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)

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.
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PMID:Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach. 838 38

The Src-related tyrosine kinase p59fyn(T) plays an important role in the generation of intracellular signals from the T-cell antigen receptor TCR zeta/CD3 complex. A key question concerns the nature and the binding sites of downstream components that interact with this Src-related kinase. p59fyn(T) contains Src-homology 2 and 3 domains (SH2 and SH3) with a capacity to bind to intracellular proteins. One potential downstream target is phosphatidylinositol 3-kinase (PI 3-kinase). In this study, we demonstrate that anti-CD3 and anti-Fyn immunoprecipitates possess PI 3-kinase activity as assessed by TLC and HPLC. Both free and receptor-bound p59fyn(T) were found to bind to the lipid kinase. Further, our results indicate that Src-related kinases have developed a novel mechanism to interact with PI 3-kinase. Precipitation using GST fusion proteins containing Fyn SH2, SH3, and SH2/SH3 domains revealed that PI 3-kinase bound principally to the SH3 domain of Fyn. Fyn SH3 bound directly to the p85 subunit of PI 3-kinase as expressed in a baculoviral system. Anti-CD3 crosslinking induced an increase in the detection of Fyn SH3-associated PI 3-kinase activity. Thus PI 3-kinase is a target of SH3 domains and is likely to play a major role in the signals derived from the TCR zeta/CD3-p59fyn complex.
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PMID:Src-homology 3 domain of protein kinase p59fyn mediates binding to phosphatidylinositol 3-kinase in T cells. 839 19

Xenopus oocytes from unprimed frogs possess insulin-like growth factor I (IGF-I) receptors but lack insulin and IGF-I receptor substrate 1 (IRS-1), the endogenous substrate of this kinase, and fail to show downstream responses to hormonal stimulation. Microinjection of recombinant IRS-1 protein enhances insulin-stimulated phosphatidylinositol (PtdIns) 3-kinase activity and restores the germinal vesicle breakdown response. Activation of PtdIns 3-kinase results from formation of a complex between phosphorylated IRS-1 and the p85 subunit of PtdIns 3-kinase. Microinjection of a phosphonopeptide containing a pYMXM motif with high affinity for the src homology 2 (SH2) domain of PtdIns 3-kinase p85 inhibits IRS-1 association with and activation of the PtdIns 3-kinase. Formation of the IRS-1-PtdIns 3-kinase complex and insulin-stimulated PtdIns 3-kinase activation are also inhibited by microinjection of a glutathione S-transferase fusion protein containing the SH2 domain of p85. This effect occurs in a concentration-dependent fashion and results in a parallel loss of hormone-stimulated oocyte maturation. These inhibitory effects are specific and are not mimicked by glutathione S-transferase fusion proteins expressing the SH2 domains of ras-GAP or phospholipase C gamma. Moreover, injection of the SH2 domains of p85, ras-GAP, and phospholipase C gamma do not interfere with progesterone-induced oocyte maturation. These data demonstrate that phosphorylation of IRS-1 plays an essential role in IGF-I and insulin signaling in oocyte maturation and that this effect occurs through interactions of the phosphorylated YMXM/YXXM motifs of IRS-1 with SH2 domains of PtdIns 3-kinase or some related molecules.
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PMID:Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase. 841 61

Binding of interferon alpha (IFN alpha) to its receptor induces activation of the Tyk-2 and Jak-1 tyrosine kinases and tyrosine phosphorylation of multiple downstream signaling elements, including the Stat components of the interferon-stimulated gene factor 3 (ISGF-3). IFN alpha also induces tyrosine phosphorylation of IRS-1, the principle substrate of the insulin receptor. In this study we demonstrate that various Type I IFNs rapidly stimulate tyrosine phosphorylation of IRS-2. This is significant since IRS-2 is the major IRS protein found in hematopoietic cells. The IFN alpha-induced phosphorylated form of IRS-2 associates with the p85 regulatory subunit of the phosphatidylinositol 3'-kinase, suggesting that this kinase participates in an IFN alpha-signaling cascade downstream of IRS-2. We also provide evidence for an interaction of IRS-2 with Tyk-2, suggesting that Tyk-2 is the kinase that phosphorylates this protein during IFN alpha stimulation. A conserved region in the pleckstrin homology domain of IRS-2 may be required for the interaction of IRS-2 with Tyk-2, as shown by the selective binding of glutathione S-transferase (GST) fusion proteins containing the IRS-2-IH1PH or IRS-1-IH1PH domains to Tyk-2 but not other Janus kinases in vitro.
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PMID:The type I interferon receptor mediates tyrosine phosphorylation of insulin receptor substrate 2. 855 May 73

