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)

Shc has two distinct domains, amino-terminal and SH2 domain, which can interact with activated growth factor receptors. Shc interacts with insulin receptor via Shc-amino-terminal (N) domain, whereas Shc associates with epidermal growth factor (EGF) receptor through both Shc-N and -SH2 domains. In accordance with the different functional roles between insulin and EGF receptors, EGF stimulated tyrosine phosphorylation of Shc faster than insulin. To clarify the functional importance of three distinct Shc domains on insulin and EGF signaling, we microinjected glutathione S-transferase (GST) fusion proteins containing the amino terminus plus collagen homology domain (NCH), collagen homology domain (CH), and Src homology 2 domain (SH2) into Rat1 fibroblasts expressing insulin receptors (HIRc). Bromodeoxyuridine (BrdUrd) incorporation into newly synthesized DNA was subsequently studied to assess the importance of the three distinct domains of Shc. Microinjection of the NCH-GST fusion protein inhibited BrdUrd incorporation induced by both EGF and insulin, whereas microinjection of the SH2-GST fusion protein inhibited EGF, but not insulin stimulation of DNA synthesis. Neither EGF- nor insulin-induced BrdUrd incorporation was inhibited by the CH-GST fusion protein. Following EGF or insulin stimulation, Shc is phosphorylated on single Tyr-317 residue serving as a docking site for Grb2. Microinjection of Shc-N+CH GST fusion protein with Tyr-317 --> Phe replacement (Y317F) also inhibited insulin stimulation of DNA synthesis. Next, we stably overexpressed wild-type Shc or Y317F mutant Shc into HIRc cells. Insulin-induced tyrosine phosphorylation of IRS-1 was compared among the transfected cell lines, since IRS-1 and Shc could competitively interact with insulin receptor. Insulin-stimulated tyrosine phosphorylation of IRS-1 was decreased in both WT-Shc and Y317F-Shc cells compared with that in HIRc cells. Furthermore, overexpression of the Shc-SH2 domain or Shc-N+CH domain with Y317F mutation interfered with EGF-stimulated endogenous Shc phosphorylation. These results suggest that the amino terminus domain of Shc is functionally important in insulin- and EGF-induced cell cycle progression and that the phosphorylation of Shc Tyr-317 residue is independent of Shc interaction with these receptors.
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PMID:Functional importance of amino-terminal domain of Shc for interaction with insulin and epidermal growth factor receptors in phosphorylation-independent manner. 870 28

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 compared the intracellular insulin-like growth factor-1 (IGF-1) and insulin signaling pathways in Rat1 fibroblasts expressing the equivalent number of insulin receptors and endogenous IGF-1 receptors. Insulin and IGF-1 stimulated tyrosine phosphorylation of IRS-1 and Shc in a similar dose- and time-dependent manner. The time course of Shc phosphorylation by both IGF-1 and insulin was slower than that of IRS-1. Both phosphorylated IRS-1 and Shc associated with Grb2.Sos complexes, leading to p21ras activation. To compare the functional importance of p21ras for IGF-1-and insulin-induced DNA synthesis, single cell microinjection studies were performed. BrdU incorporation into newly synthesized DNA was measured by immunofluorescence microscopy to assess the functional importance of p21ras. Both IGF-1 and insulin stimulated BrdU incorporation, but the effect of IGF-1 was greater. Microinjection of anti-p21ras antibody completely inhibited both IGF-1-and insulin-induced DNA synthesis, indicating the central role of p21ras in signaling by both hormones. Signal transduction from these receptors to Grb2.Sos complexes can occur through IRS-1 and/or Shc. To assess these two possible pathways, we performed Western blots for Grb2 in anti-Shc and anti-IRS-1 immunoprecipitates and found that 5-fold more Grb2 was associated with Shc than with IRS-1 after either IGF-1 or insulin stimulation. Microinjection of anti-Shc antibody inhibited IGF-1 and insulin stimulation of DNA synthesis by 78% and 74%, respectively. By microinjecting Shc subdomains of GST fusion proteins, we found that Shc N-terminus, but not the Shc SH2, was the functionally important domain through which Shc interacts with IGF-1 and insulin receptors. Insulin stimulation caused hyperphosphorylation and decreased electrophoretic mobility of Sos, and a similar effect was seen with IGF-1, although the time course was delayed compared with insulin. Finally, IGF-1 activated mitogen-activated proten kinase activity more effectively than insulin. These data indicate that Shc, rather than IRS-1, appears to be the predominant functional link to Grb2.Sos complexes from the IGF-1 receptor, as it is from the insulin receptor. Although IGF-1 and insulin stimulate cell cycle progression with similar coupling mechanisms from the receptor to Shc, to Grb2.Sos, to p21ras, the delayed IGF-1 induced mobility shift of Sos could lead to, at least in part, more efficient coupling to mitogen-activated protein kinase. These findings might explain the greater mitogenic activity of IGF-1 compared with insulin.
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PMID:Comparison of the insulin and insulin-like growth factor 1 mitogenic intracellular signaling pathways. 882 4

