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)

Monoclonal antibodies raised against the 85-kDa subunit (p85) of bovine phosphatidylinositol (PI) 3-kinase were found to recognize uncomplexed p85 or p85 in the active PI 3-kinase. Immunoprecipitation studies of Chinese hamster ovary cells, which overexpress the human insulin receptor when treated with insulin, showed increased amounts of p85 and PI 3-kinase activity immunoprecipitable with monoclonal anti-p85 antibody and no increase in the tyrosine phosphorylation of p85. Insulin also induced an association of p85 with the tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and other phosphorylated proteins ranging in size from 100 to 170 kDa but not with the activated insulin receptor. In vitro reconstitution studies were used to show p85 in the active PI 3-kinase associated with the tyrosine-phosphorylated IRS-1 but not with the activated insulin receptor. Competition studies using synthetic phosphopeptides corresponding to potential tyrosine phosphorylation sites of IRS-1 revealed that phosphopeptides containing YMXM motifs inhibited this association with different potencies, whereas nonphosphorylated analogues and a phosphopeptide containing the EYYE motif had no effect. Src homology region 2 domains of p85 expressed as glutathione S-transferase fusion proteins also bound to tyrosine-phosphorylated IRS-1. These results suggest that insulin causes the association of PI 3-kinase with IRS-1 via phosphorylated YMXM motifs of IRS-1 and Src homology region 2 domains of p85.
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PMID:Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1. 133 90

The insulin receptor is known to interact with the SH2 domain proteins p85 (the regulatory subunit of phosphatidylinositol 3-kinase), Syp (a tyrosine phosphatase), and GAP (GTPase-activating protein). In this study, we mapped the insulin receptor binding sites for each of these proteins by examining the ability of phosphopeptides, corresponding to insulin receptor phosphorylation sites, and mutant insulin receptors to inhibit an insulin receptor-SH2 domain interaction. Precipitation of partially purified insulin receptors by glutathione S-transferase fusion proteins containing the N-terminal SH2 domains of p85 and GAP and both SH2 domains of Syp was demonstrated. The effect of the addition of each phosphopeptide on insulin receptor precipitation was tested. pY1322, the C-terminal insulin receptor peptide, inhibited insulin receptor precipitation by both p85- and Syp-GST. The NPXY internalization domain peptide inhibited insulin receptor precipitation by GAP-GST. These data were confirmed by mutant insulin receptor experiments. The insulin receptor C-terminal mutants, delta CT and Y/F2, were not precipitated by p85- or Syp-GST and the NPXY mutant insulin receptors, delta Ex16 and HI delta NPEY, were not precipitated by GAP-GST. Therefore, we conclude that p85 and Syp bind to the insulin receptor C terminus at tyrosine 1322 and GAP binds to the insulin receptor NPXY domain at tyrosine 960.
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PMID:Localization of the insulin receptor binding sites for the SH2 domain proteins p85, Syp, and GAP. 752 47

Insulin activates hexose transport via at least two mechanisms: a p21ras-dependent pathway, leading to an increase in the amount of cell surface GLUT1; and a metabolic, p21ras-independent pathway, leading to translocation of the insulin-responsive transporter GLUT4 to the cell surface. Following insulin stimulation, SHPTP2, a non-transmembrane protein-tyrosine phosphatase, associates with insulin receptor substrate 1 via its Src homology 2 (SH2) domains. Microinjection of a glutathione S-transferase fusion protein encoding the N- and C-terminal SH2 domains of SHPTP2 (GST-NC-SH2) or anti-SHPTP2 antibodies into NIH-3T3 fibroblasts overexpressing the insulin receptor blocks insulin-induced DNA synthesis. Microinjection of either GST-NC-SH2 or anti-SHPTP2 antibodies into 3T3-L1 adipocytes inhibited the insulin-stimulated increase in expression of GLUT1. In contrast, translocation of GLUT4 to the cell surface was unaffected by either GST-NC-SH2 or anti-SHPTP2 antibodies. These data confirm a role for SHPTP2 in insulin-stimulated mitogenesis and indicate that whereas SHPTP2 is necessary for insulin-stimulated expression of GLUT1, it is not required for activation of the metabolic pathway leading to GLUT4 translocation.
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PMID:Different signaling roles of SHPTP2 in insulin-induced GLUT1 expression and GLUT4 translocation. 776 84