Although epidermal growth factor (EGF) activates phosphoinositide (PI) 3-kinase activity in a number of types of cells or cell lines, in most cases that we have investigated the p85 regulatory subunit of PI 3-kinase does not appear to bind directly to the EGF receptor. Previously we demonstrated that EGF-dependent activation of PI 3-kinase activity in A431 cells is accompanied by the binding of p85 to ErbB3, an EGF receptor homologue. However, this mechanism did not explain the large activation of PI 3-kinase activity that was found in PC12 and A549 cells, which possess little or no ErbB3. Here we provide evidence that the p120cbl proto-oncoprotein is an intracellular adapter protein that associates with PI 3-kinase and thus is involved in the EGF-dependent activation of this enzyme in these two cell lines. Using an anti-p120cbl antibody, we immunoprecipitated the EGF receptor from PC12 cells and PI 3-kinase activity from PC12 and A549 cells in an EGF-dependent fashion. Treatment of PC12 cells with nerve growth factor or insulin stimulated large increases in PI 3-kinase activity that was immunoprecipitated using anti-Tyr(P) antibody but not using anti-p120cbl antibody. In EGF-treated PC12 cells, the tyrosine phosphorylation of p120cbl displayed similar kinetics to the activation of PI 3-kinase as measured by both in vivo lipid production and lipid kinase assays conducted using anti-p120cbl and anti-Tyr(P) immunoprecipitates. The use of glutathione S-transferase fusion proteins of various domains of p85 demonstrated that p120cbl associated with both the SH2 and SH3 domains of p85. p120cbl was also present in A431 cells and offers an additional pathway by which EGF can activate PI 3-kinase in these cells.
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PMID:p120cbl is a cytosolic adapter protein that associates with phosphoinositide 3-kinase in response to epidermal growth factor in PC12 and other cells. 855 Jun 20

The insulin receptor, as a consequence of ligand binding, undergoes autophosphorylation of critical tyrosyl residues within the cytoplasmic portion of its beta-subunit. The 85 kDa regulatory subunit of phosphatidylinositol (PI) 3-kinase (p85), an SH2 domain protein, has been implicated as a regulatory molecule in the insulin signal transduction pathway. For the present study, glutathione S-transferase (GST) fusion proteins of p85 SH2 domains were used to determine if such motifs associate directly with the autophosphorylated human insulin receptor. The p85 N + C (amino plus carboxyl) SH2 domains were demonstrated to associate with the autophosphorylated beta-subunit, while neither the GTPase activator protein (GAP) N SH2 domain nor the phospholipase C-gamma 1 (PLC gamma 1) N + C SH2 domains exhibited measurable affinity for the activated receptor. The p85 N SH2 domain demonstrated weak association with the insulin receptor, while the p85 C SH2 domain alone formed no detectable complexes with the insulin receptor. The association of p85 N + C SH2 domains with the autophosphorylated receptor was competed efficiently by a 15-residue tyrosine-phosphorylated peptide corresponding to the carboxyl-terminal region of the insulin receptor, but not by phosphopeptides of similar length derived from the juxtamembrane or regulatory regions. The insulin receptor C domain phosphopeptide inhibited the p85 N + C SH2 domain-insulin receptor complex with an IC0.5 of 2.3 +/- 0.35 microM, whereas a 10-residue phosphopeptide derived from the insulin receptor substrate 1 (IRS-1) competed with an IC0.5 of 0.54 +/- 0.10 microM. These results demonstrate that, in vitro, there is an association between the p85 regulatory protein and the carboxyl-terminal region of the activated insulin receptor that requires the presence of both the N and C SH2 domains. Furthermore, formation of the p85/insulin receptor complex may lead to signaling pathways independent of IRS-1.
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PMID:In vitro association of the phosphatidylinositol 3-kinase regulatory subunit (p85) with the human insulin receptor. 856 77

