of a 491-nt fragment located between the two enhancer domains (nt -651 to -160) resulted in transcriptional activation in multiplying keratinocytes. This fragment interacts with <n>AP-1</n> and the YY1 transcriptional silencer. It is concluded that human <n>involucrin</n> 5'-noncoding region contains at least three regulatory domains, a distal CaCl2-responsive enhancer, a putative transcriptional silencer (that interacts with <n>AP-1</n> and YY1), and a proximal enhancer/promoter (that interacts with <n>AP-1</n>). Thus, this study demonstrates the presence of particular transcriptional factors can potentially regulate the human <n>involucrin</n> expression.
11357136><n>LDL-receptor-related protein 6</n> is a receptor for Dickkopf proteins. <n>Wnt glycoproteins</n> have been implicated in diverse processes during embryonic patterning in metazoa. They signal through frizzled-type seven-transmembrane-domain receptors to stabilize <n>beta-catenin</n>. <n>Wnt</n> signalling is antagonized by the extracellular <n>Wnt</n> inhibitor <n>dickkopf1</n> (<n>dkk1</n>), which is a member of a multigene family. <n>dkk1</n> was initially identified as a head inducer in Xenopus embryos but the mechanism by which it blocks <n>Wnt</n> signalling is unknown. <n>LDL-receptor-related protein 6</n> (<n>LRP6</n>) is required during <n>Wnt</n>/<n>beta-catenin</n> signalling in Drosophila, Xenopus and mouse, possibly acting as a co-receptor for <n>Wnt</n>. Here we show that <n>LRP6</n> (ref. 7) is a specific, high-affinity receptor for <n>Dkk1</n> and <n>Dkk2</n>. <n>Dkk1</n> blocks <n>LRP6</n>-mediated <n>Wnt</n>/<n>beta-catenin</n> signalling by interacting with domains that are distinct from those required for <n>Wnt</n>/<n>Frizzled</n> interaction. <n>dkk1</n> and <n>LRP6</n> interact antagonistically during embryonic head induction in Xenopus where <n>LRP6</n> promotes the posteriorizing role of <n>Wnt</n>/<n>beta-catenin</n> signalling. Thus, DKKs inhibit <n>Wnt</n> co-receptor function, exemplifying the modulation of LRP signalling by antagonists.
11302704>Homo-oligomerisation and nuclear localisation of <n>mouse histone deacetylase 1</n>. Reversible <n>histone</n> acetylation changes the chromatin structure and can modulate gene transcription. <n>Mammalian histone deacetylase 1</n> (<n>HDAC1</n>) is a nuclear protein that belongs to a growing family of evolutionarily conserved enzymes catalysing the removal of acetyl residues from core histones and other proteins. Previously, we have identified <n>murine HDAC1</n> as a growth factor-inducible protein in murine T-cells. Here, we characterise the molecular function of <n>mouse HDAC1</n> in more detail. Co-immunoprecipitation experiments with epitope-tagged <n>HDAC1 protein</n> reveal the association with endogenous <n>HDAC1 enzyme</n>. We show that <n>HDAC1</n> can homo-oligomerise and that this interaction is dependent on the N-terminal <n>HDAC</n> association domain of the protein. Furthermore, the same <n>HDAC1</n> domain is also necessary for in vitro binding of <n>HDAC2</n> and <n>HDAC3</n>, association with <n>RbAp48</n> and for catalytic activity of the enzyme. A lysine-rich sequence within the carboxy terminus of <n>HDAC1</n> is crucial for nuclear localisation of the enzyme. We identify a C-terminal nuclear localisation domain, which is sufficient for the transport of <n>HDAC1</n> and of reporter fusion proteins into the nucleus. Alternatively, <n>HDAC1</n> can be shuttled into the nucleus by association with another <n>HDAC1 molecule</n> via its N-terminal <n>HDAC</n> association domain. Our results define two domains, which are essential for the oligomerisation and nuclear localisation of <n>mouse HDAC1</n>. Copyright 2001 Academic Press.
11278544>Interaction of <n>p130</n> with, and consequent inhibition of, the catalytic subunit of <n>protein phosphatase 1alpha</n>. The <n>protein p130</n> was originally isolated from rat brain as an <n>inositol 1,4,5-trisphosphate-binding protein</n> with a domain organization similar to that of <n>phospholipase C-delta1</n> but which lacks <n>phospholipase C</n> activity. Yeast two-hybrid screening of a human brain cDNA library for clones that encode proteins that interact with <n>p130</n> has now led to the identification of the catalytic subunit of <n>protein phosphatase 1alpha</n> (<n>PP1calpha</n>) as a <n>p130-binding protein</n>. The association between <n>p130</n> and <n>PP1calpha</n> was also confirmed in vitro by an overlay assay, a "pull-down" assay, and surface plasmon resonance analysis. The interaction of <n>p130</n> with <n>PP1calpha</n> resulted in inhibition of the catalytic activity of the latter in a <n>p130</n> concentration-dependent manner. Immunoprecipitation and immunoblot analysis of COS-1 cells that stably express <n>p130</n> and of mouse brain extract with antibodies to <n>p130</n> and to <n>PP1calpha</n> also detected the presence of a complex of <n>p130</n> and <n>PP1calpha</n>. The activity of <n>glycogen phosphorylase</n>, which is negatively regulated by dephosphorylation by <n>PP1calpha</n>, was higher in COS-1 cells that stably express <n>p130</n> than in control COS-1 cells. These results suggest that, in addition to its role in inositol 1,4,5-trisphosphate and Ca(2+) signaling, <n>p130</n> might also contribute to regulation of protein dephosphorylation through its interaction with <n>PP1calpha</n>.
11242053>Methylation of <n>histone H3</n> lysine 9 creates a binding site for <n>HP1</n> proteins. Distinct modifications of <n>histone</n> amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. In addition to <n>histone</n> modifications that facilitate gene activity, it is of similar importance to restrict inappropriate gene expression if cellular and developmental programmes are to proceed unperturbed. Here we show that mammalian methyltransferases that selectively methylate <n>histone H3</n> on lysine 9 (<n>Suv39h HMTases</n>) generate a binding site for <n>HP1</n> proteins--a family of heterochromatic adaptor molecules implicated in both gene silencing and supra-nucleosomal chromatin structure. High-affinity in vitro recognition of a methylated <n>histone H3</n> peptide by <n>HP1</n> requires a functional chromo domain; thus, the <n>HP1</n> chromo domain is a specific interaction motif for the methyl epitope on lysine9 of <n>histone H3</n>. In vivo, heterochromatin association of <n>HP1</n> proteins is lost in <n>Suv39h</n> double-null primary mouse fibroblasts but is restored after the re-introduction of a catalytically active <n>SWUV39H1 HMTase</n>. Our data define a molecular mechanism through which the <n>SUV39H</n>-<n>HP1</n> methylation system can contribute to the propagation of heterochromatic subdomains in native chromatin.
11206060>Delineation of the <n>calcineurin</n>-interacting region of <n>cyclophilin B</n>. The immunosuppressant drug cyclosporin A (<n>CsA</n>) inhibits T-cell function by blocking the phosphatase activity of <n>calcineurin</n>. This effect is mediated by formation of a complex between the drug and <n>cyclophilin</n> (<n>CyP</n>), which creates a composite surface able to make high-affinity contacts with <n>calcineurin</n>. In vitro, the <n>CyPB</n>/<n>CsA</n> complex is more effective in inhibiting <n>calcineurin</n> than the <n>CyPA</n>/<n>CsA</n> and <n>CyPC</n>/<n>CsA</n> complexes, pointing to fine structural differences in the <n>calcineurin</n>-binding region. To delineate the <n>calcineurin</n>-binding region of <n>CyPB</n>, we mutated several amino acids, located in two loops corresponding to <n>CyPA</n> regions known to be involved, as follows: R76A, G77H, D155R, and D158R. Compared to wild-type <n>CyPB</n>, the G77H, D155R, and D158R mutants had intact isomerase and <n>CsA</n>-binding activities, indicating that no major conformational changes had taken place. When complexed to <n>CsA</n>, they all displayed only reduced affinity for <n>calcineurin</n> and much decreased inhibition of <n>calcineurin</n> phosphatase activity. These results strongly suggest that the three amino acids G77, D155, and D158 are directly involved in the interaction of <n>CyPB</n>/<n>CsA</n> with <n>calcineurin</n>, in agreement with their exposed position. The G77, D155, and D158 residues are not maintained in <n>CyPA</n> and might therefore account for the higher affinity of the <n>CyPB</n>/<n>CsA</n> complex for <n>calcineurin</n>.
11171121>Characterization and mapping of the <n>12 kDa FK506-binding protein</n> (<n>FKBP12</n>)-binding site on different isoforms of the <n>ryanodine receptor</n> and of the <n>inositol 1,4,5-trisphosphate receptor</n>. We investigated the interaction of the <n>12 kDa FK506-binding protein</n> (<n>FKBP12</n>) with two <n>ryanodine-receptor</n> isoforms (<n>RyR1</n> and <n>RyR3</n>) and with two <n>myo-inositol 1,4,5-trisphosphate (IP3) receptor</n> isoforms (<n>IP3R1</n> and <n>IP3R3</n>). Using <n>glutathione S-transferase</n> (<n>GST</n>)-<n>FKBP12</n> affinity chromatography, we could efficiently extract <n>RyR1</n> (42+/-7% of the solubilized <n>RyR1</n>) from terminal cisternae of skeletal muscle as well as <n>RyR3</n> (32+/-4% of the solubilized <n>RyR3</n>) from <n>RyR3</n>-overexpressing HEK-293 cells. These interactions were completely abolished by FK506 (20 microM) but were largely unaffected by <n>RyR-channel</n> modulators. In contrast, neither <n>IP3R1</n> nor <n>IP3R3</n> from various sources, including rabbit cerebellum, A7r5 smooth-muscle cells and <n>IP3R</n>-overexpressing Sf9 insect cells from Spodoptera frugiperda, were retained on the GST-<n>FKBP12</n> matrix. Moreover, immunoprecipitation experiments indicated a high-affinity interaction of <n>FKBP12</n> with <n>RyR1</n> but not with <n>IP3R1</n>. In order to determine the <n>FKBP12</n>-binding site, we fragmented both <n>RyR1</n> and <n>IP33R1</n> by limited proteolysis. We obtained a 45 kDa fragment of <n>RyR1</n> that bound to the GST-<n>FKBP12</n> matrix, indicating that it retained all requirements for <n>FKBP12</n> binding. This fragment was identified by its interaction with <n>antibody m34C</n> and must therefore contain its epitope (amino acids 2756-2803). However, no fragment of <n>IP3R1</n> was retained on the column. These molecular data are in agreement with the lack of correlation between <n>FKBP12</n> and <n>IP3R1</n> expression in various cell types. The observation that <n>FKBP12</n> did not affect IP3-induced Ca2+ release but reduced caffeine-induced Ca2+ release also indicated that mature <n>IP3R1</n> and <n>IP3R3</n>, in contrast to <n>RyR1</n> and <n>RyR3</n>, did not display a specific, high-affinity interaction with <n>FKBP12</n>.
11163216>A <n>Rab11</n>/<n>Rip11</n> protein complex regulates apical membrane trafficking via recycling endosomes. <n>Rab11</n> is a <n>GTPase</n> that regulates endosomal trafficking to apical plasma membrane domains in polarized epithelial cells. We report the identification of a novel <n>Rab11</n> effector, <n>Rip11</n>. <n>Rip11</n> is enriched in polarized epithelial cells where, like <n>Rab11</n>, it is localized to subapical recycling endosomes (ARE) and the apical plasma membrane. Using various transport assays, we demonstrate that <n>Rip11</n> is important for protein trafficking from ARE to the apical plasma membrane. <n>Rip11</n> is recruited to ARE by binding to <n>Rab11</n> as well as through a Mg(2+)-dependent interaction of its C2 domain with neutral phospholipids. The association of <n>Rip11</n> with membranes is regulated by a phosphorylation and dephosphorylation cycle. We propose a model whereby the <n>Rab11</n>/<n>Rip 11</n> complex regulates vesicle targeting from the ARE.
11149895><n>NOSIP</n>, a novel modulator of <n>endothelial nitric oxide synthase</n> activity. Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of <n>endothelial nitric oxide synthase</n> (<n>eNOS</n>) with effector proteins such as <n>Ca2+-calmodulin</n>, by posttranslational modifications such as phosphorylation via <n>protein kinase B</n>, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of <n>eNOS</n> is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed <n>NOSIP</n> (<n>eNOS interacting protein</n>), which avidly binds to the carboxyl-terminal region of the <n>eNOS</n> oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of <n>eNOS</n> and <n>NOSIP</n> in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the <n>caveolin-1</n> scaffolding domain. NO production was significantly reduced in <n>eNOS</n>-expressing CHO cells (CHO-<n>eNOS</n>) that transiently overexpressed <n>NOSIP</n>. Stimulation with the calcium ionophore A23187 induced the reversible translocation of <n>eNOS</n> from the detergent-insoluble to the detergent-soluble fractions of CHO-<n>eNOS</n>, and this translocation was completely prevented by transient coexpression of <n>NOSIP</n> in CHO-<n>eNOS</n>. Immunofluorescence studies revealed a prominent plasma membrane staining for <n>eNOS</n> in CHO-<n>eNOS</n> that was abolished in the presence of <n>NOSIP</n>. Subcellular fractionation studies identified <n>eNOS</n> in the <n>caveolin</n>-rich membrane fractions of CHO-<n>eNOS</n>, and coexpression of <n>NOSIP</n> caused a shift of <n>eNOS</n> to intracellular compartments. We conclude that <n>NOSIP</n> is a novel type of modulator that promotes translocation of <n>eNOS</n> from the plasma membrane to intracellular sites, thereby uncoupling <n>eNOS</n> from plasma membrane caveolae and inhibiting NO synthesis.
11115513>The <n>neuronal adaptor protein X11alpha</n> interacts with the copper chaperone for <n>SOD1</n> and regulates <n>SOD1</n> activity. The <n>neuronal adaptor protein X11alpha</n> participates in the formation of multiprotein complexes and intracellular trafficking. It contains a series of discrete protein-protein interaction domains including two contiguous C-terminal PDZ domains. We used the yeast two-hybrid system to screen for proteins that interact with the PDZ domains of human <n>X11alpha</n>, and we isolated a clone encoding domains II and III of the copper chaperone for <n>Cu,Zn-superoxide dismutase-1</n> (<n>CCS</n>). The <n>X11alpha</n>/<n>CCS</n> interaction was confirmed in coimmunoprecipitation studies plus <n>glutathione S-transferase</n> fusion protein pull-down assays and was shown to be mediated via PDZ2 of <n>X11alpha</n> and a sequence within the carboxyl terminus of domain III of <n>CCS</n>. <n>CCS</n> delivers the copper cofactor to the antioxidant <n>superoxide dismutase-1</n> (<n>SOD1</n>) enzyme and is required for its activity. Overexpression of <n>X11alpha</n> inhibited <n>SOD1</n> activity in transfected Chinese hamster ovary cells which suggests that <n>X11alpha</n> binding to <n>CCS</n> is inhibitory to <n>SOD1</n> activation. <n>X11alpha</n> also interacts with another copper-binding protein found in neurons, the <n>Alzheimer's disease amyloid precursor protein</n>. Thus, <n>X11alpha</n> may participate in copper homeostasis within neurons.
11100870>Quantitation of protein binding to the capillary wall in acidic, isoelectric buffers and means for minimizing the phenomenon. Notwithstanding the use of acidic, amphoteric, isoelectric buffers with isoelectric points (pI) in the pH 2-3 range, adsorption of proteins to the naked silica wall can be non-negligible. Two such buffers have been tested: iminodiacetic acid (IDA; pI 2.23, apparent pH 3.2 in 7 M urea) and aspartic acid (pI 2.77, apparent pH 3.7 in 7 M urea). Three potential quenchers of such interactions have been tested: hydroxyethylcellulose (HEC; number average molecular mass, Mr 27,000), TEPA (tetraethylenepentamine) and a novel, quatemarized piperazine [N(methyl-N-omega-iodobutyl)-N'-methylpiperazine] (Q-Pip), either alone or in binary and ternary mixtures. Human alpha- and beta-globin chains have been used as test proteins in capillary electrophoresis separations. It has been found that mixtures of these compounds are the worst possible remedy. E.g., a ternary mixture comprising 0.5% HEC, 0.5 mM TEPA and 1 mM Q-Pip still leaves behind 4.5% adsorbed protein onto the silica surface in runs in IDA buffer and 7 M urea (pH 3.2). Conversely, 0.5 mM TEPA or 1 mM Q-Pip, when used alone, minimize adsorption down to only 1.8% and 0.5%, respectively. When the same globin chain separations are performed in Asp and 7 M urea (pH 3.7), the situation is much worse: 44% protein is adsorbed in a ternary mixture of 0.5% HEC, 1 mM Q-Pip and 0.5 mM TEPA. However, when used alone, 0.5 mM TEPA and 1 mM Q-Pip reduce globin adsorption to levels of 8% and 5%, respectively. TEPA and Q-Pip are found to be in all cases the best quenchers of protein interaction to naked fused-silica; in addition they exhibit the unique property of smoothing the base-line and giving reproducible runs. The best method for desorbing bound protein was found to be an electrophoretic step consisting in driving sodium dodecylsulphate micelles from the cathodic reservoir.
11085749>Hematopoietic expression of <n>HOXB4</n> is regulated in normal and leukemic stem cells through transcriptional activation of the <n>HOXB4</n> promoter by <n>upstream stimulating factor (USF)-1</n> and <n>USF-2</n>. The homeobox genes encode a family of transcription factors that regulate development and postnatal tissue homeostasis. Since <n>HOXB4</n> plays a key role in regulating the balance between hematopoietic stem cell renewal and differentiation, we studied the molecular regulation of <n>HOXB4</n> expression in human hematopoietic stem cells. <n>HOXB4</n> expression in K562 cells is regulated at the level of transcription, and transient transfection defines primary <n>HOXB4</n> regulatory sequences within a 99-bp 5' promoter. Culture of highly purified human <n>CD34</n>(+) bone marrow cells in <n>thrombopoietin</n>/<n>Flt-3</n> ligand/stem cell factor induced <n>HOXB4</n> 3-10-fold, whereas culture in <n>granulocyte/macrophage colony-stimulating factor</n>, only increased <n>HOXB4</n>/<n>luciferase</n> expression 20-50%. Mutations within the <n>HOXB4</n> promoter identified a potential E box binding site (HOX response element [HXRE]-2) as the most critical regulatory sequence, and yeast one hybrid assays evaluating bone marrow and K562 libraries for HXRE-2 interaction identified <n>upstream stimulating factor (USF)-2</n> and <n>micropthalmia transcription factor</n> (<n>MITF</n>). Electrophoretic mobility shift assay with K562 extracts confirmed that these proteins, along with <n>USF-1</n>, bind to the <n>HOXB4</n> promoter in vitro. Cotransfection assays in both K562 and <n>CD34</n>(+) cells showed that <n>USF-1</n> and <n>USF-2</n>, but not <n>MITF</n>, induce the <n>HOXB4</n> promoter in response to signals stimulating stem cell self-renewal, through activation of the <n>mitogen-activated protein kinase</n> pathway. Thus hematopoietic expression of the <n>human HOXB4</n> gene is regulated by the binding of <n>USF-1</n> and <n>USF-2</n>, and this process may be favored by cytokines promoting stem cell self-renewal versus differentiation.
11063589>Critical role of a subdomain of the N-terminus of the <n>V1a vasopressin receptor</n> for binding agonists but not antagonists; functional rescue by the <n>oxytocin receptor</n> N-terminus. A fundamental issue in molecular pharmacology is to define how agonist:receptor interaction differs from that of antagonist:receptor. The <n>V(1a) receptor</n> (<n>V(1a)R</n>) is a member of a family of related G-protein-coupled receptors that are activated by the neurohypophysial peptide hormone <n>arginine-vasopressin</n> (<n>AVP</n>). Here we define a short subdomain of the N-terminus of the <n>V(1a)R</n> from Glu(37) to Asn(47) that is an absolute requirement for binding <n>AVP</n> and other agonists. In marked contrast to the situation for agonists, deleting this segment has little or no effect on the binding of either peptide or non-peptide antagonists. In addition, we established that this subdomain was crucial for receptor activation and second messenger generation. The <n>oxytocin receptor</n> (<n>OTR</n>) also binds <n>AVP</n> with high affinity but exhibits a different pharmacological profile to the <n>V(1a)R</n>. Substitution of the N-terminus of the <n>V(1a)R</n> with the corresponding sequence from the <n>OTR</n> generated a chimeric receptor (<n>OTR</n>(N)-<n>V(1a)R</n>). The presence of the <n>OTR</n> N-terminus recovered high affinity agonist binding such that the <n>OTR</n>(N)-<n>V(1a)R</n> possessed almost wild-type <n>V(1a)R</n> pharmacology and signaling. Consequently, a domain within the N-terminus is required for agonist binding but it does not provide the molecular discriminator for subtype-selective agonist recognition. Cotransfection and peptide mimetic studies demonstrated that this N-terminal subdomain had to be contiguous with the receptor polypeptide to be functional. This study establishes that a segment of the <n>V(1a)R</n> N-terminus has a pivotal role in the mechanism of agonist binding and provides molecular insight into key differences between the interaction of agonists and antagonists with a peptide receptor family.