Insulin drives the formation of a complex between tyrosine-phosphorylated IRS-1 and SH2-containing proteins. The SH2-containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild-type insulin receptor (IR) (CHO-IR cells) in a cell-free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co-immunoprecipitated with dynamin from unstimulated CHO-IR cells. After insulin treatment of CHO-IR cells, anti-dynamin antibodies co-immunoprecipitated the IR beta-subunit and IRS-1, as tyrosine-phosphorylated proteins and PI 3-kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS-1 or the p85 subunit of PI 3-kinase in vitro. Together, these results suggest that in CHO-IR cells, insulin stimulates the binding of dynamin to tyrosine-phosphorylated IRS-1 via Grb2 and that IRS-1 also associates with PI 3-kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus-expressed IRS-1, GST-Grb2 fusion proteins and dynamin peptides containing proline-rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS-1-derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin-Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment. 803 98

Several tyrosine phosphorylation sites in the insulin receptor kinase substrate IRS-1 are predicted to be within Tyr-Met-X-Met (YMXM) motifs, and synthetic peptides corresponding to these sequences are excellent substrates for the insulin receptor kinase in vitro (Shoelson, S. E., Chatterjee, S., Chaudhuri, M., and White, M. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 2027-2031). In this study, YMXM-containing peptides are shown to act as substrates for two members of the nonreceptor subfamily of tyrosine kinases, v-Src and v-Abl (the transforming gene products of Rous sarcoma virus and Abelson murine leukemia virus, respectively). For v-Src, a baculovirus expression system was used which was capable of producing milligram quantities of pure 60-kDa v-Src in Spodoptera frugiperda (Sf9) cells. The source of v-Abl was an Escherichia coli expression vector that produces a fusion protein of glutathione S-transferase with the abl catalytic domain. The synthetic YMXM-containing peptides had among the highest apparent affinities described to date for either tyrosine kinase, with Km values as low as 97 microM for v-Src and v-Abl. Comparisons with the results obtained with the insulin receptor kinase revealed differences in substrate specificity among the enzymes. In particular, v-Src was more tolerant of substitutions at the Met+1 and Met+3 positions in the YMXM motif than either v-Abl or the insulin receptor kinase but was more dependent on the presence of a preceding acidic amino acid. For v-Abl, the presence of threonine at any position in the YMXM motif caused a reduction in catalytic efficiency. Phosphorylated YMXM motifs are recognition elements for binding to the src homology 2 domains of phosphatidylinositol 3'-kinase and additional proteins; hence, differences in specificity of tyrosine kinases toward YMXM-containing proteins may have relevance to downstream signaling events.
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PMID:Phosphorylation of synthetic peptides containing Tyr-Met-X-Met motifs by nonreceptor tyrosine kinases in vitro. 822 78