Insulin receptor substrate 1 (IRS-1), and its structural relative IRS-2, are both phosphorylated on tyrosine following treatment of cells with interleukin-4 (IL-4) and insulin. We have investigated whether both IRS-1 and IRS-2 are expressed in murine lymphohemopoietic cells. T and B lymphocytes and macrophages from primary cultures expressed only IRS-2, which became phosphorylated on tyrosine following stimulation with both IL-4 and insulin. Likewise, the murine myeloid cell line FD-5 expressed only IRS-2, which was tyrosine phosphorylated in response to IL-4 and insulin, as well as interleukin-3 and granulocyte-macrophage colony stimulating factor. Neither IRS-1 nor IRS-2 were expressed at detectable levels in primary bone marrow mast cells although these cells do respond to IL-4. Moreover, a factor-dependent lymphocyte cell line, CT.4S, which grows continuously in IL-4, did not express detectable levels of IRS-1 or IRS-2. IRS-2 from FD-5 cells stimulated with either IL-4 or insulin bound to glutathione S-transferase fusion proteins of the p85 subunit of phosphoinositol 3'-kinase, Grb2, and Syp, paralleling reported associations of IRS-1 with these molecules and indicating phosphorylation of the corresponding residues on IRS-2.
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PMID:Insulin receptor substrate-2 is the major 170-kDa protein phosphorylated on tyrosine in response to cytokines in murine lymphohemopoietic cells. 899 47

The 14-3-3 proteins have been implicated as potential regulators of diverse signaling pathways. Here, using two-hybrid assays and in vitro assays of protein interaction, we show that the epsilon isoform of 14-3-3 interacts with the insulin-like growth factor I receptor (IGFIR) and with insulin receptor substrate I (IRS-1), but not with the insulin receptor (IR). Coprecipitation studies demonstrated an IGFI-dependent in vitro interaction between 14-3-3-glutathione S-transferase proteins and the IGFIR. In similar studies no interaction of 14-3-3 with the IR was observed. We present evidence to suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR. Specifically, peptide competition studies combined with mutational analysis suggested that the 14-3-3 interaction was dependent upon phosphorylation of IGFIR serine residues 1272 and/or 1283, a region which has been implicated in IGFIR-dependent transformation. Phosphorylation of these serines appears to be dependent upon prior IGFIR activation since no interaction of 14-3-3 was observed with a kinase-inactive IGFIR in the two-hybrid assay nor was any in vitro interaction with unstimulated IGFIR derived from mammalian cells. We show that the interaction of 14-3-3 with IRS-1 also appears to be phosphoserine-dependent. Interestingly, 14-3-3 appears to interact with IRS-1 before and after hormonal stimulation. In summary, our data suggest that 14-3-3 interacts with phosphoserine residues within the COOH terminus of the IGFIR and within the central domain of IRS-1. The potential functional roles which 14-3-3 may play in IGFIR and IRS-1-mediated signaling remain to be elucidated.
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PMID:14-3-3 (epsilon) interacts with the insulin-like growth factor I receptor and insulin receptor substrate I in a phosphoserine-dependent manner. 911 Oct 84

The 14-3-3 protein family has been implicated in growth factor signaling. We investigated whether 14-3-3 protein is involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the immunoprecipitate with anti-14-3-3beta antibody at the basal condition. 100 nM insulin increased the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nM wortmannin. An in vitro binding study revealed that glutathione S-transferase-14-3-3beta fusion protein directly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Because the recombinant IRS-1 was not phosphorylated on its tyrosine residues, the results suggest that serine/threonine phosphorylation of IRS-1 is responsible for the association. When the cells are treated with insulin, phosphatidylinositol 3'-kinase (PI3K) is supposed to complex either 14-3-3beta-IRS-1 or IRS-1. The 14-3-3beta-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation, first with anti-14-3-3beta and then with anti-IRS-1 antibodies. The specific activity of the PI3K in the former was approximately half of that in the latter, suggesting that 14-3-3beta protein bound to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K in 3T3L1 adipocytes.
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PMID:14-3-3beta protein associates with insulin receptor substrate 1 and decreases insulin-stimulated phosphatidylinositol 3'-kinase activity in 3T3L1 adipocytes. 942 53

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.
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PMID:Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. 944 80