11035087>Identification of a complex that binds to the <n>CD154</n> 3' untranslated region: implications for a role in message stability during T cell activation. <n>CD154</n> expression is regulated throughout a time course of <n>CD3</n>-dependent T cell activation by differential mRNA decay. To understand the molecular basis of the "stability" phase of this pathway, experiments were conducted to identify sequences and specific complexes important in this regulation. Gel retardation assays using extracts from both Jurkat T cells and <n>CD3</n>-activated <n>CD4</n>(+) T cells revealed a major complex (complex I) that bound a 65-bp highly CU-rich region of the <n>CD154</n> 3' untranslated region. The specificity of the CU-rich element for complex-I formation was confirmed by disruption of this complex by oligo(dCT) competition. Formation of complex I strongly correlated with <n>CD154</n> mRNA stability across a time course of T cell activation. UV cross-linking identified a major oligo(dCT)-sensitive species at approximately 90 kDa that showed induced and increased expression in extracts from 24- and 48-hr anti-<n>CD3</n>-activated T cells, respectively. This protein was absent in equivalent extracts from resting or 2-h-activated T cells. Using an in vitro decay assay, we found that a <n>CD154</n>-specific transcript was more rapidly degraded in 2-h-activated extract and stabilized in the 24- and 48-h extracts compared to extracts from resting T cells. Disruption of complex I resulted in the rapid decay of a <n>CD154</n>-specific transcript demonstrating a functional role for complex I in mRNA stabilization in vitro. These studies support a model of posttranscriptional regulation of <n>CD154</n> expression being controlled in part by the interaction of a poly(CU)-binding complex with a specific sequence in the 3' untranslated region.
11016930>The N-terminal end of <n>nebulin</n> interacts with <n>tropomodulin</n> at the pointed ends of the thin filaments. Strict regulation of <n>actin</n> thin filament length is critical for the proper functioning of sarcomeres, the basic contractile units of myofibrils. It has been hypothesized that a molecular template works with <n>actin</n> filament capping proteins to regulate thin filament lengths. <n>Nebulin</n> is a giant protein ( approximately 800 kDa) in skeletal muscle that has been proposed to act as a molecular ruler to specify the thin filament lengths characteristic of different muscles. <n>Tropomodulin</n> (<n>Tmod</n>), a pointed end thin filament capping protein, has been shown to maintain the final length of the thin filaments. Immunofluorescence microscopy revealed that the N-terminal end of <n>nebulin</n> colocalizes with <n>Tmod</n> at the pointed ends of thin filaments. The three extreme N-terminal modules (M1-M2-M3) of <n>nebulin</n> bind specifically to <n>Tmod</n> as demonstrated by blot overlay, bead binding, and solid phase binding assays. These data demonstrate that the N terminus of the <n>nebulin</n> molecule extends to the extreme end of the thin filament and also establish a novel biochemical function for this end. Two <n>Tmod</n> isoforms, erythrocyte <n>Tmod</n> (<n>E-Tmod</n>), expressed in embryonic and slow skeletal muscle, and <n>skeletal Tmod</n> (<n>Sk-Tmod</n>), expressed late in fast skeletal muscle differentiation, bind on overlapping sites to recombinant N-terminal <n>nebulin</n> fragments. <n>Sk-Tmod</n> binds <n>nebulin</n> with higher affinity than <n>E-Tmod</n> does, suggesting that the <n>Tmod</n>/<n>nebulin</n> interaction exhibits isoform specificity. These data provide evidence that <n>Tmod</n> and <n>nebulin</n> may work together as a linked mechanism to control thin filament lengths in skeletal muscle.
10996078><n>INCENP</n> binds the <n>Aurora-related kinase AIRK2</n> and is required to target it to chromosomes, the central spindle and cleavage furrow. Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the <n>inner centromere protein</n> (<n>INCENP</n>[2]), the <n>Aurora-related serine-threonine protein kinase AIRK2</n> [3,4] and the unidentified human autoantigen <n>TD-60</n> [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of <n>INCENP</n> function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with <n>AIRK2</n> function primarily affects cytokinesis [3,8]. Here, we report that <n>INCENP</n> is stockpiled in Xenopus eggs in a complex with <n>Xenopus AIRK2</n> (<n>XAIRK2</n>), and that <n>INCENP</n> and <n>AIRK2 kinase</n> bind one another in vitro. This association was found to be evolutionarily conserved. <n>Sli15p</n>, the binding partner of <n>yeast Aurora kinase Ipl1p</n>, can be recognized as an <n>INCENP</n> family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between <n>INCENP</n> and <n>Aurora kinase</n> was found to be biologically relevant. <n>INCENP</n> and <n>AIRK2</n> colocalized exactly in human cells, and <n>INCENP</n> was required to target <n>AIRK2</n> correctly to centromeres and the central spindle.
10975987><n>IL-5</n>-Induced <n>JAB</n>-<n>JAK2</n> interaction. Receptor activation by the haematopoietic growth factor proteins <n>interleukin 5</n> (<n>IL-5</n>) and <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) leads to phosphorylation of <n>JAK2</n> as a key trigger of signal transduction. <n>JAB</n> has recently been identified as a regulator of <n>JAK2</n> phosphorylation and activity by binding phosphorylated <n>JAK2</n> and inducing its degradation. As part of our effort to define molecular recognition networks that lead to signalling, we investigated the effect of <n>JAB</n> on both <n>JAK2</n> phosphorylation and <n>JAK2</n> interaction state that ensue upon <n>IL-5</n> stimulation in recombinant 293T cells cotransfected 293T cells with <n>IL-5R alpha</n>, <n>beta c</n> and <n>hJAK2</n> either with or without <n>JAB</n>. Without <n>JAB</n>, stimulation with wild-type and re-engineered single chain (sc) <n>IL-5</n> induced a time-dependent phosphorylation of <n>JAK2</n>. In the presence of <n>JAB</n> cotransfection, no <n>phospho-JAK2</n> was observed, and <n>JAB</n> was observed co-immunoprecipitated with non-phosphorylated <n>JAK2</n>. The time dependence of <n>JAB</n> co-immunoprecipitation correlated with the time dependence of <n>JAK2</n> phosphorylation when <n>JAB</n> was absent. Since <n>JAB</n> has already been shown to bind <n>JAK2</n> via a phosphorylated tyrosine, the current data suggest that <n>JAB</n> binds to phosphorylated <n>JAK2</n>, enhances <n>JAK2</n> dephosphorylation and remains associated in a complex, with dephosphorylated <n>JAK2</n>, that may be a precursor leading to irreversible <n>JAK2</n> degradation.
10961991>Covalent modification of <n>p73alpha</n> by <n>SUMO-1</n>. Two-hybrid screening with <n>p73</n> identifies novel <n>SUMO-1</n>-interacting proteins and a <n>SUMO-1</n> interaction motif. Two-hybrid screening in yeast with <n>p73alpha</n> isolated <n>SUMO-1</n> (<n>small ubiquitin-like modifier 1</n>), the enzyme responsible for its conjugation, <n>Ubc-9</n>, and a number of novel <n>SUMO-1</n>-interacting proteins, including thymine <n>DNA glycosylase</n>, <n>PM-Scl75</n>, <n>PIASx</n>, <n>PKY</n>, and <n>CHD3/ZFH</n>. A subset of these proteins contain a common motif, hhXSXS/Taaa, where h is a hydrophobic amino acid and a is an acidic amino acid, that is shown to interact with <n>SUMO-1</n> in the two-hybrid system. We show here that <n>p73alpha</n>, but not <n>p73beta</n>, can be covalently modified by <n>SUMO-1</n>. The major <n>SUMO-1</n>-modified residue in <n>p73alpha</n> is the C-terminal lysine (Lys(627)). The sequence surrounding this lysine conforms to a consensus <n>SUMO-1</n> modification site b(X)XXhKXE, where b is a basic amino acid. <n>SUMO-1-modified p73</n> is more rapidly degraded by the proteasome than unmodified <n>p73</n>, although <n>SUMO-1</n> modification is not required for <n>p73</n> degradation. <n>SUMO-1</n> modification does not affect the transcriptional activity of <n>p73alpha</n> on an <n>RGC-luciferase</n> reporter gene in SK-N-AS cells. Instead, <n>SUMO-1</n> modification may alter the subcellular localization of <n>p73</n>, because <n>SUMO-1-modified p73</n> is preferentially found in detergent-insoluble fractions. Alternatively, it may modulate the interaction of <n>p73</n> with other proteins that are substrates for <n>SUMO-1</n> modification or which interact with <n>SUMO-1</n>, such as those identified here.
10950948>The <n>Yersinia protein kinase A</n> is a host factor inducible <n>RhoA</n>/<n>Rac</n>-binding virulence factor. The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the <n>Yersinia protein kinase A</n> (<n>YpkA</n>), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of <n>YpkA</n>. The amino terminus, which contains the kinase domain, is sufficient to localize <n>YpkA</n> to the plasma membrane while the carboxyl terminus of <n>YpkA</n> is required for <n>YpkAs</n> morphological effects. <n>YpkAs</n> carboxyl-terminal region was found to affect the levels of <n>actin</n>-containing stress fibers as well as block the activation of the <n>GTPase RhoA</n> in Yersinia-infected cells. We show that the carboxyl-terminal region of <n>YpkA</n>, which contains sequences that bear similarity to the <n>RhoA</n>-binding domains of several <n>eukaryotic RhoA-binding kinases</n>, directly interacts with <n>RhoA</n> as well as <n>Rac</n> (but not <n>Cdc42</n>) and displays a slight but measurable binding preference for the GDP-bound form of <n>RhoA</n>. Surprisingly, <n>YpkA</n> binding to <n>RhoA(GDP)</n> affected neither the intrinsic nor <n>guanine nucleotide exchange factor</n>-mediated GDP/GTP exchange reaction suggesting that <n>YpkA</n> controls activated <n>RhoA</n> levels by a mechanism other than by simply blocking <n>guanine nucleotide exchange factor</n> activity. We go on to show that <n>YpkAs</n> kinase activity is neither dependent on nor promoted by its interaction with <n>RhoA</n> and <n>Rac</n> but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that <n>YpkA</n> possesses both similarities and differences with the <n>eukaryotic RhoA/Rac-binding kinases</n> and suggest that the yersiniae utilize the <n>Rho GTPases</n> for unique activities during their interaction with eukaryotic cells.
10930463>Specific sequence motif of 8-Cys repeats of <n>TGF-beta binding proteins</n>, <n>LTBPs</n>, creates a hydrophobic interaction surface for binding of <n>small latent TGF-beta</n>. <n>Transforming growth factor (TGF)-betas</n> are secreted in large latent complexes consisting of <n>TGF-beta</n>, its <n>N-terminal latency-associated peptide</n> (<n>LAP</n>) propeptide, and <n>latent TGF-beta binding protein</n> (<n>LTBP</n>). <n>LTBPs</n> are required for secretion and subsequent deposition of <n>TGF-beta</n> into the extracellular matrix. <n>TGF-beta1</n> associates with the 3(rd) 8-Cys repeat of <n>LTBP-1</n> by <n>LAP</n>. All <n>LTBPs</n>, as well as <n>fibrillins</n>, contain multiple 8-Cys repeats. We analyzed the abilities of <n>fibrillins</n> and <n>LTBPs</n> to bind latent <n>TGF-beta</n> by their 8-Cys repeats. 8-Cys repeat was found to interact with <n>TGF-beta1</n>*<n>LAP</n> by direct cysteine bridging. <n>LTBP-1</n> and <n>LTBP-3</n> bound efficiently all <n>TGF-beta</n> isoforms, <n>LTBP-4</n> had a much weaker binding capacity, whereas <n>LTBP-2</n> as well as <n>fibrillins -1</n> and <n>-2</n> were negative. A short, specific <n>TGF-beta</n> binding motif was identified in the <n>TGF-beta</n> binding 8-Cys repeats. Deletion of this motif in the 3(rd) 8-Cys repeat of <n>LTBP-1</n> resulted in loss of <n>TGF-beta</n>*<n>LAP</n> binding ability, while its inclusion in non-<n>TGF-beta</n> binding 3(rd) 8-Cys repeat of <n>LTBP-2</n> resulted in <n>TGF-beta</n> binding. Molecular modeling of the 8-Cys repeats revealed a hydrophobic interaction surface and lack of three stabilizing hydrogen bonds introduced by the <n>TGF-beta</n> binding motif necessary for the formation of the <n>TGF-beta</n>*<n>LAP</n> - 8-Cys repeat complex inside the cells.
10913114><n>GAS41</n>, a highly conserved protein in eukaryotic nuclei, binds to <n>NuMA</n>. The yeast two-hybrid system was used to identify binding partners of <n>NuMA</n>, a component of the nuclear matrix in interphase cells. By using the C-terminal half of <n>NuMA</n> as bait, a human cDNA sequence coding for a 223-amino acid protein with a non-helical N-terminal domain and a C-terminal alpha-helical portion was identified and fully sequenced. It was identical to <n>GAS41</n>, a sequence amplified in human gliomas. The sequence of the homologous Drosophila protein was established, and the alignment for <n>GAS41</n> from nine different species showed that <n>GAS41</n> is a general eukaryotic protein found in species as diverse as Arabidopsis, Drosophila, Caenorhabditis elegans, yeast, and man. Northern blot analysis showed a single transcript in eight human tissues. A polyclonal antibody to <n>GAS41</n> showed a dotted staining pattern in interphase nuclei and a uniform distribution in mitotic cells. A <n>GFP-GAS41 fusion protein</n> displayed equivalent patterns. In vitro <n>GAS41</n> bound to the C-terminal part of the rod region of <n>NuMA</n>, as shown by dot overlay and by surface plasmon resonance measurements. The K(d) of the complex was 2 x 10(-)(7) m. <n>GAS41</n> is related to the <n>AF-9</n> and <n>ENL</n> proteins, which are putative transcription factors found as fusion proteins in some acute leukemias. The <n>NuMA</n>/<n>GAS41</n> interaction may provide a link between nuclear structure and gene expression.
10895262>Adhesive receptors on malaria-parasitized red cells. Antigenic variation, rosetting and cytoadhesion are key determinants in the survival and virulence of the malaria parasite Plasmodium falciparum. These properties reside in a multigene protein family called <n>P. falciparum erythrocyte membrane protein I</n> (<n>PfEMPI</n>), encoded by the large and diverse var gene family. <n>PfEMPI</n> plays a central role in the biology of P. falciparum and its interaction with the human host. The molecular mechanism and the domains involved in cytoadherence, rosetting and antigenic variation are beginning to unfold. Domains mediating rosetting and adhesion to several key host receptors have already been identified. Understanding the role of <n>PfEMPI</n> in the pathogenesis and survival of malaria parasites is the key for the development of anti-adhesion vaccines and therapeutics to reduce the mortality and morbidity of P. falciparum infections.
10872826><n>Gadd45</n> family proteins are coactivators of nuclear hormone receptors. <n>Gadd45</n> family genes encode nuclear acidic proteins composed of <n>Gadd45</n>, <n>MyD118</n>, and <n>CR6</n>. Sequence analysis showed that <n>Gadd45</n> family proteins (<n>Gadd45</n>, <n>MyD118</n>, and <n>CR6</n>) contain LXXLL signature motifs considered necessary and sufficient for the binding of several coactivators to nuclear receptors. Interaction between <n>Gadd45</n> or <n>CR6</n> and <n>RXR alpha</n> was confirmed by a two-hybrid test in yeast. Results from a series of <n>GST</n> pulldown assays showed that these <n>Gadd45</n> family proteins interact with several nuclear hormone receptors including <n>RXR alpha</n>, <n>RAR alpha</n>, <n>ER alpha</n>, <n>PPAR alpha</n>, <n>PPAR beta</n>, and <n>PPAR gamma2</n> in vitro. Interaction between <n>Gadd45</n> family proteins and nuclear hormone receptors resulted in modest activation of transactivating function of nuclear hormone receptors in reporter systems. When fused to DNA binding domain of <n>GAL4</n>, <n>Gadd45</n> and <n>CR6</n> activated the UAS-mediated transcription in mammalian cells. These results suggest that <n>Gadd45</n> family proteins bind to nuclear hormone receptors and act as nuclear coactivators.
10860733>1.9 A resolution crystal structure of the <n>Saccharomyces cerevisiae Ran-binding protein Mog1p</n>. The 1.9 A resolution X-ray crystal structure of <n>Ran-binding protein Mog1p</n> shows that it has a unique fold based on a six-stranded antiparallel beta-sheet backed on both sides by an extensive alpha-helix. The topology of some elements of <n>Mog1p</n> secondary structure resemble a portion of <n>nuclear transport factor 2</n> (<n>NTF2</n>), but the hydrophobic cavity and surrounding negatively charged residues that are important in the <n>NTF2</n>-<n>RanGDP</n> interaction are not conserved in <n>Mog1p</n>. In addition to binding <n>RanGTP</n>, <n>Mog1p</n> forms a 1:1 complex with <n>RanGDP</n> and so binds <n>Ran</n> independent of its nucleotide state. <n>Mog1p</n> and <n>NTF2</n> compete for binding to <n>RanGDP</n> indicating that their binding sites on <n>RanGDP</n> are sufficiently close to prevent both proteins binding simultaneously. Although there may be some overlap between the <n>Mog1p</n> and <n>NTF2</n> binding sites on <n>RanGDP</n>, these sites are not identical. Sequence analysis of <n>Mog1p</n> homologues from Schizosaccharomyces pombe, human, and Caenorhabditis elegans in the context of the <n>Mog1p</n> crystal structure indicates the presence of a cluster of highly conserved surface residues consistent with an interaction site for <n>Ran</n>.
10838076>Role of the N-terminal region of <n>staphylokinase</n> (<n>SAK</n>): evidence for the participation of the N-terminal region of <n>SAK</n> in the enzyme-substrate complex formation. <n>Staphylokinase</n> (<n>SAK</n>) forms an inactive 1:1 complex with <n>plasminogen</n> (<n>PG</n>), which requires both the conversion of <n>PG</n> to <n>plasmin</n> (<n>Pm</n>) to expose an active site in <n>PG</n>-<n>SAK</n> activator complex and the amino-terminal processing of <n>SAK</n> to expose the positively charged (Lys-11) amino-terminus after removal of the 10 N-terminal amino acid residues from the full length protein. The mechanism by which the N-terminal segment of <n>SAK</n> affects its <n>PG</n> activation capability was investigated by generating <n>SAK</n> mutants, blocked in the native amino-terminal processing site of <n>SAK</n>, and carrying an alteration in the placement of the positively charged amino acid residue, Lys-11, and further studying their interaction with <n>PG</n>, <n>Pm</n>, miniplasmin and kringle structures. A ternary complex formation between <n>PG</n>-<n>SAK</n> <n>PG</n> was observed when an immobilized <n>PG</n>-<n>SAK</n> binary complex interacted with free radiolabelled <n>PG</n> in a sandwich binding experiment. Formation of this ternary complex was inhibited by a lysine analog, 6-aminocaproic acid (EACA), in a concentration dependent manner, suggesting the involvement of lysine binding site(s) in this process. In contrast, EACA did not significantly affect the formation of binary complex formed by native <n>SAK</n> or its mutant derivatives. Furthermore, the binary (activator) complex formed between <n>PG</n> and <n>SAK</n> mutant, <n>PRM3</n>, lacking the N-terminal lysine 11, exhibited 3-4-fold reduced binding with <n>PG</n>, <n>Pm</n> or miniplasmin substrate during ternary complex formation as compared to native <n>SAK</n>. Additionally, activator complex formed with <n>PRM3</n> failed to activate <n>miniplasminogen</n> and exhibited highly diminished activation of substrate <n>PG</n>. Protein binding studies indicated that it has 3-5-fold reduction in ternary complex formation with miniplasmin but not with the kringle structure. In aggregate, these observations provide experimental evidence for the participation of the N-terminal region of <n>SAK</n> in accession and processing of substrate by the <n>SAK</n>-<n>Pm</n> activator complex to potentiate the <n>PG</n> activation by enhancing and/or stabilizing the interaction of free <n>PG</n>.
