Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Tyrosine phosphorylation of cellular proteins is the earliest identifiable event following T-cell antigen receptor (TCR) stimulation and is essential for activating downstream signaling machinery. Two Src-family protein-tyrosine kinases, the TCR-associated p59fyn (Fyn) and the CD4/8-associated p56lck (Lck), have emerged as the likely mediators of early tyrosine phosphorylation in T cells. Here, we show direct binding of a 120-kDa TCR-induced phosphotyrosyl polypeptide, p120, to glutathione S-transferase fusion proteins of the Src homology 3 (SH3) domains of Fyn, Lck, and p60src (Src) but not other proteins. While binding of p120 to Fyn SH2 domain was phosphotyrosine-dependent as expected, its binding to the SH3 domain was independent of tyrosine phosphorylation, as shown by lack of competition with a phosphotyrosyl competitor peptide. In contrast, an SH3-specific proline-rich peptide completely abolished p120 binding to SH3. p120 was tyrosine-phosphorylated within 10 sec following stimulation of Jurkat cells with anti-CD3 monoclonal antibody, with maximal phosphorylation at 30 sec. Importantly, p120 was found associated with Fyn and Lck proteins in unstimulated Jurkat cells and served as an in vitro substrate for these kinases. These results provide evidence for a role of the SH3 domains of Fyn and Lck in the recruitment of early tyrosine-phosphorylation substrates to the TCR-associated tyrosine kinases.
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PMID:Rapid T-cell receptor-mediated tyrosine phosphorylation of p120, an Fyn/Lck Src homology 3 domain-binding protein. 751 95

Products of the crk oncogene are expressed in all tissues. Crk proteins are composed exclusively of Src homology 2 (SH2) and Src homology 3 (SH3) domains, and they have been implicated in intracellular signaling. For example, they participate as mediators of Ras activation during nerve growth factor stimulation of PC12 pheochromocytoma cells. We examined the role of Crk proteins during T cell receptor-mediated signaling and observed that Crk proteins specifically interact, via their SH2 domains, with a tyrosine-phosphorylated 116-kDa protein upon T cell activation. p116 may be related to the recently cloned fibroblast p130cas and/or p120-Cbl. In addition, we observed that GST-Crk fusion proteins and Crk-L bind, most likely via their SH3 domain, to C3G, a Ras guanine nucleotide exchange factor. Thus, the interaction of Crk with p116 and C3G strongly implicates Crk as a mediator of T cell receptor signaling, possibly involved in Ras activation.
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PMID:Crk interacts with tyrosine-phosphorylated p116 upon T cell activation. 753 94

This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products. A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-383) and highly purified (> 90%) by affinity chromatography. The affinity of Ras2p for the GST-fused Ira2p-383 was 18 microM and the maximal stimulation of the Ras2p GTPase activity 6,000 times. The Ira2p activity was confirmed to be strictly specific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase being detectable. Comparison with the GAP-like domain of mammalian p120-GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2p-383 has an affinity and turnover intermediary between GAP-334 and NF1-414. The activity of Ira2p-383 was strongly inhibited by monovalent and divalent salts. The simultaneous presence of the catalytic domains of Ira2p and the yeast GDP/GTP exchange factor Cdc25p induced on Ras2p a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, showing that it is possible to reconstitute in vitro a S. cerevisiae system suitable for the study of the regulation of the Ras2p GDP/GTP cycle. The tubulin partially inhibited (25%) the GAP activity of the Ira2p-383. A larger Ira2p catalytic fragment, Ira2p-505 (amino acids 1549-2053), that showed the same Km for Ras2p as Ira2p-383, was also inhibited by tubulin to the same extent but with a higher affinity than Ira2p-383.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties and regulation of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein of Ras2p. 757 70

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.
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PMID:Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein. 762 1

Antigenic cross-linking of the high affinity IgE receptor (Fc epsilon R1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation of the SH2 and SH3 domain containing adapter molecule Grb2 by Fc epsilon R1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S-transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL2H3. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for Fc epsilon R1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2 may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a 33-kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and Fc epsilon R1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc epsilon R1-activated PTKs and binds to Grb2 SH2 domains in Fc epsilon R1 activated but not quiescent cells. The beta subunit of the Fc epsilon R1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the Fc epsilon R1 beta chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein complexes in mast cells of which a subset are Fc epsilon R1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to Fc epsilon R1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. We propose that protein complex formation by Grb2 is an important consequence of Fc epsilon R1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to bring about optimal mast cell end function.
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PMID:Regulation of the adapter molecule Grb2 by the Fc epsilon R1 in the mast cell line RBL2H3. 772 78