Insulin and insulin-like growth factor-1 (IGF-1) treatment of cells overexpressing the insulin receptor or the IGF-1 receptor promotes phosphorylation and activation of Janus kinases JAK-1 and JAK-2 but not of TYK-2. With insulin, we observed maximal phosphorylation of JAK-1 within 2 min (5.2 +/- 0.6-fold) and maximal phosphorylation of JAK-2 within 10 min (2.4 +/- 0.6-fold). In cells incubated with IGF-1, we found maximal phosphorylation of JAK-2 within 2 min (1.9 +/- 0.2-fold) and of JAK-1 within 5 min (4.5 +/- 0.4-fold). The JAKs from insulin- or IGF-1-stimulated cells were activated, as shown by their autophosphorylation in vitro. Moreover, they were able to phosphorylate in vitro native insulin receptor substrate (IRS)-1 and a fragment of IRS-2 (GST-IRS-2591-786). Comparison of 32P-peptide maps of IRS-1 phosphorylated in vitro by the insulin receptor vs. JAK-1 showed the occurrence of different phosphopeptides, suggesting that different sites are likely to be phosphorylated by the two kinases. Finally, coprecipitation of receptors and JAK-1 was seen, and phosphorylation of both receptors was found to be necessary for receptor binding to JAK-1. Two domains of JAK- 1 are involved in the formation of the complex between receptor and JAK-1, i.e. the N-terminal portion containing JH7 and JH6 domains, and the C-terminal kinase domain (JH1 domain). Taking our data together, we conclude that: 1) insulin and IGF-1 lead to phosphorylation and activation of JAK-1 and JAK-2 in intact cells; 2) phosphorylation of IRS-I by JAK-1 seems to occur on sites different from those phosphorylated by the insulin receptor; 3) JAK-1 interacts directly with phosphorylated insulin and IGF-1 receptors; and 4) the JH7-JH6 and JH1 domains of JAK-1 are responsible for the interaction with insulin and IGF-1 receptors.
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PMID:Interaction of Janus kinases JAK-1 and JAK-2 with the insulin receptor and the insulin-like growth factor-1 receptor. 949 17

Insulin stimulation results in a considerable spectrum of cellular responses, only part of which have been firmly correlated with the activation of established insulin receptor (IR) targets such as IRS-1, IRS-2, and Shc. Many responses may be transduced by alternative direct IR targets, some of which may still be unknown, may act in parallel to but independently of IRS-1, IRS-2, and Shc, and may be members of the growing family of SH2 domain-containing signaling adaptors. An SH2 domain-coding region of a protein termed PSM was cloned based on its interaction with an activated IR cytoplasmic fragment in a yeast two-hybrid screen. When used as a hybridization probe this region led to the isolation of a protein-coding cDNA which is expressed with a wide tissue distribution and exists in several variant forms. A pleckstrin homology domain and three Pro-rich regions including a putative SH3 domain binding site were identified in addition to the SH2 domain in the deduced 756 amino acid sequence. They imply a role of PSM in tyrosine kinase and phosphatase-mediated signaling pathways. A similar sequence termed SH2-B had been reported in an earlier study, which may represent the rat homolog of PSM. A role of PSM specifically in insulin action is suggested by the interaction of its SH2 domain with an activated but not with an inactive catalytic fragment of the IR in the yeast two-hybrid system in vivo, by the insulin-dependent association of a glutathione S-transferase (GST) PSM SH2 domain fusion protein with purified IR in vitro, and by the insulin-dependent association of GST PSM SH2 with the IR in cell extracts. In contrast, PSM was not found to associate with the established IR substrate IRS-1 under any conditions and appears to act independently of IRS-1. All of our findings are compatible with a putative role of PSM in insulin action.
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PMID:PSM, an insulin-dependent, pro-rich, PH, SH2 domain containing partner of the insulin receptor. 949 52

Signaling through the insulin receptor tyrosine kinase involves its autophosphorylation in response to insulin and the subsequent tyrosine phosphorylation of substrate proteins such as insulin receptor substrate-1 (IRS-1). In basal 3T3-L1 adipocytes, IRS-1 is predominantly membrane-bound, and this localization may be important in targeting downstream signaling elements that mediate insulin action. Since IRS-1 localization to membranes may occur through its association with specific membrane proteins, a 3T3-F442A adipocyte cDNA expression library was screened with non-tyrosine-phosphorylated, baculovirus-expressed IRS-1 in order to identify potential IRS-1 receptors. A cDNA clone that encodes sigma3A, a small subunit of the AP-3 adaptor protein complex, was demonstrated to bind IRS-1 utilizing this cloning strategy. The specific interaction between IRS-1 and sigma3A was further verified by in vitro binding studies employing baculovirus-expressed IRS-1 and a glutathione S-transferase (GST)-sigma3A fusion protein. IRS-1 and sigma3A were found to co-fractionate in a detergent-resistant population of low density membranes isolated from basal 3T3-L1 adipocytes. Importantly, the addition of exogenous purified GST-sigma3A to low density membranes caused the release of virtually all of the IRS-1 bound to these membranes, while GST alone had no effect. These results are consistent with the hypothesis that sigma3A serves as an IRS-1 receptor that may dictate the subcellular localization and the signaling functions of IRS-1.
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PMID:Interaction of insulin receptor substrate-1 with the sigma3A subunit of the adaptor protein complex-3 in cultured adipocytes. 979 13


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