10823934>Specific interaction of <n>CCR5</n> amino-terminal domain peptides containing sulfotyrosines with <n>HIV-1 envelope glycoprotein gp120</n>. The <n>HIV-1 envelope glycoprotein gp120</n> interacts consecutively with <n>CD4</n> and the <n>CCR5 coreceptor</n> to mediate the entry of certain HIV-1 strains into target cells. Acidic residues and sulfotyrosines in the amino-terminal domain (Nt) of <n>CCR5</n> are crucial for viral fusion and entry. We tested the binding of a panel of <n>CCR5</n> Nt peptides to different soluble <n>gp120</n>/<n>CD4</n> complexes and anti-<n>CCR5</n> mAbs. The tyrosine residues in the peptides were sulfated, phosphorylated, or unmodified. None of the <n>gp120</n>/<n>CD4</n> complexes associated with peptides containing unmodified or phosphorylated tyrosines. The <n>gp120</n>/<n>CD4</n> complexes containing envelope glycoproteins from isolates that use <n>CCR5</n> as a coreceptor associated with Nt peptides containing sulfotyrosines but not with peptides containing sulfotyrosines in scrambled Nt sequences. Finally, only peptides containing sulfotyrosines inhibited the entry of an R5 isolate. Our data show that proper posttranslational modification of the <n>CCR5</n> Nt is required for <n>gp120</n> binding and viral entry. More importantly, the Nt domain determines the specificity of the interaction between <n>CCR5</n> and <n>gp120</n>s from isolates that use this coreceptor.
10816375>Analysis of <n>plasminogen-binding M proteins</n> of Streptococcus pyogenes. Group A streptococci are common human pathogens that cause a variety of infections. They express <n>M proteins</n> which are important cell wall-bound type-specific virulence factors. We have found that a set of strains, associated primarily with skin infections, express <n>M proteins</n> that bind <n>plasminogen</n> and <n>plasmin</n> with high affinity. The binding is mediated by a 13-amino-acid internal repeated sequence located in the N-terminal surface-exposed portion of these <n>M proteins</n>. This sequence binds to kringle 2 in <n>plasminogen</n>, a domain that is not involved in the interaction with streptokinase, a potent group A streptococcal activator of <n>plasminogen</n>. It could be demonstrated that <n>plasminogen</n>, absorbed from plasma by growing group A streptococci expressing the <n>plasminogen-binding M proteins</n>, could be activated by exogenous and endogenous <n>streptokinase</n>, thereby providing the bacteria with a surface-associated enzyme that could act on the tissue barriers in the infected host.
10799509>Identification and characterization of <n>CKIP-1</n>, a novel pleckstrin homology domain-containing protein that interacts with <n>protein kinase CK2</n>. The catalytic subunits of <n>protein kinase CK2</n>, <n>CK2alpha</n> and <n>CK2alpha'</n>, are closely related to each other but exhibit functional specialization. To test the hypothesis that specific functions of <n>CK2alpha</n> and <n>CK2alpha'</n> are mediated by specific interaction partners, we used the yeast two-hybrid system to identify <n>CK2alpha</n>- or <n>CK2alpha'</n>-binding proteins. We report the identification and characterization of a novel <n>CK2</n>-interacting protein, designated <n>CKIP-1</n>, that interacts with <n>CK2alpha</n>, but not <n>CK2alpha'</n>, in the yeast two-hybrid system. <n>CKIP-1</n> also interacts with <n>CK2alpha</n> in vitro and is co-immunoprecipitated from cell extracts with epitope-tagged <n>CK2alpha</n> and an <n>enhanced green fluorescent protein</n> fusion protein encoding <n>CKIP-1</n> (i.e. <n>EGFP</n>-<n>CKIP-1</n>) when they are co-expressed. <n>CK2</n> activity is detected in anti-<n>CKIP-1</n> immunoprecipitates performed with extracts from non-transfected cells indicating that <n>CKIP-1</n> and <n>CK2</n> interact under physiological conditions. The <n>CKIP-1</n> cDNA is broadly expressed and encodes a protein with a predicted molecular weight of 46,000. <n>EGFP</n>-<n>CKIP-1</n> is localized within the nucleus and at the plasma membrane. The plasma membrane localization is dependent on the presence of an amino-terminal <n>pleckstrin</n> homology domain. We postulate that <n>CKIP-1</n> is a non-enzymatic regulator of one isoform of <n>CK2</n> (i.e. <n>CK2alpha</n>) with a potential role in targeting <n>CK2alpha</n> to a particular cellular location.
10779362>Structure-function analysis of <n>SUV39H1</n> reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression. <n>SUV39H1</n>, a human homologue of the Drosophila position effect variegation modifier <n>Su(var)3-9</n> and of the <n>Schizosaccharomyces pombe silencing factor clr4</n>, encodes a novel heterochromatic protein that transiently accumulates at centromeric positions during mitosis. Using a detailed structure-function analysis of <n>SUV39H1</n> mutant proteins in transfected cells, we now show that deregulated <n>SUV39H1</n> interferes at multiple levels with mammalian higher-order chromatin organization. First, forced expression of full-length <n>SUV39H1</n> (412 amino acids) redistributes endogenous <n>M31</n> (<n>HP1beta</n>) and induces abundant associations with inter- and metaphase chromatin. These properties depend on the C-terminal <n>SET</n> domain, although the major portion of the <n>SUV39H1</n> protein (amino acids 89 to 412) does not display affinity for nuclear chromatin. By contrast, the <n>M31</n> interaction surface, which was mapped to the first 44 N-terminal amino acids, together with the immediately adjacent chromo domain, directs specific accumulation at heterochromatin. Second, cells overexpressing full-length <n>SUV39H1</n> display severe defects in mitotic progression and chromosome segregation. Surprisingly, whereas localization of centromere proteins is unaltered, the focal, G(2)-specific distribution of phosphorylated <n>histone H3</n> at serine 10 (phosH3) is dispersed in these cells. This <n>phosH3</n> shift is not observed with C-terminally truncated mutant <n>SUV39H1</n> proteins or with deregulated <n>M31</n>. Together, our data reveal a dominant role(s) for the <n>SET</n> domain of <n>SUV39H1</n> in the distribution of prominent heterochromatic proteins and suggest a possible link between a chromosomal <n>SU(VAR) protein</n> and <n>histone H3</n>.
10764735>Mechanisms for high affinity mannose 6-phosphate ligand binding to the <n>insulin-like growth factor II/mannose 6-phosphate receptor</n>. The two mannose 6-phosphate (Man-6-P) binding domains of the <n>insulin-like growth factor II/mannose 6-phosphate receptor</n> (<n>Man-6-P/IGF2R</n>), located in extracytoplasmic repeats 1-3 and 7-9, are capable of binding Man-6-P with low affinity and glycoproteins that contain more than one Man-6-P residue with high affinity. High affinity multivalent ligand binding sites could be formed through two possible mechanisms: the interaction of two Man-6-P binding domains within one <n>Man-6-P/IGF2R</n> molecule or by receptor oligomerization. To discriminate between these mechanisms, truncated <n>FLAG</n> epitope-tagged <n>Man-6-P/IGF2R</n> constructs, containing one or both of the Man-6-P binding domains, were expressed in 293T cells, and characterized for binding of pentamannose phosphate-<n>bovine serum albumin</n> (PMP-<n>BSA</n>), a pseudoglycoprotein bearing multiple Man-6-P residues. A construct containing all 15 repeats of the <n>Man-6-P/IGF2R</n> extracytoplasmic domain bound PMP-<n>BSA</n> with the same affinity as the full-length receptor (K(d) = 0.54 nm) with a curvilinear Scatchard plot. The presence of excess unlabeled PMP-<n>BSA</n> increased the dissociation rate of pre-formed (125)I-PMP-<n>BSA</n>/receptor complexes, suggesting negative cooperativity in multivalent ligand binding and affirming the role of multiple <n>Man-6-P/IGF2R</n> binding domains in forming high affinity binding sites. Truncated receptors containing only one Man-6-P binding domain and mutant receptor constructs, containing an Arg(1325) --> Ala mutation that eliminates binding to the repeats 7-9 binding domain, formed high affinity PMP-<n>BSA</n> binding, but with reduced stoichiometries. Collectively, these observations suggest that alignment of Man-6-P binding domains of separate <n>Man-6-P/IGF2R</n> molecules is responsible for the formation of high affinity Man-6-P binding sites and provide functional evidence for <n>Man-6-P/IGF2R</n> oligomerization.
10749676><n>Thrombospondin-1</n> binds to polyhistidine with high affinity and specificity. <n>Thrombospondin-1</n> (<n>TSP1</n>) is a secreted trimeric glycoprotein of 450 kDa with demonstrated effects on cell growth, adhesion and migration. Its complex biological activity is attributed to its ability to bind to cell-surface receptors, growth factors and extracellular-matrix proteins. In this study, we used a (125)I solid-phase binding assay to demonstrate that <n>TSP1</n> binds specifically to proteins containing polyhistidine stretches. Based on studies with three different six-histidine-containing recombinant proteins, we derived an average dissociation constant of 5 nM. The binding of (125)I-labelled <n>TSP1</n> to these proteins was inhibited by peptides containing histidine residues, with the degree of competition being a function of the number of histidines within the peptide. Binding was not inhibited by excess histidine or imidazole, indicating that the imidazole ring is not sufficient for recognition by <n>TSP1</n>. Heparin was a potent inhibitor of binding with a K(i) of 50 nM, suggesting that the heparin-binding domain of <n>TSP1</n> may be involved in this interaction. This was confirmed by the ability of a recombinant heparin-binding domain of <n>TSP1</n> to directly compete for <n>TSP1</n> binding to polyhistidine-containing proteins. Affinity chromatography with a polyhistidine-containing peptide immobilized on agarose revealed that <n>TSP1</n> in platelet releasates is the major polypeptide retained on the six-histidine-peptide column. We conclude that <n>TSP1</n> contains a high-affinity binding site for polyhistidine and this is likely to be the molecular basis for the observed binding of <n>TSP1</n> to histidine-rich glycoprotein. The possibility that other polyhistidine-containing proteins also interact with <n>TSP1</n> warrants further study.
10732979>Generation and evaluation of putative neuroregenerative drugs. Part 2: screening virtual libraries of novel polyketides which possess the binding domain of rapamycin. The use of computational methods to direct engineered biosynthesis toward candidates based on the desired properties of the target compounds has been explored. The objective for this study has been the modification of rapamycin in order to eliminate its immunosuppressive activity and retain its neuroregenerative abilities. We have designed analogues of rapamycin which have truncated effector domains but retain the ability to bind to <n>FKBP</n> proteins, which is a prerequisite for the neuroregenerative abilities of the drugs. The procedures described here consist of the screening of large virtual libraries of molecules which retain the binding domain of rapamycin but in which different substitute ketide units replace the effector domain. These methods have provided analogues of rapamycin that cannot retain the immunosuppressive abilities of rapamycin, have a binding affinity to <n>FKBP12</n> identical to that of rapamycin (by linear interaction energy calculations), and are suitable for synthesis by modified polyketide synthases.
10713102><n>TrkA</n> amino acids controlling specificity for <n>nerve growth factor</n>. <n>Neurotrophins</n> are important for the development and maintenance of the vertebrate nervous system, mediating their signal into the cell by specific interaction with tyrosine kinase receptors of the <n>Trk</n> family. The extracellular portion of the <n>Trk receptors</n> has been previously proposed to consist of a cysteine-rich motif, a leucine-rich motif, a second cysteine-rich motif followed by two immunoglobulin-like domains. Earlier studies have shown that a major <n>neurotrophin</n>-binding site in the <n>Trk</n> receptors resides in the second <n>immunoglobulin</n>-like domain. Although the individual amino acids in <n>TrkA</n> involved in binding to <n>nerve growth factor</n> (<n>NGF</n>) and those in <n>TrkC</n> involved in binding to <n>neurotrophin-3</n> have been mapped in this domain, the <n>Trk</n> amino acids that provide specificity remained unclear. In this study, a minimum set of residues in the human <n>TrkC</n> second <n>immunoglobulin</n>-like domain, which does not bind <n>nerve growth factor</n> (<n>NGF</n>), were substituted with those from <n>human TrkA</n>. The resulting Trk variant recruited binding of <n>NGF</n> equivalent to <n>TrkA</n>, maintained <n>neurotrophin-3</n> binding equivalent to <n>TrkC</n>, and also bound brain-derived <n>neurotrophin</n>, although with lower affinity compared with <n>TrkB</n>. This implies that the amino acids in the second <n>immunoglobulin</n>-like domain that determine <n>Trk</n> specificity are distinct for each <n>Trk</n>.
10692436>Ca(2+)-dependent and Ca(2+)-independent <n>calmodulin</n> binding sites in <n>erythrocyte protein 4.1</n>. Implications for regulation of <n>protein 4.1</n> interactions with transmembrane proteins. In vitro protein binding assays identified two distinct <n>calmodulin</n> (<n>CaM</n>) binding sites within the NH(2)-terminal 30-kDa domain of <n>erythrocyte protein 4.1</n> (<n>4.1R</n>): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound <n>CaM</n> in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to <n>CaM</n>. Thus, <n>4.1R</n> is a unique <n>CaM</n>-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity <n>CaM</n> binding sites. <n>CaM</n> bound to <n>4.1R</n> at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one <n>CaM</n> molecule binds to two distinct sites in the same molecule of <n>4.1R</n>. Interactions of <n>4.1R</n> with membrane proteins such as band 3 is regulated by Ca(2+) and <n>CaM</n>. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both <n>CaM</n> binding sites, the ability of Ca(2+)/<n>CaM</n> to down-regulate <n>4. 1R</n>-band 3 interaction was abrogated by such deletions. Thus, regulation of <n>protein 4.1</n> binding to membrane proteins by Ca(2+) and <n>CaM</n> requires binding of <n>CaM</n> to both Ca(2+)-independent and Ca(2+)-dependent sites in <n>protein 4.1</n>.
10668799>Interaction cloning and characterization of <n>RoBPI</n>, a novel protein binding to <n>human Ro ribonucleoproteins</n>. <n>Human Ro ribonucleoproteins</n> (<n>RNPs</n>) are autoantigenic particles of unknown function(s) that consist of a 60-kDa protein (<n>Ro60</n>) associated with one hY RNA (hY1-5). Using a modified yeast three-hybrid system, named <n>RNP</n> interaction trap assay (RITA), we cloned a novel <n>Ro RNP-binding protein</n> (<n>RoBPI</n>), based on its property to interact in vivo in yeast with an <n>RNP</n> complex made of recombinant <n>Ro60</n> (<n>rRo60</n>) protein and hY5 (rhY5) RNA. <n>RoBPI</n> cDNA contains three conserved RNA recognition motifs (RRM) and is present as a family of isoforms differing slightly at their 5' end. The 2.0-kb <n>RoBPI</n> mRNA was detected in all human tissues tested. Highly homologous cDNA sequences were found in banks of expressed sequence tags (ESTs) from mice. Two-hybrid, three-hybrid, and RITA experiments respectively established that <n>60 kDa RoBPI</n> did not interact in yeast with <n>rRo60</n> alone, with rhY5 RNA alone, or with bait <n>RNPs</n> consisting of <n>rRo60</n> and recombinant hY1, hY3, or hY4 RNAs. <n>RoBPI</n> coimmunoprecipitated with <n>Ro RNPs</n> from HeLa cell extracts and partially colocalized with <n>Ro60</n> in nuclei of cultured cells. Because hY5 RNA and RohY5 RNPs are recent evolutionary additions seen only in primates, but <n>RoBPI</n> seems more conserved, their interaction may represent a gain of function for <n>Ro RNPs</n>. Alternatively, interaction of <n>RohY5 RNPs</n> with <n>RoBPI</n> may have no functional bearing, but may underlie some of the unique biochemical and immunological properties of these <n>RNPs</n>.
10636863>Direct interaction of the <n>CD38</n> cytoplasmic tail and the <n>Lck</n> SH2 domain. <n>Cd38</n> transduces T cell activation signals through associated <n>Lck</n>. <n>CD38</n> ligation has been shown to induce activation of intracellular signaling cascade in T lymphocytes through a <n>Lck</n>-dependent pathway. However, it is not clear how <n>Lck</n> initiates the <n>CD38</n>-mediated signaling process. In the present study, we showed that <n>CD38</n> and <n>Lck</n> were physically associated through the cytoplasmic tail and the <n>Src</n> homology 2 domain, respectively. This was evidenced by coimmunoprecipitation of <n>Lck</n> with <n>CD38</n> and <n>Lck</n> with isolated <n>CD38</n> cytoplasmic domain from T cell lysate, cell lysate of COS-7 cells cotransfected with cDNAs of <n>Lck</n> and <n>CD38</n>, or a mixture of in vitro translated <n>CD38</n> and <n>Lck</n>. Because the <n>CD38</n> cytoplasmic domain does not contain any tyrosine residue, the interaction should be independent of phosphotyrosine. The interaction was further confirmed by in vitro interaction between a purified <n>Lck</n> <n>Src</n> homology 2 domain and a nonphosphosynthetic peptide corresponding to the membrane proximal region of the <n>CD38</n> cytoplasmic domain. In addition, <n>CD38</n> ligation resulted in an elevated tyrosine kinase activity of the <n>CD38</n>-associated <n>Lck</n> and ultimate activation of <n>interleukin-2</n> gene transcription. Furthermore, expression of a kinase-deficient <n>Lck</n> mutant suppressed <n>interleukin-2</n> gene activation in a dose-dependent manner. These results strongly suggested that <n>CD38</n> ligation indeed tranduced signals for T cell activation using its associated <n>Lck</n>.
10611293>The alpha-helical FXXPhiPhi motif in <n>p53</n>: TAF interaction and discrimination by <n>MDM2</n>. Transcriptional activation domains share little sequence homology and generally lack folded structures in the absence of their targets, aspects that have rendered activation domains difficult to characterize. Here, a combination of biochemical and nuclear magnetic resonance experiments demonstrates that the activation domain of the tumor suppressor <n>p53</n> has an FXXPhiPhi motif (F, Phe; X, any amino acids; Phi, hydrophobic residues) that folds into an alpha-helix upon binding to one of its targets, <n>hTAF(II)31</n> (a <n>human TFIID TATA box-binding protein-associated factor</n>). <n>MDM2</n>, the cellular attenuator of <n>p53</n>, discriminates the FXXPhiPhi motif of <n>p53</n> from those of <n>NF-kappaB</n> <n>p65</n> and <n>VP16</n> and specifically inhibits <n>p53</n> activity. Our studies support the notion that the FXXPhiPhi sequence is a general alpha-helical recognition motif for <n>hTAF(II)31</n> and provide insights into the mechanistic basis for regulation of <n>p53</n> function.
10591185>The inhibitory receptor <n>LIR-1</n> uses a common binding interaction to recognize class I MHC molecules and the viral homolog <n>UL18</n>. <n>LIR-1</n> is a class I MHC receptor related to <n>natural killer inhibitory receptors</n> (<n>KIRs</n>). Binding of <n>LIR-1</n> or <n>KIRs</n> to class I molecules results in inhibitory signals. Unlike individual <n>KIRs</n>, <n>LIR-1</n> recognizes many class I alleles and also binds <n>UL18</n>, a human cytomegalovirus class I MHC homolog. Here, we show that <n>LIR-1</n> interacts with the relatively nonpolymorphic alpha3 domain of class I proteins and the analogous region of <n>UL18</n> using its N-terminal <n>immunoglobulin</n>-like domain. The >1000-fold higher affinity of <n>LIR-1</n> for <n>UL18</n> than for class I illustrates how a viral protein competes with host proteins to subvert the host immune response. <n>LIR-1</n> recognition of class I molecules resembles the <n>CD4</n>-class II MHC interaction more than the <n>KIR</n>-class I interaction, implying a functional distinction between <n>LIR-1</n> and <n>KIRs</n>.