Protein tyrosine phosphatase 1B (PTP1B) is a protein tyrosine phosphatase of unknown function, although increasing evidence supports a role for this phosphatase in insulin action. We have investigated the interaction of PTP1B with the insulin receptor using a PTP1B glutathione S-transferase (GST) fusion protein with a point mutation in the enzyme's catalytic domain. This fusion protein is catalytically inactive, but the phosphatase's phosphotyrosine binding site is maintained. The activated insulin receptor was precipitated from purified receptor preparations and whole-cell lysates by the inactive PTP1B-GST, demonstrating a direct association between the insulin receptor and PTP1B. A p120 of unknown identity was also precipitated from whole-cell lysates by the PTP1B fusion protein, but IRS-1 (pp185) was not. A catalytically inactive [35S]PTP1B-fusion protein bound directly to immobilized insulin receptor kinase domains and was displaced in a concentration-dependent manner. Finally, tyrosine-phosphorylated PTP1B was precipitated from whole-cell lysates by an anti-insulin receptor antibody after insulin stimulation. The site of interaction between PTP1B and the insulin receptor was studied using phosphopeptides modeled after the receptor's kinase domain, the NPXY domain, and the COOH-terminal. Each phosphopeptide inhibited the PTP1B-GST:insulin receptor interaction. Study of mutant insulin receptors demonstrated that activation of the kinase domain is necessary for the PTP1B:insulin receptor interaction, but receptors with deletion of the NPXY domain or of the COOH-terminal can still bind to the PTP1B-GST. We conclude that PTP1B can associate directly with the activated insulin receptor at multiple different phosphotyrosine sites and that dephosphorylation by PTP1B may play a significant role in insulin receptor signal transduction.
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PMID:Protein tyrosine phosphatase 1B interacts with the activated insulin receptor. 882 75

We have used two approaches to identify possible substrates of the insulin receptor (IR) tyrosine kinase. First, we used a potent tyrosine phosphatase inhibitor, phenylarsine oxide (PAO), which is reported to be specific for the insulin-induced signal transduction route, to augment tyrosine phosphorylation. Second, we used src homology 2 (SH2) domains fused to glutathione S-transferase as high affinity binding agents for tyrosine-phosphorylated proteins. Using the SH2 domain-containing region of p120 GTPase-activating protein and growth factor-bound protein 2, we observed a tyrosine-phosphorylated M(r) 70,000 protein in insulin- plus PAO-treated NIH3T3 cells overexpressing the IR. This M(r) 70,000 protein, which migrated as a doublet on SDS-polyacrylamide gels, efficiently bound to polyuridylic acid-Sepharose but is distinct from similar-size RNA-binding proteins such as p62 (sam68) and heterogeneous nuclear ribonucleoproteins I, K, L, and M. In addition, it differs from other M(r) 70,000 tyrosine-phosphorylated proteins, such as SH2-containing tyrosine phosphatase, raf1, and paxillin. Tyrosine phosphorylation of this protein was hardly observed after epidermal growth factor treatment. This suggests that the M(r) 70,000 protein is a novel and specific substrate for the IR kinase or an insulin-induced tyrosine kinase. The requirement for PAO to identify this tyrosine phosphorylation indicates a high turnover rate of the tyrosine phosphate.
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PMID:Insulin-induced tyrosine phosphorylation of a M(r) 70,000 protein revealed by association with the Src homology 2 (SH2) and SH3 domains of p120GAP and Grb2. 905 95