p56lck is a potential in vivo substrate for the tyrosine-specific phosphatase, CD45. In this study, recombinant purified p56lck was found to specifically associate with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of another related tyrosine phosphatase, receptor protein-tyrosine phosphatase alpha. Under equilibrium binding conditions, the binding was saturable and occurred at a 1:1 molar stoichiometry. A fusion protein containing only the amino-terminal region of p56lck (residues 34-150) also bound to recombinant CD45, and further analysis of this region indicated that glutathione S-transferase fusion proteins of the unique amino-terminal region and the SH2 domain, but not the SH3 domain of p56lck, bound to recombinant CD45. The SH2 domain protein bound with a higher affinity than the amino-terminal region, but both were able to compete for the binding of p56lck to CD45, and when added together worked synergistically to compete for p56lck binding. The SH2 domain interaction with CD45 was specific as glutathione S-transferase-SH2 fusion proteins from p85 alpha subunit of phosphatidylinositol 3-kinase and SHC did not bind to CD45. In addition, this interaction occurred in the absence of any detectable tyrosine phosphorylation on CD45, suggesting a nonconventional SH2 domain interaction.
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PMID:Demonstration of a direct interaction between p56lck and the cytoplasmic domain of CD45 in vitro. 857 15

Development of phosphotyrosyl (pTyr) mimetics which are stable to protein-tyrosine phosphatases (PTPs), yet can retain biological potency when incorporated into peptides, is an active area of drug development. Since a majority of pTyr mimetics derive their "phosphofunctionality" from phosphorus-containing moieties, such as phosphonates, evolution of new inhibitors and modes of prodrug derivatization have been restricted to chemistries appropriate for phosphorus-containing moieties. A new, nonphosphorus-containing pTyr mimetic has recently been reported, L-O-(2-malonyl)tyrosine (OMT,5), which can be incorporated into peptides that exhibit good PTP and Src homology 2 (SH2) domain inhibitory potency. For phosphonate-based pTyr mimetics such as phosphonomethyl phenylalanine (Pmp,2) introduction of fluorines alpha to the phosphorus has provided higher affinity pTyr mimetics. This strategy has now been applied to OMT, and herein is reported 4'-O-[2-(2-fluoromalonyl)]-L-tyrosine (FOMT,6) a new fluorine-containing nonphosphorus pTyr mimetic. Incorporation of FOMT into appropriate peptides results in good inhibition of both PTP and SH2 domains. In an assay measuring the inhibition of PTP 1B-mediated dephosphorylation of phosphorylated insulin receptor, the peptide Ac-D-A-D-E-X-L-amide exhibited a 10-fold enhancement in inhibitory potency for X = FOMT (19) (IC(50) = 10 microM) relative to the unfluorinated peptide, X = OMT (18) (IC(50) = 10 microM. Molecular modeling indicated that this increased affinity may be attributable to new hydrogen-bonding interactions between the fluorine and the enzyme catalytic site, and not due to lowering of pKa values. In a competition binding assay using the p85 PI 3-kinase C-terminal SH2 domain GST fusion construct, the inhibitory peptide, Ac-D-X-V-P-M-L-amide, showed no enhancement of inhibitory potency for X = FOMT (22) (IC(50) = 18 microM) relative to the unfluorinated peptide, X = OMT (21) (IC(50) = 14 microM). The use of FOMT would therefore appear to have particular potential for the development of PTP inhibitors.
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PMID:4'-O-[2-(2-fluoromalonyl)]-L-tyrosine: a phosphotyrosyl mimic for the preparation of signal transduction inhibitory peptides. 867 36