10567369>The N-terminal <n>ERK</n>-binding site of <n>MEK1</n> is required for efficient feedback phosphorylation by <n>ERK2</n> in vitro and <n>ERK</n> activation in vivo. An <n>ERK2</n>-binding site at the N terminus of <n>MEK1</n> was reported to mediate their stable association. We examined the importance of this binding site in the feedback phosphorylation of <n>MEK1</n> on Thr(292) and Thr(386) by <n>ERK2</n>, the phosphorylation and activation of <n>ERK2</n> by <n>MEK1</n>, and the interaction of <n>MEK1</n> with <n>ERK2</n> and <n>Raf-1</n>. Deletion of the binding site from <n>MEK1</n> reduced its phosphorylation by <n>ERK2</n>, but had no effect on its phosphorylation by <n>p21-activated protein kinase-1</n> (<n>PAK1</n>). A <n>MEK1</n> N-terminal peptide containing the binding site inhibited <n>MEK1</n> phosphorylation by <n>ERK2</n>. However, it did not affect <n>MEK1</n> phosphorylation by <n>p21-activated protein kinase</n> or <n>myelin basic protein</n> phosphorylation by <n>ERK2</n>. Deletion of the N-terminal <n>ERK</n>-binding domain of <n>MEK1</n> also reduced its ability to phosphorylate <n>ERK2</n> in vitro, to co-immunoprecipitate with <n>ERK2</n>, and to stimulate <n>ERK2</n> activation in transfected cells, but it did not alter the association with endogenous <n>Raf-1</n>. Using <n>ERK2</n>-<n>p38</n> chimeras and an <n>ERK2</n> deletion mutant, a <n>MEK1</n>-binding site of <n>ERK2</n> was localized to its N terminus. 
10536032>Further characterization of the combining sites of <n>Bandeiraea (Griffonia) simplicifolia lectin-I</n>, <n>isolectin A(4)</n>. <n>Bandeiraea (Griffonia) simplicifolia lectin-I</n>, <n>isolectin A(4)</n>(<n>GS I-A(4)</n>), which is cytotoxic to the human colon cancer cell lines, is one of two lectin families derived from its seed extract. It contains only a homo-oligomer of subunit A, and is most specific for GalNAcalpha1. In order to elucidate the <n>GS I-A(4)</n>-glycoconjugate interactions in greater detail, the combining site of this <n>lectin</n> was further characterized by enzyme linked lectino-sorbent assay (ELLSA) and by inhibition of <n>lectin</n>-glycoprotein interactions. This study has demonstrated that the Tn-containing glycoproteins tested, consisting of mammalian salivary glycoproteins (armadillo, asialo-hamster sublingual, asialo-ovine, -bovine, and -porcine submandibular), are bound strongly by <n>GS I-A(4)</n>. Among monovalent inhibitors so far tested, p-NO2-phenylalphaGalNAc is the most potent, suggesting that hydrophobic forces are important in the interaction of this <n>lectin</n>. <n>GS I-A(4)</n>is able to accommodate the monosaccharide GalNAc at the nonreducing end of oligosaccharides. This suggests that the combining site of the lectin is a shallow cavity. Among oligosaccharides and monosaccharides tested as inhibitors of the binding of <n>GS I-A(4)</n>, the hierarchy of potencies are: GalNAcalpha1-->3GalNAcbeta1-->3Galalpha1-->4Galbeta 1-->4Glc (Forssman pentasaccharide) > GalNAcalpha1-->3(LFucalpha1-->2)Gal (blood group A)()> GalNAc > Galalpha1-->4Gal > Galalpha1-->3Gal (blood group B-like)> Gal.
10513896>Interaction of newly synthesized <n>apolipoprotein B</n> with <n>calnexin</n> and <n>calreticulin</n> requires glucose trimming in the endoplasmic reticulum. <n>Apolipoprotein B</n> (<n>ApoB</n>) is the only protein component of the low density lipoproteins (LDL) in plasma. It is a glycoprotein with a molecular mass of about 550 kDa (4536 amino acids) containing 16 N-glycans. We have studied the interaction of <n>ApoB</n> with two <n>lectin</n>-like chaperones of the Endoplasmic Reticulum (ER)--<n>Calnexin</n> (<n>CN</n>) and <n>Calreticulin</n> (<n>CR</n>). Using a co-immunoprecipitation approach we observed that newly synthesized <n>ApoB</n> associates with <n>CN</n> and <n>CR</n>. The interaction was transient; within 30-60 min after synthesis bulk of newly formed <n>ApoB</n> dissociated. Using McA Rh7777 cells expressing an N-terminal fragment of <n>ApoB</n> we found that inhibition of glucosidases in the ER prevented the association of <n>CN</n> and <n>CR</n> to newly synthesized <n>ApoB</n>. The results showed that like for association with other glycoprotein substrates, trimming of glucose residues was essential for <n>ApoB</n> binding to <n>CN</n> and <n>CR</n>.
10493212>Monoclonal antibody specific for <n>tissue factor pathway inhibitor</n>-<n>factor Xa</n> complex: its characterization and application to plasmas from patients with disseminated intravascular coagulation and pre-disseminated intravascular coagulation. <n>Tissue factor pathway inhibitor</n> (<n>TFPI</n>), a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2 and K3), inhibits the initial reactions of the extrinsic blood coagulation pathway through its K1 and K2 domains. We prepared and characterized a monoclonal antibody (<n>Mab8-1</n>) against <n>TFPI</n>-<n>factor Xa</n> (<n>TFPI</n>-<n>Xa</n>) complex. The reactivities of <n>Mab8-1</n> toward <n>TFPI</n>-<n>Xa</n> complex, <n>TFPI</n> without C-terminal (<n>TFPI</n>-C)-<n>Xa</n> complex, K1K2-<n>Xa</n> complex and K2K3-<n>Xa</n> complex were examined using a surface plasmon resonance analysis (Biacore). The Biacore system allowed a quantitative analysis of antibody-antigen interaction, in real time, from which the association and dissociation rate constants could readily be obtained. The bindings of <n>Mab8-1</n> to <n>TFPI</n>-<n>Xa</n> complex, <n>TFPI</n>-C-<n>Xa</n> complex and K2K3-Xa complex were each concentration-dependent. However, no binding of <n>Mab8-1</n> to the K1K2-Xa complex was observed. The binding of <n>Mab8-1</n> to <n>TFPI</n> or <n>Xa</n> was also not observed. These results suggested that the epitope for <n>Mab8-1</n> was exposed in the K3 domain of <n>TFPI</n>, which was generated by the conformational change after the formation of <n>TFPI</n>-<n>Xa</n> complex. We then developed an enzyme-linked immunosorbent assay method specific for <n>TFPI</n>-<n>Xa</n> complex using <n>Mab8-1</n>, and we used this assay to measure plasma levels of <n>TFPI</n>-<n>Xa</n>. The normal range assessed from analyses of plasma from 30 normal healthy volunteers was 17.7-66.7 with a mean of 35.5 +/- 11.7 pmol/l. In order to asses the clinical implication of <n>TFPI</n>-<n>Xa</n> complex in the plasma of patients with thrombotic disorders, plasma concentrations were measured in 37 patients with disseminated intravascular coagulation (DIC) caused by a variety of underlying diseases. The <n>TFPI</n>-<n>Xa</n> antigen levels were significantly higher in the patients with DIC (51.9 +/- 21.6 pmol/l) and the 36 patients with pre-DIC (55.1 +/- 20.2 pmol/l) than in the 137 non-DIC patients (37.9 +/- 13.1 pmol/l). In the patients with DIC or pre-DIC, there was no significant correlation between <n>TFPI</n>-<n>Xa</n> complex and the elevated levels of <n>thrombin</n>-<n>antithrombin</n> complex, <n>plasmin</n>-<n>alpha2 plasmin inhibitor</n> complex, D-dimer, soluble <n>fibrin</n> monomer, soluble <n>thrombomodulin</n> or <n>tissue factor</n>. These data indicate that the plasma level of <n>TFPI</n>-<n>Xa</n> seems to be a novel independent molecular marker of DIC and pre-DIC.
10469658>Control of glycosylation of MHC class II-associated invariant chain by translocon-associated <n>RAMP4</n>. Protein translocation across the membrane of the endoplasmic reticulum (ER) proceeds through a proteinaceous translocation machinery, the translocon. To identify components that may regulate translocation by interacting with nascent polypeptides in the translocon, we used site-specific photo-crosslinking. We found that a region C-terminal of the two N-glycosylation sites of the MHC class II-associated invariant chain (Ii) interacts specifically with the <n>ribosome-associated membrane protein 4</n> (<n>RAMP4</n>). <n>RAMP4</n> is a small, tail-anchored protein of 66 amino acid residues that is homologous to the <n>yeast YSY6 protein</n>. <n>YSY6</n> suppresses a secretion defect of a <n>secY</n> mutant in Escherichia coli. The interaction of <n>RAMP4</n> with Ii occurred when nascent Ii chains reached a length of 170 amino acid residues and persisted until Ii chain completion, suggesting translocational pausing. Site-directed mutagenesis revealed that the region of Ii interacting with <n>RAMP4</n> contains essential hydrophobic amino acid residues. Exchange of these residues for serines led to a reduced interaction with <n>RAMP4</n> and inefficient N-glycosylation. We propose that <n>RAMP4</n> controls modification of Ii and possibly also of other secretory and membrane proteins containing specific <n>RAMP4</n>-interacting sequences. Efficient or variable glycosylation of Ii may contribute to its capacity to modulate antigen presentation by MHC class II molecules.
10438481><n>beta(1) integrin</n> binds the 16-kDa subunit of <n>vacuolar H(+)-ATPase</n> at a site important for <n>human papillomavirus E5</n> and <n>platelet-derived growth factor</n> signaling. <n>Integrins</n> mediate adhesive interactions between cells and the extracellular matrix, and play a role in cell migration, proliferation, differentiation, cytoskeletal organization, and signal transduction. We have identified an interaction between the <n>beta(1) integrin</n> and the 16-kDa subunit of <n>vacuolar H(+)-ATPase</n> (16K). This interaction was first isolated in a yeast two-hybrid screen and confirmed by coimmunoprecipitation and in in vitro binding assays using bacterially expressed proteins. Immunofluorescent studies performed in L6 myoblasts expressing both native and epitope-tagged 16K demonstrate co-localization with <n>beta(1) integrin</n> in focal adhesions. Deletion of the fourth of four transmembrane helices in 16K results in loss of interaction with <n>beta(1) integrin</n> in vitro and in the two-hybrid system, and less prominent staining in focal adhesions. This helix is also required for ligand-independent activation of <n>platelet-derived growth factor-beta receptor</n> signaling by the <n>human papillomavirus E5 oncoprotein</n>. Overexpression of 16K or expression of 16K lacking this helix alters the morphology of myoblasts and fibroblasts, suggesting that the interaction of 16K with integrins could be important for cell growth control. We also discuss the possible role 16K might play in integrin movement.
10409738><n>Human TAF(II)55</n> interacts with the vitamin D(3) and <n>thyroid hormone receptors</n> and with derivatives of the <n>retinoid X receptor</n> that have altered transactivation properties. We have identified novel interactions between the human <n>(h)TATA-binding protein-associated factor TAF(II)55</n> and the ligand-binding domains (LBDs) of the nuclear receptors for vitamin D(3) (<n>VDR</n>) and thyroid hormone (<n>TRalpha</n>). Following expression in Cos cells, <n>hTAF(II)55</n> interacts with the <n>VDR</n> and <n>TRalpha</n> LBDs in a ligand-independent manner whereas no interactions with the <n>retinoid X receptors</n> (<n>RXRs</n>) or with other receptors were observed. Deletion mapping indicates that <n>hTAF(II)55</n> interacts with a 40-amino-acid region spanning alpha-helices H3 to H5 of the <n>VDR</n> and <n>TRalpha</n> LBDs but not with the equivalent highly related region of <n>RXRgamma</n>. <n>TAF(II)55</n> also interacts with chimeric receptors in which the H3-to-H5 region of <n>RXRgamma</n> has been replaced with that of the <n>VDR</n> or <n>TRalpha</n>. Furthermore, replacement of two single amino acids of the <n>RXRgamma</n> LBD with their <n>VDR</n> counterparts allows the <n>RXRgamma</n> LBD to interact with <n>hTAF(II)55</n> while the corresponding double substitution allows a much stronger interaction. In transfection experiments, the single mutated <n>RXRgamma</n> LBDs activate transcription to fivefold higher levels than wild-type <n>RXRgamma</n> while the double mutation activates transcription to a level comparable to that observed with the <n>VDR</n>. There is therefore a correlation between the ability of the modified <n>RXRs</n> to interact with <n>hTAF(II)55</n> and transactivation. These results strongly suggest that the <n>TAF(II)55</n> interactions with the modified <n>RXR</n> LBDs modulate transcriptional activation.
10391250>Interaction of <n>c-Abl</n> and <n>p73alpha</n> and their collaboration to induce apoptosis. <n>c-Abl</n>, a non-receptor tyrosine kinase, is activated by agents that damage DNA. This activation results in either arrest of the cell cycle in phase G1 or apoptotic cell death, both of which are dependent on the kinase activity of <n>c-Abl</n>. <n>p73</n>, a member of the <n>p53</n> family of tumour-suppressor proteins, can also induce apoptosis. Here we show that the apoptotic activity of <n>p73alpha</n> requires the presence of functional, kinase-competent <n>c-Abl</n>. Furthermore, <n>p73</n> and <n>c-Abl</n> can associate with each other, andthis binding is mediated by a PxxP motif in <n>p73</n> and the SH3 domain of <n>c-Abl</n>. We find that <n>p73</n> is a substrate of the <n>c-Abl kinase</n> and that the ability of <n>c-Abl</n> to phosphorylate <n>p73</n> is markedly increased by gamma-irradiation. Moreover, <n>p73</n> is phosphorylated in vivo in response to ionizing radiation. These findings define a pro-apoptotic signalling pathway involving <n>p73</n> and <n>c-Abl</n>.
10366588><n>Transportin-SR</n>, a nuclear import receptor for SR proteins. The SR proteins, a group of abundant arginine/serine (RS)-rich proteins, are essential pre-mRNA splicing factors that are localized in the nucleus. The RS domain of these proteins serves as a nuclear localization signal. We found that RS domain-bearing proteins do not utilize any of the known nuclear import receptors and identified a novel nuclear import receptor specific for SR proteins. The SR protein import receptor, termed <n>transportin-SR</n> (<n>TRN-SR</n>), binds specifically and directly to the RS domains of <n>ASF/SF2</n> and <n>SC35</n> as well as several other SR proteins. The nuclear transport regulator <n>RanGTP</n> abolishes this interaction. Recombinant <n>TRN-SR</n> mediates nuclear import of RS domain- bearing proteins in vitro. <n>TRN-SR</n> has amino acid sequence similarity to several members of the <n>importin beta</n>/<n>transportin</n> family. These findings strongly suggest that <n>TRN-SR</n> is a nuclear import receptor for the SR protein family.
10330192>Identification of <n>CHIP</n>, a novel tetratricopeptide repeat-containing protein that interacts with heat shock proteins and negatively regulates chaperone functions. The chaperone function of the <n>mammalian 70-kDa heat shock proteins Hsc70</n> and <n>Hsp70</n> is modulated by physical interactions with four previously identified chaperone cofactors: <n>Hsp40</n>, <n>BAG-1</n>, the <n>Hsc70-interacting protein Hip</n>, and the <n>Hsc70-Hsp90-organizing protein Hop</n>. <n>Hip</n> and <n>Hop</n> interact with <n>Hsc70</n> via a tetratricopeptide repeat domain. In a search for additional tetratricopeptide repeat-containing proteins, we have identified a novel 35-kDa cytoplasmic protein, <n>carboxyl terminus of Hsc70-interacting protein</n> (<n>CHIP</n>). <n>CHIP</n> is highly expressed in adult striated muscle in vivo and is expressed broadly in vitro in tissue culture. <n>Hsc70</n> and <n>Hsp70</n> were identified as potential interaction partners for this protein in a yeast two-hybrid screen. In vitro binding assays demonstrated direct interactions between <n>CHIP</n> and both <n>Hsc70</n> and <n>Hsp70</n>, and complexes containing <n>CHIP</n> and <n>Hsc70</n> were identified in immunoprecipitates of human skeletal muscle cells in vivo. Using <n>glutathione S-transferase</n> fusions, we found that <n>CHIP</n> interacted with the carboxy-terminal residues 540 to 650 of <n>Hsc70</n>, whereas <n>Hsc70</n> interacted with the amino-terminal residues 1 to 197 (containing the tetratricopeptide domain and an adjacent charged domain) of <n>CHIP</n>. Recombinant <n>CHIP</n> inhibited <n>Hsp40-stimulated ATPase</n> activity of <n>Hsc70</n> and <n>Hsp70</n>, suggesting that <n>CHIP</n> blocks the forward reaction of the <n>Hsc70</n>-<n>Hsp70</n> substrate-binding cycle. Consistent with this observation, both <n>luciferase</n> refolding and substrate binding in the presence of <n>Hsp40</n> and <n>Hsp70</n> were inhibited by <n>CHIP</n>. Taken together, these results indicate that <n>CHIP</n> decreases net ATPase activity and reduces chaperone efficiency, and they implicate <n>CHIP</n> in the negative regulation of the forward reaction of the <n>Hsc70</n>-<n>Hsp70</n> substrate-binding cycle.
10225901>Molecular interactions of Porphyromonas gingivalis fimbriae with host proteins: kinetic analyses based on surface plasmon resonance. Fimbriae of Porphyromonas gingivalis are thought to play an important role in the colonization and invasion of periodontal tissues. In this study, we analyzed the interactions of P. gingivalis fimbriae with <n>human hemoglobin</n>, <n>fibrinogen</n>, and salivary components (i.e., <n>proline-rich protein</n> [<n>PRP</n>], <n>proline-rich glycoprotein</n> [<n>PRG</n>], and <n>statherin</n>) based on surface plasmon resonance (SPR) spectroscopy with a biomolecular interaction analyzing system (BIAcore). The real-time observation showed that the fimbriae interacted more quickly with <n>hemoglobin</n> and <n>PRG</n> than with other proteins and more intensely with <n>fibrinogen</n>. The significant association constant (ka) values obtained by BIAcore demonstrated that the interactions between fimbriae and these host proteins are specific. These estimated Ka values were not too different; however, the Ka values for <n>hemoglobin</n> (2.43 x 10(6)) and <n>fibrinogen</n> (2.16 x 10(6)) were statistically greater than those for the salivary proteins (1.48 x 10(6) to 1.63 x 10(6)). The Ka value of anti-fimbriae <n>immunoglobulin G</n> for fimbriae was estimated to be 1. 22 x 10(7), which was 6.55-fold higher than the mean Ka value of the host proteins. Peptide PRP-C, a potent inhibitor of <n>PRP</n>-fimbriae interaction, dramatically inhibited fimbrial association to <n>PRP</n> and <n>PRG</n> and was also inhibitory against other host proteins by BIAcore. The binding of fimbriae to these proteins was also evaluated by other methods with hydroxyapatite beads or polystyrene microtiter plates. The estimated binding abilities differed considerably, depending on the assay method that was used. It was noted that the binding capacity of <n>PRP</n> was strongly diminished by immobilization on a polystyrene surface. Taken together, these findings suggest that P. gingivalis fimbriae possess a strong ability to interact with the host proteins which promote bacterial adherence to the oral cavity and that SPR spectroscopy is a useful method for analyzing specific protein-fimbriae interactions.
10212261>Inhibition of <n>Ets-1</n> DNA binding and ternary complex formation between <n>Ets-1</n>, <n>NF-kappaB</n>, and DNA by a designed DNA-binding ligand. Sequence-specific pyrrole-imidazole polyamides can be designed to interfere with transcription factor binding and to regulate gene expression, both in vitro and in living cells. Polyamides bound adjacent to the recognition sites for <n>TBP</n>, <n>Ets-1</n>, and <n>LEF-1</n> in the human immunodeficiency virus, type 1 (HIV-1), long terminal repeat inhibited transcription in cell-free assays and viral replication in human peripheral blood lymphocytes. The DNA binding activity of the transcription factor <n>Ets-1</n> is specifically inhibited by a polyamide bound in the minor groove. <n>Ets-1</n> is a member of the winged-helix-turn-helix family of transcription factors and binds DNA through a recognition helix bound in the major groove with additional phosphate contacts on either side of this major groove interaction. The inhibitory polyamide possibly interferes with phosphate contacts made by <n>Ets-1</n>, by occupying the adjacent minor groove. Full-length <n>Ets-1</n> binds the HIV-1 enhancer through cooperative interactions with the p50 subunit of <n>NF-kappaB</n>, and the Ets-inhibitory polyamide also blocks formation of ternary <n>Ets-1</n>. <n>NF-kappaB</n>.DNA complexes on the HIV-1 enhancer. A polyamide bound adjacent to the recognition site for <n>NF-kappaB</n> also inhibits <n>NF-kappaB</n> binding and ternary complex formation. These results broaden the application range of minor groove-binding polyamides and demonstrate that these DNA ligands are powerful inhibitors of DNA-binding proteins that predominantly use major groove contacts and of cooperative protein-DNA ternary complexes.