We have shown previously that insulin stimulated the tyrosine phosphorylation of the alpha-type 85-kDa subunit (p85) of phosphatidylinositol (PI) 3-kinase in vitro and in vivo. In the present work, we identified the major tyrosine phosphorylation sites of the alpha-type p85 by the insulin receptor. [32P]Phosphopeptides obtained from lysylendopeptidase digestion of phosphorylated alpha-type p85 in intact cells after insulin treatment were analyzed using reverse-phase high performance liquid chromatography and thin layer electrophoresis. The tyrosine phosphorylation sites of alpha-type p85 in vivo were assigned to three major phosphopeptides, designated p1, p2, and p3. Highly purified insulin receptor also phosphorylated the purified p85 of PI 3-kinase from the bovine thymus at p1. The purified glutathione S-transferase (GST)-p85 (alpha-type) fusion protein and its truncated proteins from Escherichia coli were also phosphorylated by the purified insulin receptor at p1, p2, and p3 in vitro. Analysis of [32P]phosphopeptide of the truncated GST-p85 (alpha-type) fusion proteins and radiosequence analysis revealed that the p1, p2, and p3 phosphopeptides were phosphorylated at tyrosines 607, 580, and 368, respectively. In addition, phenylalanine substitutions at tyrosine 607 and 580 reduced the p1 and p2 phosphopeptides in vivo, respectively. We conclude that the alpha-type p85 of PI 3-kinase was phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor in vivo.
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PMID:The alpha-type 85-kDa subunit of phosphatidylinositol 3-kinase is phosphorylated at tyrosines 368, 580, and 607 by the insulin receptor. 838 99

The polymerase chain reaction was used to amplify protein tyrosine phosphatase (PTPase)-related cDNA from a template of total RNA isolated from human skeletal muscle. A novel PTPase, which we term PTP-PEST, was detected by this method. The polymerase chain reaction fragment was used to screen two different HeLa cell libraries to obtain full length cDNA clones. The cDNA predicts a protein of 510 amino acids, approximately 60 kDa, that does not contain an obvious signal sequence or transmembrane segment suggesting it is a nonreceptor type enzyme. The PTPase domain is located in the N-terminal portion of the molecule and displays approximately 35% identity to other members of this family of enzymes. The C-terminal segment is rich in Pro, Glu, Asp, Ser, and Thr residues, possessing features of PEST motifs which have previously been identified in proteins with very short intracellular half-lives. The protein was expressed in Escherichia coli as a fusion product with glutathione S-transferase. Intrinsic activity was demonstrated in vitro against a variety of phosphotyrosine-containing substrates including BIRK, the autophosphorylated cytoplasmic kinase domain of the insulin receptor beta subunit. It did not dephosphorylate phosphoseryl-phosphorylase a. PTP-PEST mRNA is broadly distributed in a variety of cell lines. Stimulation of human rhabdomyosarcoma A204 cells, a transformed muscle line, with insulin led to an approximately 4-fold induction of PTP-PEST mRNA within 36 h.
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PMID:Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase. 834 45

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

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

Insulin receptor substrate 2 (IRS-2) has recently been shown to be a substrate of the insulin receptor (IR). In this study we utilize the yeast two-hybrid system and assays of in vitro interaction to demonstrate that IRS-2 interacts directly with the IR and the insulin-like growth factor I receptor. We show that, like IRS-1, the region of IRS-2 that contains the putative phosphotyrosine binding and SAIN elements (188-591) is sufficient for receptor interaction and that this interaction is dependent upon the NPX(p)Y (where (p)Y is phosphotyrosine) motifs within the juxtamembrane domains of the receptors. In addition to this amino-terminal NPX(p)Y-binding domain, an additional domain of strong interaction was identified in the central region of IRS-2 and was localized between amino acids 591 and 733. This interaction was found to be dependent upon receptor phosphorylation but was NPX(p)Y-independent. This region does not appear to have either an SH2 or a phosphotyrosine binding domain. Both of the interactions could also be demonstrated in vitro using IRS-2 glutathione S-transferase fusion proteins. We conclude that IRS-2, unlike IRS-1, can interact with tyrosine-phosphorylated receptors such as the IR and insulin-like growth factor I receptor via multiple independent binding motifs. Our findings suggest the existence of a previously unidentified phosphotyrosine-dependent binding domain within the central region of IRS-2.
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PMID:Interaction of insulin receptor substrate-2 (IRS-2) with the insulin and insulin-like growth factor I receptors. Evidence for two distinct phosphotyrosine-dependent interaction domains within IRS-2. 866 6


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