Alternative splicing of the T-cell protein tyrosine phosphatase (TCPTP) 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 GST-TCPTP fusion proteins, we have isolated three cytoplasmic proteins of 120, 116, and 97 kDa that interact with TCPTP. The p120 protein associated with residues 377-415 from the C terminus of the 48-kDa form of TCPTP, whereas the recognition site for p97 and p116 was mapped to residues 350-381 encompassing the TCPTP nuclear localization sequence (NLS). The TCPTP 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 p97 and p116, for efficient nuclear translocation. The interaction between p97, p116, and the TCPTP NLS appeared unique in that these proteins did not form a stable interaction with the classical NLS of SV40 large T antigen or the standard bipartite NLS of nucleoplasmin. Sequence analysis of p97 identified it as the nuclear import factor p97 (importin-beta), which is an essential component of the nuclear import machinery. In assays in vitro in permeabilized cells, p97 was necessary but not sufficient for optimal nuclear import of TCPTP. We found that TCPTP co-immunoprecipitated with the nuclear import factor p97 from cell lysates and that purified recombinant p97 and TCPTP interacted directly in vitro. These results indicate selectivity in the binding of p97 and p116 to the TCPTP NLS and suggest that p97 may mediate events that are distinct from the classical nuclear import process. Moreover, these results demonstrate that the C-terminal segment of TCPTP contains docking sites for interaction with proteins that may function to target the enzyme to defined intracellular locations and in the process regulate TCPTP function.
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PMID:Association of the T-cell protein tyrosine phosphatase with nuclear import factor p97. 926 Nov 75

Human proliferation-associated protein p120 has previously been shown to localize to the nucleolus, and several functional domains of p120 have been elucidated. By using a nitrocellulose filter binding assay and a Northwestern blotting procedure this study shows that recombinant p120 binds to an rRNA fragment in vitro with a dissociation constant of 4 nM. The specific RNA-binding region of p120 (residues 1-57) was identified with glutathione S-transferase-fused p120 deletion constructs and Northwestern blotting procedures. This RNA-binding region of p120, which includes the nucleolar localization signal of p120, is similar to the arginine-rich RNA-binding regions found in other RNA-binding proteins such as HIV Rev and Tat. Experiments in vivo with HeLa cell nucleolar extracts showed that p120 was associated with the 60-80S pre-ribosomal particles. This association is disrupted by treatment with either RNase A or buffer of high ionic strength. These results suggest that p120 might be involved in rRNA/ribosome maturation, consistent with the role of the yeast homologue Nop2p in rRNA biogenesis.
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PMID:Nucleolar protein p120 contains an arginine-rich domain that binds to ribosomal RNA. 953 75

p190 RhoGAP is a 190-kDa protein that stably associates with p120 RasGAP and regulates actin dynamics through members of the Rho family of small GTPases. Previous studies have indicated a direct relationship between levels of p190 tyrosine phosphorylation, the extent and kinetics of epidermal growth factor (EGF)-induced actin rearrangements, and EGF-induced cell cycle progression, suggesting that p190 links Ras-mediated mitogenic signaling with signaling through the actin cytoskeleton. Determining which tyrosine residues in p190 are phosphorylated, what factors regulate phosphorylation of these sites, and what effect tyrosine phosphorylation has on p190 function is key to understanding the role(s) that p190 may play in these processes. To begin investigating these questions, we used biochemical approaches to characterize the number and relative levels of in vivo-phosphorylated tyrosine residues on endogenous p190 from C3H10T1/2 murine fibroblasts. Only two tryptic phosphopeptides containing phosphotyrosine (p-Tyr), a major site, identified as Y1105, and a minor, unidentified site, were detected. Phosphorylation of Y1105, but not the minor site, was modulated in vivo to a greater extent by overexpression of c-Src than by the EGF receptor and was efficiently catalyzed by c-Src in vitro, indicating that Y1105 is a selective and preferential target of c-Src both in vitro and in vivo. In vitro and in vivo coprecipitation analysis using glutathione S-transferase (GST) fusion proteins containing wild-type and Y1105F variants of the p190 middle domain, variants of full-length p190 ectopically expressed in COS-7 cells, and endogenous p190 and p120 in C3H10T1/2 cells revealed that p190 could bind to p120 in the presence and absence of p190 tyrosine phosphorylation. p-Tyr-independent complexes comprised 10 to 20% of the complexes formed in the presence of p-Tyr. Mutation of Y1105 from Tyr to Phe resulted in complete loss of p-Tyr-dependent complex formation, indicating that p-Y1105 was the sole p-Tyr residue mediating binding to p120. These studies describe a specific mechanism by which c-Src can regulate p190-p120 association and also document a significant role for p-Tyr-independent means of p190-p120 binding.
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PMID:Phosphotyrosine (p-Tyr)-dependent and -independent mechanisms of p190 RhoGAP-p120 RasGAP interaction: Tyr 1105 of p190, a substrate for c-Src, is the sole p-Tyr mediator of complex formation. 981 92


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