10187830>Mechanisms for <n>GroEL</n>/<n>GroES</n>-mediated folding of a large 86-kDa fusion polypeptide in vitro. Our understanding of mechanisms for <n>GroEL</n>/<n>GroES</n>-assisted protein folding to date has been derived mostly from studies with small proteins. Little is known concerning the interaction of these chaperonins with large multidomain polypeptides during folding. In the present study, we investigated <n>chaperonin</n>-dependent folding of a large 86-kDa fusion polypeptide, in which the mature <n>maltose-binding protein</n> (<n>MBP</n>) sequence was linked to the N terminus of the alpha subunit of the <n>decarboxylase</n> (<n>E1</n>) component of the <n>human mitochondrial branched-chain alpha-ketoacid dehydrogenase</n> complex. The fusion polypeptide, <n>MBP-alpha</n>, when co-expressed with the beta subunit of <n>E1</n>, produced a chimeric protein <n>MBP</n>-<n>E1</n> with an (<n>MBP-alpha</n>)2beta2 structure, similar to the alpha2 beta2 structure in native <n>E1</n>. Reactivation of <n>MBP</n>-<n>E1</n> denatured in 8 M urea was absolutely dependent on <n>GroEL</n>/<n>GroES</n> and Mg2+-ATP, and exhibited strikingly slow kinetics with a rate constant of 376 M-1 s-1, analogous to denatured untagged <n>E1</n>. <n>Chaperonin</n>-mediated refolding of the <n>MBP-alpha</n> fusion polypeptide showed that the folding of the <n>MBP</n> moiety was about 7-fold faster than that of the alpha moiety on the same chain with rate constants of 1.9 x 10(-3) s-1 and 2.95 x 10(-4) s-1, respectively. This explained the occurrence of an <n>MBP-alpha</n>. <n>GroEL</n> binary complex that was isolated with amylose resin from the refolding mixture and transformed Escherichia coli lysates. The data support the thesis that distinct functional sequences in a large polypeptide exhibit different folding characteristics on the same <n>GroEL</n> scaffold. Moreover, we show that when the <n>alpha.GroEL</n> complex (molar ratio 1:1) was incubated with <n>GroES</n>, the latter was capable of capping either the very ring that harbored the 48-kDa (His)6-alpha polypeptide (in cis) or the opposite unoccupied cavity (in trans). In contrast, the <n>MBP-alpha.GroEL</n> (1:1) complex was capped by <n>GroES</n> exclusively in the trans configuration. These findings suggest that the productive folding of a large multidomain polypeptide can only occur in the <n>GroEL</n> cavity that is not sequestered by <n>GroES</n>.
10074138>Interaction of the human immunodeficiency virus type 1 nucleocapsid with <n>actin</n>. The nucleocapsid (NC) domain of the <n>retrovirus Gag protein</n> plays several important roles in the viral life cycle, including virus assembly, viral genomic RNA encapsidation, primer tRNA placement, and enhancement of viral reverse transcription. In this study, deletion of NC domain of <n>human immunodeficiency virus type 1 (HIV-1) Gag</n> was found to drastically reduce virus particle production in <n>CD4</n>(+) T cells. Cellular fractionation experiments showed that although most of the uncleaved wild-type <n>HIV-1 Gag</n>, unmyristylated <n>Gag</n>, and p6(<n>Gag</n>) domain-truncated <n>Gag</n> molecules copurified with the host cell cytoskeleton, most of the mutant <n>Gag</n> molecules lacking both the NC and p6(<n>Gag</n>) domains failed to cofractionate with cytoskeleton. In wild-type virus-infected cells, in which the viral protease was active, the cleaved NCp7 copurified with the cytoskeleton, whereas most of the MAp17 and CAp24 did not. Monoclonal antibody against <n>actin</n> coimmunoprecipitated full-length <n>Gag</n> and p6(<n>Gag</n>) domain-truncated <n>Gag</n> molecules from cell lysates but failed to precipitate the truncated mutant <n>Gag</n> molecules lacking NC plus p6(<n>Gag</n>). Purified recombinant NCp7, but not CAp24, was able to bind <n>F-actin</n> in cosedimentation experiments. Furthermore, wild-type NCp7 and a zinc finger mutant NCp7(F16A), like a cellular <n>actin</n>-binding protein (<n>the villin headpiece</n>), bound <n>F-actin</n> in a dose-dependent fashion in vitro. Taken together, these results suggest that HIV-1 NCp7 can bind <n>F-actin</n> directly and that interaction between <n>HIV-1 Gag</n> and the <n>actin</n> cytoskeleton through the NC domain may play an important role in HIV-1 assembly and/or other steps of the viral life cycle.
10026279>Importance of <n>factor VIIa</n> Gla-domain residue Arg-36 for recognition of the macromolecular substrate <n>factor X</n> Gla-domain. Macromolecular substrate docking with coagulation enzyme-cofactor complexes involves multiple contacts distant from the enzyme's catalytic cleft. Here we characterize the binding of the Gla-domain of macromolecular substrate <n>coagulation factor X</n> to the complex of <n>tissue factor</n> (<n>TF</n>) and <n>VIIa</n>. Site-directed mutagenesis of charged residue side chains in the VIIa Gla-domain identified Arg-36 as being important for macromolecular substrate docking. Ala substitution for Arg-36 resulted in an increased KM and a decreased rate of X activation. X with a truncated Gla-domain was activated by mutant and wild-type VIIa at indistinguishable rates, demonstrating that Arg-36 interactions require a properly folded Gla-domain of the macromolecular substrate. VIIa Arg-36 was also required for effective docking of the X Gla-domain in the absence of phospholipid, demonstrating that the Gla-domain of VIIa participates in protein-protein interactions with X. In the absence of <n>TF</n>, the mutant VIIa had essentially normal function, indicating that the cofactor positions VIIa's Gla-domain for optimal macromolecular substrate docking. Computational docking suggests multiple charge complementary contacts of the X Gla-domain with <n>TF</n>.VIIa. A prominent interaction is made by the functionally important X residue Gla-14 with the center of the extended docking site created by residues in the carboxyl module of <n>TF</n> and the contiguous VIIa Gla-domain. These data demonstrate the functional importance of interactions of the Gla-domains of enzyme and substrate, and begin to elucidate the molecular details of the ternary <n>TF</n>.VIIa.X complex.
9920917><n>Smad1</n> recognition and activation by the <n>ALK1</n> group of <n>transforming growth factor-beta</n> family receptors. Two structural elements, the L45 loop on the kinase domain of the <n>transforming growth factor-beta</n> (<n>TGF-beta</n>) family type I receptors and the L3 loop on the MH2 domain of <n>Smad</n> proteins, determine the specificity of the interactions between these receptors and <n>Smad</n> proteins. The L45 sequence of the <n>TGF-beta type I receptor</n> (<n>TbetaR-I</n>) specifies <n>Smad2</n> interaction, whereas the related L45 sequence of the <n>bone morphogenetic protein (BMP) type I receptor</n> (<n>BMPR-I</n>) specifies <n>Smad1</n> interactions. Here we report that members of a third receptor group, which includes <n>ALK1</n> and <n>ALK2</n> from vertebrates and <n>Saxophone</n> from Drosophila, specifically phosphorylate and activate <n>Smad1</n> even though the L45 sequence of this group is very divergent from that of <n>BMPR-I</n>. We investigated the structural elements that determine the specific recognition of <n>Smad1</n> by <n>ALK1</n> and <n>ALK2</n>. In addition to the receptor L45 loop and the <n>Smad1</n> L3 loop, the specificity of this recognition requires the alpha-helix 1 of <n>Smad1</n>. The alpha-helix 1 is a conserved structural element located in the vicinity of the L3 loop on the surface of the <n>Smad</n> MH2 domain. Thus, <n>Smad1</n> recognizes two distinct groups of receptors, the <n>BMPR-I</n> group and the <n>ALK1</n> group, through different L45 sequences on the receptor kinase domain and a differential use of two surface structures on the <n>Smad1</n> MH2 domain.
94129004>Retinoic acid downmodulates erythroid differentiation and <n>GATA1</n> expression in purified adult-progenitor culture. All-trans retinoic acid (RA) is an important morphogen in vertebrate development, a normal constituent in human adult blood and is also involved in the control of cell growth and differentiation in acute promyelocytic leukemia. We have examined the effects of RA on normal hematopoiesis by using early hematopoietic progenitor cells (HPC) stringently purified from adult peripheral blood. In clonogenetic fetal calf serum-supplemented (FCS+) or -nonsupplemented (FCS-) culture treated with saturating levels of <n>interleukin-3</n> (<n>IL-3</n>) <n>granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>) and <n>erythropoietin</n> (<n>Ep</n>) (combined with c-kit ligand in FCS(-)-culture conditions), RA induces a dramatic dose-dependent shift from erythroid to granulomonocytic colony formation, the latter colonies being essentially represented by granulocytic clones. This shift is apparently not caused by a recruitment phenomenon, because in FCS+ culture, the total number of colonies is not significantly modified by RA addition. In FCS-liquid-suspension culture supplemented with saturating <n>Ep</n> level and low-dose <n>IL-3</n>/<n>GM-CSF</n>, adult HPC undergo unilineage erythropoietic differentiation: Here again, treatment with high-dose RA induces a shift from the erythroid to granulocytic differentiation pathway. Studies on RA time-response or pulse treatment in semisolid or liquid culture show that early RA addition is most effective, thus indicating that early but not late HPC are sensitive to its action. We then analyzed the expression of the master <n>GATA1</n> gene, which encodes a finger transcription factor required for normal erythroid development; addition of RA to HPC stimulated into unilineage erythropoietic differentiation in liquid culture caused a virtually complete inhibition of <n>GATA1</n> mRNA induction. These results indicate that RA directly inhibits the erythroid differentiation program at the level of early adult HPC, and may lead to a shift from the erythroid to granulocytic differentiation pathway. This phenomenon is correlated with inhibition of <n>GATA1</n> induction in the early stages of erythropoietic differentiation.
97175006>Pancreatic development and maturation of the islet B cell. Studies of pluripotent islet cultures. Pancreas organogenesis is a highly regulated process, in which two anlage evaginate from the primitive gut. They later fuse, and, under the influence of the surrounding mesenchyme, the mature organ develops, being mainly composed of ductal, exocrine and endocrine compartments. Early buds are characterized by a branching morphogenesis of the ductal epithelium from which endocrine and exocrine precursor cells bud to eventually form the two other compartments. The three compartments are thought to be of common endodermal origin; in contrast to earlier hypotheses, which suggested that the endocrine compartment was of neuroectodermal origin. It is thus generally believed that the pancreatic endocrine-lineage possesses the ability to mature along a differentiation pathway that shares many characteristics with those of neuronal differentiation. During recent years, studies of <n>insulin</n>-gene regulation and, in particular, the tissue-specific transcriptional control of <n>insulin</n>-gene activity have provided information on pancreas development in general. The present review summarizes these findings, with a special focus on our own studies on pluripotent endocrine cultures of rat pancreas.
97087515>Tissue and cell-type specific expression of the <n>tuberous sclerosis gene</n>, <n>TSC2</n>, in human tissues. <n>TSC2</n> is a gene on chromosome 16p13.3 associated with the autosomal dominant neurocutaneous disorder, tuberous sclerosis complex (TSC). By using a partial nucleotide sequence from the cloned <n>TSC2</n> and polymerase chain reaction methodology, we constructed a digoxigenin-labeled complementary DNA probe to examine <n>TSC2</n> gene expression in autopsy- or biopsy-derived human tissues by in situ hybridization. <n>TSC2</n> messenger RNA was widely expressed in various cell types throughout the body, including epithelia, lymphocytes, and cells with endocrine functions, e.g., adrenal cortex and anterior pituitary. It was prominently and selectively (within the central nervous system) expressed in pyramidal cells of the cerebral cortex and other motor neurons, e.g., in spinal cord and brainstem nuclei. Visceral <n>TSC2</n> expression was comparable in autopsy tissues from patients with and without TSC; <n>TSC2</n> messenger RNA expression was most prominent in cells with a rapid mitotic rate and turnover, e.g., epithelia and lymphocytes, with central nervous system pyramidal cells and other neurons being an obvious exception, and/or in cells with important secretory/transport functions. This widespread expression of the <n>TSC2</n> gene supports the view that it encodes a protein vital to cell growth and metabolism or one that functions as a tumor/growth suppressor.
94110342>Steroid-resistant asthma. Cellular mechanisms contributing to inadequate response to glucocorticoid therapy. The current study examined whether alterations in <n>glucocorticoid receptor</n> (<n>GR</n>) binding contribute to poor response to glucocorticoid therapy in asthma. 29 asthma patients with forced expiratory volume in 1 s (FEV1) 30% after a 1-wk course of oral prednisone 20 mg twice daily and steroid resistant (SR) if they failed to increase > 15%. PBMC obtained from these two groups, 17 SR and 12 SS, as well as 12 normal controls were analyzed. SR patients had two distinguishable GR binding abnormalities: 15 of the 17 SR patients demonstrated a significantly reduced GR binding affinity, as compared with SS patients (P = 0.0001) and normal controls (P = 0.0001). This defect was localized to T cells and reverted to normal after 48 h in culture media. However, incubation with a combination of <n>IL-2</n> and <n>IL-4</n> sustained this abnormality. The other two SR patients had an abnormally low GR number with normal binding affinity that was not limited to T cells. Furthermore, GR number failed to normalize after incubation in media alone or <n>IL-2</n> and <n>IL-4</n>. Therefore, SR asthma may be due to more than one abnormality, the majority related to a reversible cytokine-induced reduction in GR binding affinity and the second related to an irreversible reduction in GR number. These findings may have important implications for the design of alternative treatment approaches for recalcitrant asthma.
97098661><n>Elf-1</n> and <n>Stat5</n> bind to a critical element in a new enhancer of the human <n>interleukin-2 receptor alpha</n> gene [published erratum appears in Mol Cell Biol 1997 Apr;17(4):2351] The <n>interleukin 2 receptor alpha</n>-chain (<n>IL-2R alpha</n>) gene is a key regulator of lymphocyte proliferation. <n>IL-2R alpha</n> is rapidly and potently induced in T cells in response to mitogenic stimuli. <n>Interleukin 2</n> (<n>IL-2</n>) stimulates <n>IL-2R alpha</n>. transcription, thereby amplifying expression of its own high-affinity receptor. <n>IL-2R alpha</n> transcription is at least in part controlled by two positive regulatory regions, PRRI and PRRII. PRRI is an inducible proximal enhancer, located between nucleotides -276 and -244, which contains <n>NF-kappaB</n> and SRE/CArG motifs. PRRII is a T-cell-specific enhancer, located between nucleotides -137 and -64, which binds the T-cell-specific <n>Ets protein Elf-1</n> and <n>HMG-I(Y)</n> proteins. However, none of these proximal regions account for the induction of <n>IL-2R alpha</n> transcription by <n>IL-2</n>. To find new regulatory regions of the <n>IL-2R alpha</n> gene, 8.5 kb of the 5' end noncoding sequence of the <n>IL-2R alpha</n> gene have been sequenced. We identified an 86-nucleotide fragment that is 90% identical to the recently characterized murine <n>IL-2</n>-responsive element (mIL-2rE). This putative human IL-2rE, designated PRRIII, confers <n>IL-2</n> responsiveness on a heterologous promoter. PRRIII contains a Stat protein binding site that overlaps with an EBS motif (GASd/EBSd). These are essential for <n>IL-2</n> inducibility of PRRIII/CAT reporter constructs. <n>IL-2</n> induced the binding of <n>Stat5a</n> and <n>b</n> proteins to the human GASd element. To confirm the physiological relevance of these findings, we carried out in vivo footprinting experiments which showed that stimulation of <n>IL-2R alpha</n> expression correlated with occupancy of the GASd element. Our data demonstrate a major role of the GASd/EBSd element in <n>IL-2R alpha</n> regulation and suggest that the T-cell-specific <n>Elf-1</n> factor can serve as a transcriptional repressor.
94149008><n>rel</n> Is rapidly tyrosine-phosphorylated following <n>granulocyte-colony stimulating factor</n> treatment of human neutrophils. Stimulation of neutrophils with <n>granulocyte-colony stimulating factor</n> (<n>G-CSF</n>) results in an enhanced respiratory burst, prolonged survival, and increased tumor cell killing. The effects of <n>G-CSF</n> are mediated by binding to specific, high affinity receptors. <n>G-CSF receptors</n> lack intrinsic tyrosine kinase activity, but activation of the receptor results in the rapid induction of tyrosine kinase activity. Antiphosphotyrosine immunoblots of whole cell lysates prepared from neutrophils show that the <n>G-CSF</n> rapidly induces prominent tyrosine phosphorylation of a protein of a relative molecular mass of 80 kDa. Using monospecific antibodies, the 80-kDa tyrosine-phosphorylated protein has been shown to be <n>p80c-rel</n>, a proto-oncogene belonging to a family of transcriptional regulators which include <n>NF-kB</n>. The induction of tyrosine phosphorylation of <n>p80c-rel</n> was unique to <n>G-CSF</n> in that <n>granulocyte-macrophage colony stimulating factor</n> which also stimulates neutrophils and induces tyrosine phosphorylation does not result in tyrosine phosphorylation of <n>p80c-rel</n>. The consequences of <n>p80c-rel</n> tyrosine phosphorylation are not yet known; however, tyrosine-phosphorylated <n>p80c-rel</n> is capable of binding to DNA, and <n>G-CSF</n> stimulation results in an increase in the amount of <n>p80c-rel</n> which binds to DNA. These results demonstrate that one of the first biochemical events which occurs in neutrophils following <n>G-CSF</n> stimulation, activation of a tyrosine kinase, leads directly to the tyrosine phosphorylation of <n>p80c-rel</n>. Thus, the tyrosine kinase activated by <n>G-CSF</n> appears to directly transduce a signal to a protein which functions as a transcriptional regulator.
97115792>Mutation of tyrosines 492/493 in the kinase domain of <n>ZAP-70</n> affects multiple T-cell receptor signaling pathways. The protein-tyrosine kinase <n>ZAP-70</n> is implicated, together with the <n>Src kinase p56(lck)</n>, in controlling the early steps of the <n>T-cell antigen receptor</n> (<n>TCR</n>) signaling cascade. To help elucidate further the mechanism by which <n>ZAP-70</n> regulates these initial events, we used a dominant-negative mutant approach. We overexpressed in the Jurkat T-cell line <n>ZAP-70</n> mutated on Tyr-492 and Tyr-493 in the putative regulatory loop of its kinase domain. This mutant inhibited <n>TCR</n>-induced activation of <n>nuclear factor of activated T cells</n> by interfering with both intracellular calcium increase and <n>Ras</n>-regulated activation of extracellular signal-regulated kinases. Moreover, <n>TCR</n>-induced phosphorylation of <n>pp36-38</n>, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wild-type <n>ZAP-70</n> induced constitutive activation of <n>nuclear factor of activated T cells</n>. The <n>ZAP-70</n> mutant studied here could be phosphorylated on tyrosine when associated to the <n>TCR</n> zeta chain and was able to bind <n>p56(lck)</n>. This result demonstrates that Tyr-492 and Tyr-493 are not responsible for the <n>Src</n> homology domain 2-mediated association of <n>p56(lck)</n> with <n>ZAP-70</n>. Our data are most consistent with a model in which recruitment to the <n>TCR</n> allows <n>ZAP-70</n> autophosphorylation and binding to <n>p56(lck)</n>, which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the <n>ZAP-70</n> kinase activity. <n>ZAP-70</n> will then be able to effectively control phosphorylation of its substrates and lead to gene activation.