Tyrosine phosphorylation of cellular proteins is an early and key step after activation of the insulin receptor kinase (IRK). The study of the properties of these proteins should contribute to our understanding of insulin action. In rat hepatoma cells overexpressing human insulin receptors (HTC-IR), insulin treatment resulted in rapid tyrosine phosphorylation of proteins of 180, 94, 68, and 60 kDa. When lysates from insulin-treated cells were immunoprecipitated with anti-Syp antibody, subsequent immunoblotting identified p65 and p68, which reacted with anti-Syp, and p6O and p68, which reacted with antiphosphotyrosine antibody. Thus, insulin treatment yielded tyrosine phosphorylation of both Syp and a Syp-associated p6O molecule. When lysates from insulin-treated cells were adsorbed with a glutathione S-transferase (GST)-Syp-Src homology-2 (SH2) fusion protein, tyrosine- phosphorylated p6O was sequestered. After subjecting lysates to SDS-PAGE, the GST-SypSH2 fusion protein was found to bind to p18O, p94, and p6O. Thus, Syp associates directly with a 60-kDa IRK substrate via its SH2 domains. Syp-associated p6O differed from the 60- to 62-kDa proteins, associating with ras guanosine triphosphatase-activating protein, which also underwent modest tyrosine phosphorylation in response to insulin. Preadsorption of cell lystates with antibody against the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase substantially reduced the amount of p60 subsequently immunoprecipitated by anti-Syp. Thus, p60 associates with both Syp and p85. The amount of tyrosine-phosphorylated p60 exceeded that of p180 in anti-Syp immunoprecipitates, whereas their proportion was comparable in anti-p85 immunoprecipitates. Grb2 was also observed in the anti-Syp immunoprecipitates. When lysates from insulin-treated cells were adsorbed with GST-p85SH2 domains or GST-Grb2, the subsequent eluates contained tyrosine-phosphorylated p60, as determined by immunoblotting with antiphosphotyrosine. Membrane binding assays using GST fusion proteins showed that these associations were direct. Studies in rat liver, muscle, and adipose tissue identified insulin-dependent association of Syp, Grb2, and p85 with tyrosine-phosphorylated p60 in adipose tissue only. We conclude that insulin treatment of HTC-IR cells and rat adipose tissue results in the tyrosine phosphorylation of p60, which might participate in the recruitment of downstream effectors involved in insulin signal transduction.
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PMID:A 60-kilodalton protein in rat hepatoma cells overexpressing insulin receptor was tyrosine phosphorylated and associated with Syp, phophatidylinositol 3-kinase, and Grb2 in an insulin-dependent manner. 877 Aug 81

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high affinity binding sites for cellular proteins with Src homology 2 (SH2) domains that are involved in various signal cascades. Tryptic digestion of the autophosphorylated v-Fms and of its cellular counterpart, the feline c-Fms polypeptide, gave rise to at least six common major phosphopeptides, four of which have been characterized previously. Employing site-directed mutagenesis and phosphopeptide mapping of in vitro phosphorylated glutathione S-transferase v-Fms fusion proteins as well as full-length v-Fms molecules expressed in various cells, we show here that Tyr543 of the juxtamembrane domain and Tyr696 of the kinase insert domain constitute major autophosphorylation sites. Recombinant fusion proteins containing the tyrosine-phosphorylated kinase insert domain bind the growth factor receptor bound protein 2 and the p85 and p110 subunits of phosphatidylinositol 3'-kinase. In contrast, fusion proteins containing the juxtamembrane domain phosphorylated on Tyr543 fail to bind any of the known SH2 domain-containing cellular proteins but associate specifically with an as yet undefined 55-kDa cellular protein that by itself is phosphorylated on tyrosine.
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PMID:Tyrosine phosphorylation of the juxtamembrane domain of the v-Fms oncogene product is required for its association with a 55-kDa protein. 879 7


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