97066996><n>Stat3</n> recruitment by two distinct ligand-induced, tyrosine-phosphorylated docking sites in the <n>interleukin-10 receptor</n> intracellular domain. Recent work has shown that <n>IL-10</n> induces activation of the <n>JAK</n>-<n>STAT</n> signaling pathway. To define the mechanism underlying <n>signal transducer and activator of transcription</n> (<n>STAT</n>) protein recruitment to the <n>interleukin 10 (IL-10) receptor</n>, the <n>STAT</n> proteins activated by <n>IL-10</n> in different cell populations were first defined using electrophoretic mobility shift assays. In all cells tested, <n>IL-10</n> activated <n>Stat1</n> and <n>Stat3</n> and induced the formation of three distinct DNA binding complexes that contained different combinations of these two transcription factors. <n>IL-10</n> also activated <n>Stat5</n> in Ba/F3 cells that stably expressed the <n>murine IL-10 receptor</n>. Using a structure-function mutagenesis approach, two tyrosine residues (Tyr427 and Tyr477) in the intracellular domain of the <n>murine IL-10 receptor</n> were found to be redundantly required for receptor function and for activation of <n>Stat3</n> but not for <n>Stat1</n> or <n>Stat5</n>. Twelve amino acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated <n>Stat3</n> but not <n>Stat1</n> and blocked <n>IL-10</n>-induced <n>Stat3</n> phosphorylation in a cell-free system. In contrast, tyrosine-phosphorylated peptides containing Tyr374 or Tyr396 did not interact with <n>Stat3</n> or block <n>Stat3</n> activation. These data demonstrate that <n>Stat3</n> but not <n>Stat1</n> or <n>Stat5</n> is directly recruited to the ligand-activated <n>IL-10 receptor</n> by binding to specific but redundant receptor intracellular domain sequences containing phosphotyrosine. This study thus supports the concept that utilization of distinct <n>STAT</n> proteins by different cytokine receptors is dependent on the expression of particular ligand-activatable, tyrosine-containing <n>STAT</n> docking sites in receptor intracellular domains.
96420243><n>Interleukin-6</n> promotes multiple myeloma cell growth via phosphorylation of <n>retinoblastoma protein</n>. <n>Interleukin-6</n> (<n>IL-6</n>) mediates autocrine and paracrine growth of multiple myeloma (MM) cells and inhibits tumor cell apoptosis. Abnormalities of <n>retinoblastoma protein</n> (<n>pRB</n>) and mutations of <n>RB</n> gene have been reported in up to 70% of MM patients and 80% of MM-derived cell lines. Because dephosphorylated (activated) <n>pRB</n> blocks transition from G1 to S phase of the cell cycle whereas phosphorylated (inactivated) <n>pRB</n> releases this growth arrest, we characterized the role of <n>pRB</n> in <n>IL-6</n>-mediated MM cell growth. Both phosphorylated and dephosphorylated <n>pRB</n> were expressed in all serum-starved MM patient cells and MM-derived cell lines, but <n>pRB</n> was predominantly in its phosphorylated form. In MM cells that proliferated in response to <n>IL-6</n>, exogenous <n>IL-6</n> downregulated dephosphorylated <n>pRB</n> and decreased dephosphorylated <n>pRB</n>-<n>E2F</n> complexes. Importantly, culture of MM cells with <n>RB</n> antisense, but not <n>RB</n> sense, oligonucleotide (ODN) triggered <n>IL-6</n> secretion and proliferation in MM cells; however, proliferation was only partially inhibited by neutralizing <n>anti-IL-6 monoclonal antibody</n> (<n>MoAb</n>). In contrast to MM cells, normal splenic B cells express dephosphorylated <n>pRB</n>. Although <n>CD40</n> ligand (CD40L) triggers a shift from dephosphorylated to phosphorylated <n>pRB</n> and proliferation of B cells, the addition of exogenous <n>IL-6</n> to CD40L-treated B cells does not alter either <n>pRB</n> or proliferation, as observed in MM cells. These results suggest that phosphorylated <n>pRB</n> is constitutively expressed in MM cells and that <n>IL-6</n> further shifts <n>pRB</n> from its dephosphorylated to its phosphorylated form, thereby promoting MM cell growth via two mechanisms; by decreasing the amount of E2F bound by dephosphorylated <n>pRB</n> due to reduced dephosphorylated <n>pRB</n>, thereby releasing growth arrest; and by upregulating <n>IL-6</n> secretion by MM cells and related <n>IL-6</n>-mediated autocrine tumor cell growth.
97031819>Regulation of <n>interferon-gamma</n> gene expression. <n>Interferon-gamma</n> (<n>IFN-gamma</n>), also known as <n>type II interferon</n>, is an important immunoregulatory gene that has multiple effects on the development, maturation, and function of the immune system. <n>IFN-gamma</n> mRNA and protein are expressed predominantly by T cells and large granular lymphocytes. The <n>IFN-gamma</n> mRNA is induced/inhibited in these cell types by a wide variety of extracellular signals, thus implicating a number of diverse, yet convergent signal transduction pathways in its transcriptional control. In this review, I describe how DNA methylation and specific DNA binding proteins may regulate transcription of the <n>IFN-gamma</n> gene in response to extracellular signals.
97028169>Dual action of retinoic acid on human embryonic/fetal hematopoiesis: blockade of primitive progenitor proliferation and shift from multipotent/erythroid/monocytic to granulocytic differentiation program. In preliminary studies, we have analyzed the <n>hematopoietic growth factor</n> (<n>HGF</n>) requirement of hematopoietic progenitor cells (HPCs) purified from embryonic-fetal liver (FL) and grown in fetal calf serum-supplemented (FCS+) clonogenic culture. The key role of <n>erythropoietin</n> (<n>Epo</n>) for colony formation by early erythroid progenitors (burst-forming units-erythroid [BFU-E]) has been confirmed. Furthermore, in the absence of exogenous <n>HGFs</n>, FL monocytic progenitors (colony-forming unit monocyte [CFU-M]) generate large colonies exclusively composed of monocytes-macrophages; these colonies are absent in FCS- clonogenic culture. On this basis, we have investigated the role of all-trans retinoic acid (ATRA) and its isomer 9-cis RA in FL hematopoiesis. Both compounds modulate the growth of purified FL HPCs, which show a dose-dependent shift from mixed/erythroid/ monocytic to granulocytic colony formation. Studies on unicellular and paired daughter cell culture unequivocally indicate that the shift is mediated by modulation of the HPC differentiation program to the granulopoietic pathway (rather than RA-induced down-modulation of multipotent/ erythroid/monocytic HPC growth coupled with recruitment of granulocytic HPCs). ATRA and 9-cis RA also exert their effect on the proliferation of primitive HPCs (high-proliferative potential colony-forming cells [HPP-CFCs]) and putative hematopoietic stem cells (HSCs; assayed in Dexter-type long-term culture). High concentrations of either compound (1) drastically reduced the number of primary HPP-CFC colonies and totally abolished their recloning capacity and (2) inhibited HSC proliferation. It is crucial that these results mirror recent observations indicating that murine adult HPCs transduced with dominant negative <n>ATRA receptor</n> (<n>RAR</n>) gene are immortalized and show a selective blockade of granulocytic differentiation. Altogether, these results suggest that ATRA/9-cis RA may play a key role in FL hematopoiesis via a dual effect hypothetically mediated by interaction with the <n>RAR</n>/<n>RXR</n> heterodimer, ie, inhibition of HSC/ primitive HPC proliferation and induction of CFU-GEMM/ BFU-E/CFU-M shift from the multipotent/erythroid/monocytic to the granulocytic-neutrophilic differentiation program.
96355009>Defective transcription of the <n>IL-2</n> gene is associated with impaired expression of <n>c-Fos</n>, <n>FosB</n>, and <n>JunB</n> in anergic T helper 1 cells. Anergic <n>CD4</n>+ Th cells do not produce <n>IL-2</n> when challenged with Ag-pulsed accessory cells because of a transcriptional defect. In this work, we report that these anergic T cells are defective in their ability to up-regulate protein binding and transactivation at two critical <n>IL-2</n> DNA enhancer elements: <n>NF-AT</n> (<n>nuclear factor of activated T cells</n>; a sequence that binds a heterotrimeric <n>NFATp</n>, <n>Fos</n>, and <n>Jun</n> protein complex) and <n>Activator Protein-1</n> (<n>AP-1</n>) (that binds <n>Fos</n> and <n>Jun</n> heterodimers). Western blot analysis of nuclear extracts showed that the impaired DNA-protein interactions in anergic T cells were associated with poor expression of the inducible <n>AP-1</n> family members <n>c- Fos</n>, <n>FosB</n>, and <n>JunB</n>. However, the reduced expression of these proteins was not the result of a global <n>TCR</n>/<n>CD3</n>-signaling defect because <n>CD3</n> cross-linking induced an equivalent increase in intracellular-free calcium ions, as well as <n>NFATp</n> dephosphorylation, translocation to the nucleus, and DNA binding in both normal and anergic T cells.Thus, defective <n>IL-2</n> gene transcription appears to be due, at least in part, to a selective block in the expression of the <n>AP-1 Fos</n> and <n>Jun</n> family members in anergic T cells.
96405269>Regulation of <n>GM-CSF</n> gene transcription by <n>core-binding factor</n>. <n>GM-CSF</n> gene activation in T cells is known to involve the transcription factors <n>nuclear factor-kappa B</n>, <n>AP-1</n>, <n>NFAT</n>, and <n>Sp1</n>. Here we demonstrate that the <n>human GM-CSF</n> promoter and enhancer also encompass binding sites for <n>core-binding factor</n> (<n>CBF</n>). Significantly, the <n>CBF</n> sites are in each case contained within the minimum essential core regions required for inducible activation of transcription. Furthermore, these core regions of the enhancer and promoter each encompass closely linked binding sites for <n>CBF</n>, <n>AP-1</n>, and <n>NFATp</n>. The <n>GM-CSF</n> promoter <n>CBF</n> site TGTGGTCA is located 51 bp upstream of the transcription start site and also overlaps a YY-1 binding site. A 2-bp mutation within the CBF site resulted in a 2-3-fold decrease in the activities of both a 69-bp proximal promoter fragment and a 627-bp full-length promoter fragment. Stepwise deletions into the proximal promoter also revealed that the <n>CBF</n> site, but not the YY-1 site, was required for efficient induction of transcriptional activation. The <n>AML1</n> and <n>CBF beta</n> genes that encode <n>CBF</n> each have the ability to influence cell growth and differentiation and have been implicated as proto-oncogenes in acute myeloid leukemia. This study adds <n>GM-CSF</n> to a growing list of cytokines and receptors that are regulated by <n>CBF</n> and which control the growth, differentiation, and activation of hemopoietic cells. The <n>GM-CSF</n> locus may represent one of several target genes that are dysregulated in acute myeloid leukemia.
96427516>Cytomegalovirus modulates <n>interleukin-6</n> gene expression. Complications after lung transplantation include the development of rejection and an increased incidence of infection, particularly with cytomegalovirus (CMV). Several recent studies have suggested that <n>interleukin (IL)-6</n> may be used to detect both infection and rejection after lung transplantation. In addition, <n>IL-6</n> may play a role in the development of bronchiolitis obliterans after transplantation. Because CMV is also associated with the development of bronchiolitis obliterans after transplantation, we determined whether CMV induces <n>IL-6</n> gene expression. We demonstrated that CMV infection increased both <n>IL-6</n> protein and mRNA in peripheral blood mononuclear cells. We also demonstrated that the <n>CMV immediate early 1 gene product</n> increased expression of the <n>IL-6</n> promoter. This effect of the <n>CMV immediate early 1 gene product</n> was dependent upon the presence of specific transcription factor binding sites in the <n>IL-6</n> promoter. These studies demonstrate that CMV may be an important cofactor in the development of rejection and infection after transplantation through its effects on <n>IL-6</n>.
96186497>Effects of <n>interleukin-10</n> on human peripheral blood mononuclear cell responses to Cryptococcus neoformans, Candida albicans, and lipopolysaccharide. Deactivation of mononuclear phagocytes is critical to limit the inflammatory response but can be detrimental in the face of progressive infection. We compared the effects of the deactivating cytokine <n>interleukin 10</n> (<n>IL-10</n>) on human peripheral blood mononuclear cell (PBMC) responses to lipopolysaccharide (LPS), Cryptococcus neoformans, and Candida albicans. <n>IL-10</n> effected dose-dependent inhibition of <n>tumor necrosis factor alpha</n> (<n>TNF-alpha</n>) release in PBMC stimulated by LPS and C. neoformans, with significant inhibition seen with 0.1 U/ml and greater than 90% inhibition noted with 10 U/ml. In contrast, even at doses as high as 100 U/ml, <n>IL-10</n> inhibited <n>TNF-alpha</n> release in response to C. albicans by only 50%. <n>IL-10</n> profoundly inhibited release of <n>IL-1beta</n> from PBMC stimulated by all three stimuli. <n>TNF-alpha</n> mRNA and release was inhibited even if <n>IL-10</n> was added up to 8 h after cryptococcal stimulation. In contrast, inhibition of <n>IL-1 beta</n> mRNA was of lesser magnitude and occurred only when <n>IL-10</n> was added within 2 h of cryptococcal stimulation. <n>IL-10</n> inhibited translocation of <n>NF-kappaB</n> in response to LPS but not the fungal stimuli. All three stimuli induced <n>IL-10</n> production in PBMC, although over 10-fold less <n>IL-10</n> was released in response to C. neoformans compared with LPS and C. albicans. Thus, while <n>IL-10</n> has deactivating effects on PBMC responses to all three stimuli, disparate stimulus- and response-specific patterns of deactivation are seen. Inhibition by <n>IL-10</n> of proinflammatory cytokine release appears to occur at the level of gene transcription for <n>TNF-alpha</n> and both transcriptionally and posttranscriptionally for <n>IL-1beta</n>.
96279111>Multiple transcription factors are required for activation of <n>human interleukin 9</n> gene in T cells. The genetic elements and regulatory mechanisms responsible for <n>human interleukin 9</n> (<n>IL-9</n>) gene expression in a human T cell leukemia virus type I-transformed human T cell line, C5MJ2, were investigated. We demonstrated that <n>IL-9</n> gene expression is controlled, at least in part, by transcriptional activation. Transient expression of the <n>luciferase</n> reporter gene linked to serially deleted sequences of the 5'-flanking region of the <n>IL-9</n> gene has revealed several positive and negative regulatory elements involved in the basal and inducible expression of the <n>IL-9</n> gene in C5MJ2 cells. An <n>AP-1</n> site at -146 to -140 was shown to be involved in the expression of the <n>IL-9</n> gene. A proximal region between -46 and -80 was identified as the minimum sequence for the basal and inducible expression of the <n>IL-9</n> gene in C5MJ2 cells. Within this region, an <n>NF-kappaB</n> site at -59 to -50 and its adjacent 20-base pair upstream sequence were demonstrated to play a critical role for the <n>IL-9</n> promoter activity. DNA-protein binding studies indicated that <n>NF-kappaB</n>, <n>c-Jun</n>, and potentially novel proteins (around 35 kDa) can bind to this important sequence. Mutations at different sites within this proximal promoter region abolished the promoter activity as well as the DNA binding. Taken together, these results suggest that the cooperation of different transcription factors is essential for <n>IL-9</n> gene expression in T cells.
96198880>Soluble <n>tumor necrosis factor</n> receptors inhibit phorbol myristate acetate and cytokine-induced HIV-1 expression chronically infected U1 cells. Recombinant <n>human tumor necrosis factor (TNF) binding protein-1</n> (<n>r-h TBP-1</n>) and recombinant <n>human soluble dimeric TNF receptor</n> (<n>rhu TNFR:Fc</n>) were used to determine the relative contributions of <n>TNF</n> to phorbol myristate acetate (PMA) and cytokine-induced human immunodeficiency virus type 1 (HIV-1) replication in chronically infected cell lines. Treatment of HIV-1-infected promonocytic U1 cells with <n>r-h-TBP-1</n> or <n>rhu TNFR:Fc</n> reduced PMA-induced <n>HIV-1 p24 antigen</n> production in a concentration-dependent manner, with a maximal inhibition of approximately 90%. Maximal inhibition of <n>p24 antigen</n> production in T-lymphocytic ACH-2 cells was 47% with <n>r-hTBP-1</n> and 42% with <n>rhu TNFR:Fc</n>. <n>r-hTBP-1</n> and <n>rhu TNFR:Fc</n> also decreased <n>p24 antigen</n> synthesized by U1 cells in response to other stimuli, including <n>phytohemagglutinin</n> (<n>PHA</n>)-induced supernatant, <n>granulocyte-macrophage colony-stimulating factor</n>, <n>interleukin-6</n>, and <n>TNF</n>. Addition of <n>r-hTBP-1</n> to U1 cells during the last 4 h of a 24 h incubation with PMA still inhibited <n>p24 antigen</n> production by 15%. U1 cells stimulated with 10(-7) M PMA released approximately 1 ng/ml endogenous <n>TBP-1</n> with an initial peak observed at 1 h and a second peak at 24 h after PMA stimulation. <n>r-hTBP-1</n> also partially reversed inhibition of U1 cellular proliferation caused by PMA. Both <n>r-hTBP-1</n> and <n>rhu TNFR:Fc</n> blocked PMA induction of <n>nuclear factor (NK)-kappa B</n> DNA-binding activity in U1 cells in association with decreases in HIV-1 replication. We conclude that soluble <n>TNF</n> receptors can inhibit stimuli-induced HIV-1 expression and <n>NK-kappa B</n> DNA-binding activity in chronically infected U1 cells.
96216453><n>Granulocyte-macrophage colony-stimulating factor</n> stimulates <n>JAK2</n> signaling pathway and rapidly activates <n>p93fes</n>, <n>STAT1 p91</n>, and <n>STAT3 p92</n> in polymorphonuclear leukocytes. <n>Granulocyte-macrophage colony-stimulating factor</n> (<n>GM-CSF</n>), supports proliferation, differentiation, and functional activation of hemopoietic cells by its interaction with a heterodimeric receptor. Although <n>GM-CSF receptor</n> is devoid of tyrosine kinase enzymatic activity, <n>GM-CSF</n>-induced peripheral blood polymorphonuclear leukocytes (PMN) functional activation is mediated by the phosphorylation of a large number of intracellular signaling molecules. We have previously shown that <n>JAK2</n> becomes tyrosine-phosphorylated in response to <n>GM-CSF</n> in PMN. In the present study we demonstrate that also the <n>signal transducers and activators of transcription</n> (<n>STAT</n>) family members <n>STAT1 p91</n> and <n>STAT3 p92</n> and the product of the <n>c-fps</n>/<n>fes</n> protooncogene become tyrosine-phosphorylated upon <n>GM-CSF</n> stimulation and physically associated with both <n>GM-CSF receptor</n> beta common subunit and <n>JAK2</n>. Moreover <n>GM-CSF</n> was able to induce <n>JAK2</n> and <n>p93fes</n> catalytic activity. We also demonstrate that the association of the <n>GM-CSF receptor beta</n> common subunit with <n>JAK2</n> is ligand-dependent. Finally we demonstrate that <n>GM-CSF</n> induces a DNA-binding complex that contains both <n>p91</n> and <n>p92</n>. These results identify a new signal transduction pathway activated by <n>GM-CSF</n> and provide a mechanism for rapid activation of gene expression in <n>GM-CSF</n>-stimulated PMN.
96189089>Inactivation of <n>IkappaBbeta</n> by the <n>tax protein</n> of human T-cell leukemia virus type 1: a potential mechanism for constitutive induction of <n>NF-kappaB</n>. In resting T lymphocytes, the transcription factor <n>NF-kappaB</n> is sequestered in the cytoplasm via interactions with members of the <n>I kappa B</n> family of inhibitors, including <n>IkappaBalpha</n> and <n>IkappaBbeta</n>. During normal T-cell activation, <n>IkappaBalpha</n> is rapidly phosphorylated, ubiquitinated, and degraded by the 26S proteasome, thus permitting the release of functional <n>NF-kappaB</n>. In contrast to its transient pattern of nuclear induction during an immune response, <n>NF-kappaB</n> is constitutively activated in cells expressing the <n>Tax transforming protein</n> of human T-cell leukemia virus type I (HTLV-1). Recent studies indicate that <n>HTLV-1 Tax</n> targets <n>IkappaBalpha</n> to the ubiquitin-proteasome pathway. However, it remains unclear how this viral protein induces a persistent rather than transient <n>NF-kappaB</n> response. In this report, we provide evidence that in addition to acting on <n>IkappaBalpha</n>, <n>Tax</n> stimulates the turnover Of <n>IkappaBbeta</n> via a related targeting mechanism. Like <n>IkappaBalpha</n>, Tax-mediated breakdown of <n>IkappaBbeta</n> in transfected T lymphocytes is blocked either by cell-permeable proteasome inhibitors or by mutation Of <n>IkappaBbeta</n> at two serine residues present within its N-terminal region. Despite the dual specificity of <n>HTLV-1 Tax</n> for <n>IkappaBalpha</n> and <n>IkappaBbeta</n> at the protein level, <n>Tax</n> selectively stimulates <n>NF-kappaB</n>-directed transcription of the <n>IkappaBalpha</n> gene. Consequently, <n>IkappaBbeta</n> protein expression is chronically downregulated in HTLV-1-infected T lymphocytes. These findings with <n>IkappaBbeta</n> provide a potential mechanism for the constitutive activation of <n>NF-kappaB</n> in <n>Tax</n>-expressing cells.
96182568>A model of latent adenovirus 5 infection in the guinea pig (Cavia porcellus). A model of adenovirus 5 (Ad5) infection was developed in guinea pigs to begin to study its role in the pathogenesis of peripheral lung inflammation. Forty animals were inoculated intranasally with 10(7.0) pfu of Ad5/animal, and 15 animals inoculated with sterile culture media served as controls. Viral titres were 10(4.4), 10(6.1), 10(5.2), and 10(2.9) pfu/animal, on days 1, 3, 4, and 7 after infection, respectively. In situ hybridization to viral DNA and immunocytochemistry for <n>Ad5 E1A protein</n> localized the virus to airway and alveolar epithelial cells. Histologic examination showed an extensive inflammatory cell infiltration around the airways, with epithelial necrosis and an alveolar exudate that caused localized alveolar collapse in the infected areas. Immunocytochemistry identified the cells in the infiltrate as cytotoxic T cells. Although all animals 20 and 47 days after infection had seroconverted to Ad5, virus was not detected in these groups either by viral plaque assay or in situ hybridization. <n>Ad5 E1A</n> DNA was detected by polymerase chain reaction in five of six animals 20 days after infection and in five of five animals 47 days after infection. In these same animals, <n>E1A protein</n> was detected 20 days after infection in two and 47 days after infection in one while persistent bronchiolitis was observed in four and three animals 20 and 47 days after infection, respectively. These results demonstrate that the guinea pig provides a useful model to study the role of Ad5 infection in chronic airway inflammation.
96278860>In vivo protein-DNA interactions of the human <n>beta-globin</n> locus in erythroid cells expressing the fetal or the adult <n>globin</n> gene program. To characterize the protein-DNA interactions important for the developmental control of the human <n>beta-globin</n> locus, we analyzed by in vivo dimethyl sulfate footprinting erythroid cells expressing either the fetal or the adult <n>globin</n> developmental program. In the locus control region (LCR) of the <n>beta-globin</n> locus, in vivo footprints on <n>NF-E2</n> (or <n>AP-1</n>) and <n>GATA-1</n> motifs remained the same regardless of whether the fetal or the adult <n>globin</n> genes are expressed. In contrast, in vivo footprints on GT (CACCC) motifs differed between the cells expressing the fetal or the adult <n>globin</n> program. In promoter regions, the actively transcribed genes demonstrated extensive and consistent footprints over the canonical elements, such as CACCC and CCAAT motifs. The adult <n>globin</n> expressing cells displayed more extensive footprints than the fetal <n>globin</n> expressing cells in the 3' regulatory sequences of both the <n>Agamma</n>- and the <n>beta-globin</n> genes, suggesting a role of these 3' elements in <n>beta-globin</n> gene expression. Our results suggest that the bulk of protein-DNA interactions that underlies the developmental control of <n>globin</n> genes takes place in the <n>gamma-</n> and <n>beta-globin</n> gene promoters, and that GT motifs of the <n>beta-globin</n> locus LCR may play a role in the developmental regulation of <n>human beta-globin</n> gene expression, perhaps by increasing the probability of interaction of the LCR holocomplex with the fetal or the adult <n>globin</n> gene.
99401563>Polyamines in human breast cancer and its relations to classical prognostic features: clinical implications. Experimental evidence suggest an important role of polyamines in breast cancer development. Polyamines have been determined in tissue and erythrocyte samples from 100 patients with primary invasive breast cancer and 30 patients with fibroadenomas. Statistical analysis was performed in order to determine the prognostic value of the polyamine patterns of tumor tissues and erythrocytes in comparison with clinical and histological prognostic factors. In malignant tissues, polyamine levels were significantly higher than in benign tissues. They correlated with markers of tumor aggressivity (axillary node involvement and especially with markers of high mitotic rate as Ki-67 staining, histological grade). No correlation was found between estrogen and progesterone status, tumor size and polyamine concentrations. Erythrocyte polyamines levels were identical between cancer patients and controls. The knowledge of the polyamine pattern in breast cancer could become useful in clinical practice particularly if polyamine metabolism is targeted as a therapeutic approach. 
99353322>Peptide binding affinity and pH variation establish functional thresholds for activation of HLA-DQ-restricted T cell recognition. Peptides derived from the <n>HSV-2 VP16 protein</n> were utilized for studies of peptide binding to DQ0302 molecules and T cell activation at both neutral and acidic pH. The native peptide <n>VP16</n> 430-444 contains an Asp at position 442, binds to DQ0302 strongly, with a Kd value of 50nM at acidic pH and very weakly, with a Kd value of greater than 10 microM at neutral pH. A truncated version of 430-444, i.e., <n>VP16</n> 433-442, binds with an affinity 10-fold lower compared to 430-444 at acidic pH, and binding at neutral pH was barely detectable. The homologous peptide 430-444,442A has an Asp to Ala substitution at position 442 and binds to DQ0302 with a Kd similar to 433-442. The short truncated analog 433-442A binds very poorly at both acidic and neutral pH. Both the wild type 430-444 and 433-442 peptides stimulated a HSV-specific T cell clone after a brief incubation with antigen presenting cells (APC) expressing DQ0302 at acidic pH. Much higher concentrations of wild type peptides were needed to activate T cells at neutral pH. In contrast, APC pulsed with Ala-substituted peptides 430-444,442A or 433-442A at neutral pH failed to stimulate the T cell clone, while APC pulsed at acidic pH and subsequently washed led to successful T cell activation. The Ala-substituted peptide was recognized by the T cell clone at neutral pH only when it was present in the APC culture throughout the stimulation process. While the MHC-peptide complexes formed with the native peptide are stable, complexes formed with the Ala-substituted peptide had a functional t1/2 of less than 4 hr at neutral pH.
96320762>Involvement of tyrosine phosphorylation in <n>endothelial adhesion molecule</n> induction. Induction of <n>endothelial adhesion molecules</n> by the cytokine <n>tumor necrosis factor-alpha</n> (<n>TNF</n>) can occur independently of <n>protein kinase C</n> and activation of a protein tyrosine kinase (PTK) has recently been implicated in the upregulation of <n>vascular cell adhesion molecule 1</n> (<n>VCAM-1</n>) by <n>interleukin-4</n> (<n>IL-4</n>) on endothelial cells. We demonstrate that the PTK inhibitors herbimycin A or genistein suppress induction of endothelial <n>VCAM-1</n> and <n>E-selectin</n>, as well as subsequent monocytic cell adhesion to endothelial cells stimulated by <n>TNF</n>. Inhibition studies indicate that specific tyrosine phosphorylation following PTK activation is involved in the mobilization of the transcription factor, <n>nuclear factor kappa B</n>, and <n>VCAM-1</n> mRNA expression. This may have implications for pathophysiological conditions that involve the upregulation of these molecules (e.g. inflammation and atherosclerosis).
99376409>Dendritic cells and the pathogenesis of rheumatoid arthritis. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease in which unknown arthrogenic autoantigen is presented to <n>CD4</n>+ T cells. The strong association of the disease with an epitope within the <n>HLA-DR</n> chain shared between various alleles of <n>HLA-DR4</n> and <n>DR1</n> emphasizes the importance of antigen presentation. This immune response predominantly occurs in the synovial tissue and fluid of the joints and autoreactive T cells are readily demonstrable in both the synovial compartment and blood. Circulating dendritic cells (DC) are phenotypically and functionally identical with normal peripheral blood (PB) DC. In the synovial tissue, fully differentiated perivascular DC are found in close association with T cells and with B cell follicles, sometimes containing follicular DC. These perivascular DC migrate across the activated endothelium from blood and receive differentiative signals within the joint from monocyte-derived cytokines and <n>CD40</n>-ligand+ T cells. In the SF, DC manifest an intermediate phenotype, similar to that of monocyte-derived DC in vitro. Like a delayed-type hypersensitivity response, the rheumatoid synovium represents an effector site. DC at many effector sites have a characteristic pattern of infiltration and differentiation. It is important to note that the effector response is not self-limiting in RA autoimmune inflammation. In this article, we argue that the presentation of self-antigen by DC and by autoantibody-producing B cells is critical for the perpetuation of the autoimmune response. Permanently arresting this ongoing immune response with either pharmaceutical agents or immunotherapy is a major challenge for immunology.
96144699>Inhibition of <n>NF-AT</n> signal transduction events by a dominant-negative form of <n>calcineurin</n>. An inhibitory, "dominant-negative," form of the <n>calcineurin</n> catalytic (A) subunit was prepared, which lacks the <n>calmodulin</n>-binding domain, autoinhibitory domain and most of its catalytic core but possesses the regulatory (B) subunit binding domain. When tested for its ability to block <n>calcineurin</n>-dependent signaling in Jurkat cells, expression of this "B-subunit knock-out" (BKO) construct suppressed reporter gene activity driven by <n>NF-AT</n>, the pivotal promoter element for <n>interleukin (IL)-2</n> gene induction. Immunoprecipitation of epitope-labeled BKO demonstrated for the formation of a tight complex with endogenous B subunit in Jurkat cells, consistent with an inhibitory mechanism that involves the sequestration of the B subunit. Furthermore, the sharply reduced <n>NF-AT</n> activity produced by co-transfecting BKO could be "rescued" by overexpression of transfected B subunit, suggesting that depletion of this subunit was responsible for the inhibition. These data suggest the potential utility of agents that disrupt <n>calcineurin</n>-mediated signal transduction pathways by blocking formation of the catalytically active dimer of <n>calcineurin</n> A and B subunits.
99409030><n>c-Maf</n> induces monocytic differentiation and apoptosis in bipotent myeloid progenitors. The transcriptional mechanisms that drive colony-forming unit granulocyte-macrophage (CFU-GM) myeloid progenitors to differentiate into cells of either the granulocytic or monocytic lineage are not fully understood. We have shown that the <n>c-Maf</n> and <n>c-Myb</n> transcription factors physically interact in myeloid cells to form inhibitory complexes that hinder transactivation of <n>c-Myb</n> target genes through direct binding to <n>Myb</n> consensus sites. These complexes arise in a developmentally regulated pattern, peaking at the promyelocyte stage, or in cell model systems, appearing soon after the induction of monocytic differentiation. We wished to determine if this developmentally related interaction is a consequence of myeloid differentiation or an intrinsic differentiating stimulus. Because the elevated <n>Myb</n>:<n>Maf</n> status seen in differentiating cells can be recapitulated by overexpression of <n>c-Maf</n> in myeloid cell lines, we inducibly expressed the <n>c-Maf</n> cDNA in 2 bipotent human myeloid progenitor cells. Elevated levels of <n>c-Maf protein</n> led to marked increases in <n>Myb</n>:<n>Maf</n> complexes and the accumulation of monocyte/macrophage cells, followed by eventual programmed cell death. Analysis of targets that could mediate these phenotypic changes indicated that <n>c-Maf</n> likely plays a key role in myeloid cell development through dual mechanisms; inhibition of a select set of <n>c-Myb</n> regulated targets, such as <n>Bcl-2</n> and <n>CD13/APN</n>, coupled with the activation of as yet undefined differentiation-promoting genes.
99357866>Host defense mechanisms triggered by microbial lipoproteins through <n>toll-like receptors</n>. The generation of cell-mediated immunity against many infectious pathogens involves the production of <n>interleukin-12</n> (<n>IL-12</n>), a key signal of the innate immune system. Yet, for many pathogens, the molecules that induce <n>IL-12</n> production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of <n>IL-12</n> production by human macrophages, and that induction is mediated by <n>Toll-like receptors</n> (<n>TLRs</n>). Several lipoproteins stimulated <n>TLR</n>-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of <n>TLRs</n> by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.
93268332>Regulation of the <n>interleukin-1 beta</n> (<n>IL-1 beta</n>) gene by mycobacterial components and lipopolysaccharide is mediated by two <n>nuclear factor-IL6</n> motifs. The cytokines <n>interleukin-1 beta</n> (<n>IL-1 beta</n>) and <n>tumor necrosis factor alpha</n> (<n>TNF-alpha</n>) are released by mononuclear phagocytes in vitro after stimulation with mycobacteria and are considered to mediate pathophysiologic events, including granuloma formation and systemic symptoms. We demonstrated that the Mycobacterium tuberculosis cell wall component lipoarabinomannan (LAM) is a very potent inducer of <n>IL-1 beta</n> gene expression in human monocytes and investigated the mechanism of this effect. We localized the LAM-, lipopolysaccharide (LPS)-, and <n>TNF-alpha</n>-inducible promoter activity to a -131/+15 (positions -131 to +15) DNA fragment of the <n>IL-1 beta</n> gene by deletion analysis and <n>chloramphenicol acetyltransferase</n> assay. Within this DNA fragment, there were two novel 9-bp motifs (-90/-82 and -40/-32) with high homology to the <n>nuclear factor-IL6</n> (<n>NF-IL6</n>) binding site. Site-directed mutagenesis demonstrated that the two <n>NF-IL-6</n> motifs could be independently activated by LAM, LPS, or <n>TNF-alpha</n> and that they acted in an orientation-independent manner. DNA mobility shift assay revealed specific binding of nuclear protein(s) from LAM-, LPS-, or <n>TNF-alpha</n>-stimulated THP-1 cells to the <n>NF-IL6</n> motifs. We conclude that the two <n>NF-IL6</n> sites mediate induction of <n>IL-1 beta</n> in response to the stimuli LAM, LPS, and <n>TNF-alpha</n>.
93264305>Activation of primary human T-lymphocytes through <n>CD2</n> plus <n>CD28</n> adhesion molecules induces long-term nuclear expression of <n>NF-kappa B</n>. Stimulation of highly purified human T-cells via <n>CD2</n> and <n>CD28</n> adhesion molecules induces and maintains proliferation for more than 3 weeks. This potent <n>interleukin 2</n> (<n>IL-2</n>)-dependent activation does not require monocytes or accessory cells. Long-lasting <n>IL-2</n> receptivity is associated with high-level expression of the inducible <n>IL-2 receptor alpha</n> chain (<n>IL-2R alpha</n>) gene that is regulated at both transcriptional and posttranscriptional levels. Increase of <n>IL-2R alpha</n> gene transcription involves the enhanced binding of the transcription factor <n>NF-kappa B</n> to its consensus sequence in the 5'-regulatory region of the <n>IL-2R alpha</n> gene. To dissect the molecular basis for the unusually persistent transcription of the <n>IL-2R alpha</n> gene, we analyzed nuclear <n>NF-kappa B</n> binding to a radiolabeled <n>IL-2R alpha</n> kappa B-specific oligonucleotide probe during the time course of <n>CD2</n> + <n>CD28</n> activation. Resting T-cell nuclear extracts contained <n>KBF1/p50</n> homodimer. After stimulation, two new kappa B-specific complexes were identified as <n>NF-kappa B p50-p65</n> heterodimer and putative <n>c-Rel</n> homodimer or <n>c-Rel</n>-<n>p65</n> heterodimer. Both inducible complexes persisted for at least 3 weeks. Their relative levels were very similar for the duration of proliferation. In parallel, <n>CD2</n> + <n>CD28</n> activation triggered a significant intracellular thiol decrease, suggesting that oxygen radicals are involved in the signaling pathway of adhesion molecules. Finally, micromolar amounts of pyrrolidine dithiocarbamate, an oxygen radical scavenger that efficiently blocked the nuclear appearance of <n>NF-kappa B</n> in T-lymphocytes, also inhibited <n>IL-2</n> secretion, <n>IL-2</n>R alpha cell surface expression, and T-cell proliferation. Together, these results suggest that <n>NF-kappa B</n> plays an important role in long-term activation of human primary T-lymphocytes via <n>CD2</n> + <n>CD28</n>.
93320446>Antisense oligonucleotides to the <n>p65</n> subunit of <n>NF-kappa B</n> block <n>CD11b</n> expression and alter adhesion properties of differentiated HL-60 granulocytes. <n>NF-kappa B</n> is a pleiotropic regulator of a variety of genes implicated in the cellular response to injury. This function has been attributed to the coordinated binding of subunits of <n>NF-kappa B</n> to distinct regions of the promoter elements of numerous genes, including cytokines, growth factor receptors, and adhesion molecules. Antisense phosphorothioate oligonucleotides to the <n>p50</n> and <n>p65</n> subunits of the <n>NF-kappa B</n> complex were used to define the physiologic role of this transcription factor in resting and stimulated granulocytes. A reduction in the expression of <n>p65</n> was produced by treatment with the phosphorothioate antisense oligodeoxynucleotide. This reduction was accompanied by rapid changes in the cellular adhesion of dimethyl sulfoxide-differentiated HL-60 leukemia cells stimulated by 12-O-tetradecanoylphorbol 13-acetate (TPA). These effects were characterized by a marked reduction in <n>CD11b integrin</n> expression on the surface of treated cells. Furthermore, the <n>p65</n> antisense oligomer effectively abolished an upregulation of <n>CD11b</n> that was produced by formyl-met-leu-phe and TPA. However, the <n>p65</n> antisense phosphorothioate oligodeoxynucleotide had no significant effect on the production of reactive oxygen intermediates or on phagocytosis by these cells. These findings indicate that antisense oligomers to <n>p65</n> can be used to define the role of <n>NF-kappa B</n> in the activation pathways of neutrophils.
93224733>A protein of the <n>AP-1</n> family is a component of <n>nuclear factor of activated T cells</n>. <n>Nuclear factor of activated T cells</n> (<n>NF-AT</n>) is a transcriptional activator involved in the induction of <n>IL-2</n> gene expression. The response element for <n>NF-AT</n> is a sequence localized between -285/-254 in the <n>IL-2</n> regulatory region. The composition of <n>NF-AT protein</n> is still not fully elucidated. We demonstrate that, in normal human T cells, an <n>AP-1 protein</n> is a component of the <n>NF-AT protein</n> complex. This was evidenced by the ability of the <n>AP-1</n> site to compete with the <n>NF-AT</n> site for binding to <n>NF-AT</n> and by the capacity of immobilized anti-<n>Jun</n> and anti-<n>Fos</n> antibodies to deplete <n>NF-AT</n>-binding activity from nuclear extracts of activated T cells. There was no detectable binding of in vitro translated <n>Jun</n>/<n>Fos</n> heterodimer (<n>AP-1</n>) to the <n>NF-AT</n> sequence, and the <n>NF-AT</n> sequence was unable to inhibit the binding of <n>Jun</n>/<n>Fos</n> to the <n>AP-1</n> sequence. The presence of an <n>AP-1 protein</n> in the <n>NF-AT protein</n> complex may regulate <n>NF-AT</n>-binding activity through protein-protein interaction.
93152834>Expression of <n>tal-1</n> and <n>GATA-binding proteins</n> during human hematopoiesis. <n>Tal-1</n> rearrangements are associated with nearly 30% of human T acute lymphoblastic leukemia. <n>Tal-1</n> gene encodes a putative transcription factor with a basic helix-loop-helix domain and is known to be predominantly expressed in hematopoietic cells. We investigated the pattern of <n>tal-1</n> expression in purified human hematopoietic cells by in situ hybridization and reverse transcriptase polymerase chain reaction analysis. Both methods demonstrated that the <n>tal-1</n> gene is expressed in megakaryocytes and erythroblasts as well as in basophilic granulocytes. In addition, our results indicate that the <n>tal-1</n> 1A promoter, which contains two consensus GATA-binding sites, is active mainly in these lineages. Because the <n>GATA-1</n> gene is known to transactivate several genes specific for the erythroid, megakaryocytic, and mastocytic/basophilic lineages, we studied <n>GATA-1</n> expression in these purified hematopoietic cells. We found that <n>GATA-1</n> and <n>tal-1</n> genes are coexpressed in these three lineages. Remarkably, the expression of both genes is downmodulated during erythroid and megakaryocytic terminal maturation. In immature hematopoietic cells, <n>tal-1</n> and <n>GATA-1</n> genes are coexpressed in committed progenitors cells (<n>CD34</n>+/<n>CD38</n>(2+)), whereas they are not detectable in the most primitive cells (<n>CD34</n>(2+)/<n>CD38</n>-). In contrast, <n>GATA-2</n> is strongly expressed in both most primitive and committed progenitors cells, whereas <n>GATA-3</n> is mostly detected in most primitive ones. Altogether our results strongly suggest that <n>GATA-1</n> modulates the transcription of <n>tal-1</n> during the differentiation of the erythroid, megakaryocytic, and basosophilic lineages.
93350203>Cytokine modulation of HIV expression. Cytokines, the peptide hormones which control the homeostasis of the immune system and also play a fundamental role in inflammatory and immune mediated reactions, have been involved at multiple levels in the pathogenesis of the acquired immune deficiency syndrome (AIDS). Infection with the human immunodeficiency virus (HIV) has been shown to induce production of several cytokines both in vitro and in vivo. Conversely, several cytokines modulate the levels of HIV expression in infected cells of both T lymphocytic and mononuclear phagocytic lineage. Activated mononuclear cells, particularly B cells which are in a state of chronic activation in HIV infected individuals, release HIV-inductive cytokines and thus play a potentially important role in the pathogenesis of HIV infection.
92193465>Mineralocorticoids and <n>mineralocorticoid receptors</n> in mononuclear leukocytes in patients with pregnancy-induced hypertension. To examine the role of mineralocorticoids in the pathophysiology of pregnancy-induced hypertension (PIH), we studied plasma aldosterone and 18-hydroxycorticosterone levels in 25 women with PIH and 25 normal pregnant women, as controls. Furthermore, we evaluated the <n>mineralocorticoid receptor</n> (<n>MR</n>) status in mononuclear leukocytes in the 2 groups. <n>MR</n> count was significantly (P less than 0.0005) decreased in the PIH group (148 +/- 9 binding sites/cell) compared with the control group (300 +/- 17 binding sites/cell; mean +/- SEM). Plasma aldosterone in women with PIH was 281 +/- 61 pmol/L; in normal pregnant women it was 697 +/- 172 pmol/L (P less than 0.025). Plasma 18-hydroxycorticosterone was also significantly (P less than 0.025) lower (PIH, 1071 +/- 149 pmol/L; controls, 1907 +/- 318 pmol/L). These values were determined at the onset of clinical symptoms of PIH. These results cannot be explained by receptor down-regulation due to higher levels of mineralocorticoids in PIH; a hitherto unknown mineralocorticoid may, thus, be responsible for the hypertension and altered <n>MR</n> status.
93129248>Alpha-lipoic acid is a potent inhibitor of <n>NF-kappa B</n> activation in human T cells. Acquired immunodeficiency syndrome (AIDS) results from infection with a human immunodeficiency virus (HIV). The long terminal repeat (LTR) region of HIV proviral DNA contains binding sites for <n>nuclear factor kappa B</n> (<n>NF-kappa B</n>), and this transcriptional activator appears to regulate HIV activation. Recent findings suggest an involvement of reactive oxygen species (ROS) in signal transduction pathways leading to <n>NF-kappa B</n> activation. The present study was based on reports that antioxidants which eliminate ROS should block the activation of <n>NF-kappa B</n> and subsequently HIV transcription, and thus antioxidants can be used as therapeutic agents for AIDS. Incubation of Jurkat T cells (1 x 10(6) cells/ml) with a natural thiol antioxidant, alpha-lipoic acid, prior to the stimulation of cells was found to inhibit <n>NF-kappa B</n> activation induced by <n>tumor necrosis factor</n>-alpha (25 ng/ml) or by phorbol 12-myristate 13-acetate (50 ng/ml). The inhibitory action of alpha-lipoic acid was found to be very potent as only 4 mM was needed for a complete inhibition, whereas 20 mM was required for N-acetylcysteine. These results indicate that alpha-lipoic acid may be effective in AIDS therapeutics.
93041375>Activation of <n>NF-kappa B</n> by <n>interleukin 2</n> in human blood monocytes. We report here that <n>interleukin 2</n> (<n>IL-2</n>) acts on human blood monocytes by enhancing binding activity of the transcription factor <n>NF-kappa B</n> to its consensus sequence in the 5' regulatory enhancer region of the <n>IL-2 receptor alpha chain</n> (<n>p55</n>). Similarly, <n>IL-2</n> activates <n>NF-kappa B</n> in the human monocytic cell line U 937, but not in resting human T-cells. This effect is detectable within 15 min and peaks 1 h after exposure to <n>IL-2</n>. Enhanced <n>NF-kappa B</n> binding activity is followed by functional activation in that inducibility of the <n>IL-2 receptor alpha chain</n> is mediated by enhanced <n>NF-kappa B</n> binding and that a heterologous promoter containing the <n>NF-kappa B</n> consensus sequence (-291 to -245) of the <n>IL-2 receptor alpha chain</n> gene is activated. In addition, <n>IL-2</n> is capable of increasing transcript levels of the <n>p50</n> gene coding for the <n>p50</n> subunit of the <n>NF-kappa B transcription factor</n>, whereas mRNA levels of the <n>p65 NF-kappa B</n> gene remained unchanged.
92195648>Modulation of normal erythroid differentiation by the endogenous thyroid hormone and retinoic acid receptors: a possible target for <n>v-erbA</n> oncogene action. The <n>v-erbA</n> oncogene, a mutated version of the <n>thyroid hormone receptor alpha</n> (<n>c-erbA</n>/<n>TR-alpha</n>), inhibits erythroid differentiation and constitutively represses transcription of certain erythrocyte genes, suggesting a normal function of the proto-oncogene <n>c-erbA</n> in erythropoiesis. Here we demonstrate that the endogenous <n>thyroid hormone receptor alpha</n> (<n>c-erbA</n>/<n>TR-alpha</n>) and the closely related <n>retinoic acid receptor alpha</n> (<n>RAR-alpha</n>) play a role in the regulation of normal erythroid differentiation. Retinoic acid (RA) distinctly modulated the erythroid differentiation program of normal erythroid progenitors and erythroblasts reversibly transformed by a conditional tyrosine kinase oncogene. When added pulsewise to immature cells, differentiation was accelerated while more mature cells underwent premature cell death. Thyroid hormone (T3) alone caused similar but weaker effects. Interestingly, T3 strongly enhanced the action of RA, suggesting cooperative action of the two receptors in modulating erythroid differentiation. Expression of the human <n>RAR-alpha</n> in receptor-negative erythroblasts conferred RA-induced regulation of differentiation to the otherwise unresponsive cells, thus showing that the <n>RAR-alpha</n> is essential for the RA effect. Likewise, enhanced expression of exogenous <n>c-erbA</n>/TR-alpha in erythroblasts rendered them susceptible to modulation of differentiation by T3, suggesting a similar function of both receptors.
93011470>Leukotriene B4 transcriptionally activates <n>interleukin-6</n> expression involving <n>NK-chi B</n> and <n>NF-IL6</n>. Leukotriene B4 (LTB4) is a notable participant in inflammation and chemotaxis. It is, however, still unclear whether LTB4 acts in this regard directly or indirectly by stimulating the release of chemotactic and inflammatory cytokines. Here we report that LTB4 induces synthesis of <n>interleukin (IL)-6</n> by human blood monocytes through transcriptional activation of the <n>IL-6</n> gene. We furthermore demonstrate that this process involves activation of the <n>transcription factor NF-chi B</n> and, to a lesser extent, of <n>NF-IL6</n>, while the activity of the <n>transcription factor AP-1</n>, shown to otherwise confer <n>IL-6</n> inducibility, appeared to be unaffected by LTB4. Involvement of <n>NF-chi B</n> and <n>NF-IL6</n> in induction of <n>IL-6</n> transcription by monocytes was demonstrated using deleted forms of the <n>IL-6</n> promoter. Activation of the <n>IL-6</n> promoter by LTB4 was not only associated with accumulation of the respective transcripts but resulted in synthesis of functional <n>IL-6</n> protein as well. In addition, LTB4 mediated transactivation of a heterologous promoter construct containing the <n>NF-chi B</n> or the <n>NF-IL6</n> enhancer, but not the <n>AP-1</n> enhancer. The signaling events mediating this effect appeared to involve the release of H2O2, since LTB4 failed to induce <n>NF-chi B</n> or <n>NF-IL6</n> in the presence of the scavenger of H2O2, N-acetyl-L-cysteine.
92195320>A lymphoid cell-specific nuclear factor containing <n>c-Rel</n>-like proteins preferentially interacts with <n>interleukin-6 kappa B</n>-related motifs whose activities are repressed in lymphoid cells. The <n>proto-oncoprotein c-Rel</n> is a member of the <n>nuclear factor kappa B transcription factor</n> family, which includes the <n>p50</n> and <n>p65</n> subunits of <n>nuclear factor kappa B</n>. We show here that <n>c-Rel</n> binds to <n>kappa B</n> sites as homodimers as well as heterodimers with <n>p50</n>. These homodimers and heterodimers show distinct DNA-binding specificities and affinities for various <n>kappa B</n> motifs. In particular, the <n>c-Rel</n> homodimer has a high affinity for <n>interleukin-6</n> (<n>IL-6</n>) and <n>beta interferon kappa B</n> sites. In spite of its association with <n>p50</n> in vitro, however, we found a lymphoid cell-specific nuclear factor in vivo that contains <n>c-Rel</n> but not <n>p50</n> epitopes; this factor, termed <n>IL-6 kappa B binding factor II</n>, appears to contain the <n>c-Rel</n> homodimer and preferentially recognizes several <n>IL-6 kappa B</n>-related <n>kappa B</n> motifs. Although it has been previously shown that the <n>IL-6 kappa B</n> motif functions as a potent <n>IL-1</n>/<n>tumor necrosis factor</n>-responsive element in nonlymphoid cells, its activity was found to be repressed in lymphoid cells such as a Jurkat T-cell line. We also present evidence that <n>IL-6 kappa B binding factor II</n> functions as a repressor specific for <n>IL-6 kappa B</n>-related <n>kappa B</n> motifs in lymphoid cells.
93087498>Transcription of the hypersensitive site HS2 enhancer in erythroid cells. In the human genome, the erythroid-specific hypersensitive site HS2 enhancer regulates the transcription of the downstream <n>beta-like globin</n> genes 10-50 kilobases away. The mechanism of HS2 enhancer function is not known. The present study employs RNA protection assays to analyze the transcriptional status of the HS2 enhancer in transfected recombinant chloramphenicol acetyltransferase (CAT) plasmids. In erythroid K562 cells in which the HS2 enhancer is active, the HS2 sequence directs the synthesis of long enhancer transcripts that are initiated apparently from within the enhancer and elongated through the intervening DNA into the cis-linked CAT gene. In nonerythroid HL-60 cells in which the HS2 enhancer is inactive, long enhancer transcripts are not detectable. Splitting the HS2 enhancer between two tandem <n>Ap1</n> sites abolishes the synthesis of a group of long enhancer transcripts and results in loss of enhancer function and transcriptional silencing of the cis-linked CAT gene. In directing the synthesis of RNA through the intervening DNA and the gene by a tracking and transcription mechanism, the HS2 enhancer may (i) open up the chromatin structure of a gene domain and (ii) deliver enhancer binding proteins to the promoter sequence where they may stimulate the transcription of the gene at the cap site.
91292530>Characterization of an <n>immediate-early</n> gene induced in adherent monocytes that encodes <n>I kappa B</n>-like activity. We have cloned a group of cDNAs representing mRNAs that are rapidly induced following adherence of human monocytes. One of the induced transcripts (<n>MAD-3</n>) encodes a protein of 317 amino acids with one domain containing five tandem repeats of the <n>cdc10</n>/<n>ankyrin</n> motif, which is 60% similar (46% identical) to the <n>ankyrin</n> repeat region of the precursor of <n>NF-kappa B</n>/<n>KBF1 p50</n>. The C-terminus has a putative <n>protein kinase C</n> phosphorylation site. In vitro translated <n>MAD-3</n> protein was found to specifically inhibit the DNA-binding activity of the <n>p50</n>/<n>p65 NF-kappa B</n> complex but not that of the <n>p50</n>/<n>p50 KBF1 factor</n> or of other DNA-binding proteins. The <n>MAD-3</n> cDNA encodes an <n>I kappa B</n>-like protein that is likely to be involved in regulation of transcriptional responses to <n>NF-kappa B</n>, including adhesion-dependent pathways of monocyte activation.
91282927>Towards a molecular understanding of T-cell differentiation. Lymphoid differentiation is one of the best studied examples of mammalian development. Here Hans Clevers and Michael Owen describe how the cloning of the genes that encode T-cell-specific membrane proteins allows the identification of transcription factors that control the expression of these T-cell genes. Such transcription factors play a key role in the development of the mature T-cell phenotype by functioning as 'master regulators of T-cell differentiation'.
92042309>Inhibition of phorbol ester-induced monocytic differentiation by dexamethasone is associated with down-regulation of <n>c-fos</n> and <n>c-jun</n> (<n>AP-1</n>). Previous studies have shown that treatment of human myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with induction of monocytic differentiation and expression of the <n>c-jun</n> and <n>c-fos</n> early response genes. The present work demonstrates that the glucocorticoid dexamethasone inhibits TPA-induced increases in <n>c-jun</n> and <n>c-fos</n> mRNA levels in U-937 leukemia cells. These findings were associated with a block in appearance of the monocytic phenotype, including inhibition of TPA-induced increases in <n>lamin A</n>, <n>lamin C</n>, and <n>vimentin</n> transcripts. Other studies have demonstrated that TPA-induced monocytic differentiation and expression of the <n>c-jun</n> and <n>c-fos</n> genes in myeloid leukemia cells are regulated by <n>protein kinase C</n> (<n>PKC</n>). The finding that dexamethasone has no effect on TPA-induced activation of <n>PKC</n> suggests that this glucocorticoid inhibits signals downstream or parallel to this enzyme. Nuclear run-on assays demonstrate that: (1) induction of <n>c-jun</n> and <n>c-fos</n> expression by TPA is regulated by transcriptional mechanisms, (2) TPA-induced expression of <n>c-jun</n> and <n>c-fos</n> does not require protein synthesis, and (3) TPA-induced expression of both genes is inhibited at the transcriptional level by dexamethasone. To further define the effects of dexamethasone at the molecular level, we prepared a series of deleted <n>c-jun</n> promoter fragments linked to the <n>chloramphenicol acetyltransferase</n> (<n>CAT</n>) gene. Increases in <n>CAT</n> activity during transient expression of these constructs in TPA-treated U-937 cells could be assigned to the region (-97 to -20) of the promoter that contains the <n>AP-1</n> binding site. This induction of <n>CAT</n> activity was sensitive to dexamethasone. These findings suggest that dexamethasone down-regulates TPA-induced transcription of the <n>c-jun</n> gene during monocytic differentiation by inhibiting activation of the <n>AP-1</n> site.
91321794>Inhibition of HIV-1 replication and <n>NF-kappa B</n> activity by cysteine and cysteine derivatives. HIV-1 proviral DNA contains two binding sites for the <n>transcription factor NF-kappa B</n>. HIV-1-infected individuals have, on average, abnormally high levels of <n>tumour necrosis factor alpha</n> (<n>TNF alpha</n>) and abnormally low plasma cysteine levels. We therefore investigated the effects of cysteine and related thiols on HIV-1 replication and <n>NF-kappa B</n> expression. The experiments in this report show that cysteine or N-acetylcysteine (NAC) raise the intracellular glutathione (GSH) level and inhibit HIV-1 replication in persistently infected Molt-4 and U937 cells. However, inhibition of HIV-1 replication appears not to be directly correlated with GSH levels. Cysteine and NAC also inhibit <n>NF-kappa B</n> activity as determined by electrophoretic mobility shift assays and <n>chloramphenicol acetyl-transferase</n> (<n>CAT</n>) gene expression under control of <n>NF-kappa B</n> binding sites in uninfected cells. This suggests that the cysteine deficiency in HIV-1-infected individuals may cause an over-expression of <n>NF-kappa B</n>-dependent genes and enhance HIV-1 replication. NAC may be considered for the treatment of HIV-1-infected individuals.
92068245>Constitutive activation of <n>NF-kB</n> in human thymocytes. <n>NF-kB</n> is a eukaryotic transcription regulatory factor. In T cells and T cell lines, <n>NF-kB</n> is bound to a cytoplasmic proteic inhibitor, the <n>IkB</n>. Treatment of T cells with mitogens (phorbol esters) or cytokines (<n>TNF alpha</n>) induces <n>NF-kB</n> nuclear translocation and the subsequent expression of <n>NF-kB</n> dependent T cell genes. Here we examined the activation of <n>NF-kB</n> in human T cell thymic progenitors. We report differences in (Ca2+)i requirement for <n>NF-kB</n> activation in thymocytes as compared to mature T cells. Furthermore, our results indicated that thymocytes have a constitutively active form of <n>NF-kB</n>, suggesting that they are activated in vivo.
92049367>Evaluation of the role of ligand and thermal activation of specific DNA binding by in vitro synthesized <n>human glucocorticoid receptor</n>. We have used a DNA-binding/immunoprecipitation assay to analyze the capacity of <n>human glucocorticoid receptor</n> (<n>hGR</n>), generated in rabbit reticulocyte lysates, to bind DNA. In vitro translated <n>hGR</n> was indistinguishable from native <n>hGR</n>, as determined by migration on sodium dodecyl sulfate-polyacrylamide gels, sedimentation on sucrose density gradients, and reactivity with antipeptide antibodies generated against <n>hGR</n>. In addition, cell-free synthesized <n>hGR</n> was capable of specific binding to glucocorticoid response element (GRE)-containing DNA fragments. Using this assay system, we have evaluated the contributions of ligand binding and heat activation to DNA binding by these <n>glucocorticoid receptors</n>. In vitro translated <n>hGR</n> was capable of selective DNA binding even in the absence of glucocorticoid. Treatment with dexamethasone or the antiglucocorticoid RU486 had no additional effect on the DNA-binding capacity when receptor preparations were maintained at 0 C (no activation). In contrast, addition of either ligand or antagonist in combination with a heat activation step promoted DNA binding by approximately 3-fold over that of heat-activated unliganded receptors. Agonist (dexamethasone) was slightly more effective in supporting specific DNA binding than antagonist (RU486). DNA binding by in vitro synthesized <n>GR</n> was blocked by the addition of sodium molybdate to the receptor preparations before steroid addition and thermal activation. Addition of KCl resulted in less DNA binding either due to blockage of DNA-receptor complex formation or disruption of the complexes. The specificity of DNA binding by cell-free synthesized <n>hGR</n> was analyzed further by examining the abilities of various DNAs to compete for binding to a naturally occurring GRE found in the mouse mammary tumor virus-long terminal repeat. Oligonucleotides containing the consensus GRE were the most efficient competitors, and fragments containing regulatory sequences from glucocorticoid-repressible genes were somewhat competitive, whereas single stranded oligonucleotides were unable to compete for mouse mammary tumor virus-long terminal repeat DNA binding, except when competitor was present at extremely high concentrations. Together these studies indicate that <n>hGR</n> synthesized in rabbit reticulocyte lysates displays many of the same properties, including GRE-specific DNA binding, observed for <n>glucocorticoid receptor</n> present in cytosolic extracts of mammalian cells and tissues. Similarities between the effects of dexamethasone and RU486 suggest that the antiglucocorticoid properties of RU486 do not occur at the level of specific DNA binding.
91132035><n>Platelet-activating factor</n> induces phospholipid turnover, calcium flux, arachidonic acid liberation, eicosanoid generation, and oncogene expression in a human B cell line. <n>Platelet-activating factor</n> is a potent mediator of the inflammatory response. Studies of the actions of <n>platelet-activating factor</n> have centered mainly around neutrophils, monocytes, and platelets. In this report we begin to uncover the influence of <n>platelet-activating factor</n> on B lymphocytes. Employing the EBV-transformed human B cell line SKW6.4, we demonstrate that <n>platelet-activating factor</n> significantly alters membrane phospholipid metabolism indicated by the incorporation of 32P into phosphatidylcholine, phosphatidylinositol, and phosphatidic acid but not significantly into phosphatidylethanolamine at concentrations ranging from 10(-9) to 10(-6) M. The inactive precursor, <n>lyso-platelet-activating factor</n>, at a concentration as high as 10(-7) M had no effect on any of the membrane phospholipids. We also show that <n>platelet-activating factor</n> from 10(-12) to 10(-6) M induced rapid and significant elevation in intracellular calcium levels, whereas <n>lyso-platelet-activating factor</n> was again ineffective. We further demonstrate the impact of <n>platelet-activating factor</n> binding to B cells by measuring <n>platelet-activating factor</n> induced arachidonic acid release and 5-hydroxyeicosatetraenoic acid production. Moreover, <n>platelet-activating factor</n> was capable of inducing transcription of the nuclear proto-oncogenes <n>c-fos</n> and <n>c-jun</n>. Finally we explored the possible role of 5-hydroxyeicosatetraenoic acid as a regulator of arachidonic acid liberation demonstrating that endogenous 5-lipoxygenase activity modulates <n>platelet-activating factor</n> induced arachidonic acid release perhaps acting at the level of phospholipase A2. In summary, <n>platelet-activating factor</n> is shown here to have a direct and profound effect on a pure B cell line.