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 placenta possesses the ability to metabolize a number of xenobiotics and endogenous compounds by processes similar to those seen in the liver. Animal and in vivo studies have observed that the presence of diabetes alters the expression of hepatic metabolizing enzymes (cytochrome P450 and glutathione S-transferase); however, it is unknown whether similar alterations occur in the human placenta. To evaluate whether diabetes has any effect of placental xenobiotic metabolizing activity, the catalytic activities of 7-ethoxyresorufin O-deethylation (EROD, CYP1A1), chlorzoxazone 6-hydroxylation (CYP2E1), dextromethorphan N-demethylation (CYP3A4), dextromethorphan O-demethylation (CYP2D6), and 1-chloro-2, 4-dinitrobenzene (CDNB) conjugation with glutathione (glutathione S-transferase, GST) from placentas of diet (class A1) and insulin-dependent (class A2) gestational diabetics and overt diabetics were compared with matched controls. EROD activity (CYP1A1) ranged from 0.29 to 2.67 pmol/min/mg protein. However, no differences were observed among overt or gestational diabetics and their respective matched controls. CDNB conjugation (GST) ranged from 0.275 to 1.65 units/min/mg protein. In contrast to that observed with CYP1A1, a small but statistically significant reduction in GST activity was noted in overt diabetics as compared with their matched controls and gestational diabetics. CYP2E1, 2D6, and 3A4 enzymatic activities were not detected in human placental tissue. GST protein was detectable in all tissues studied, but no CYP protein could be detected in any of the tissues. Thus, it seems that pregnant women with overt diabetes have reduced GST activity in the placenta, which could potentially result in the exposure of the fetus to harmful electrophiles. However, the full clinical significance of this finding remains to be elucidated.
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PMID:Effects of gestational and overt diabetes on human placental cytochromes P450 and glutathione S-transferase. 953 26

SHPS-1 is a receptor-like protein that undergoes tyrosine phosphorylation and binds SHP-2, an SH2 domain-containing protein tyrosine phosphatase, in response to insulin and other mitogens. The overexpression of wild-type SHPS-1, but not of a mutant SHPS-1 in which all four tyrosine residues in its cytoplasmic region were mutated to phenylalanine, markedly enhanced insulin-induced activation of mitogen-activated protein kinase in Chinese hamster ovary cells that overexpress the human insulin receptor. Mutation of each tyrosine residue individually revealed that the major sites of tyrosine phosphorylation of SHPS-1 in response to insulin are Tyr449 and Tyr473. In addition, mutation of either Tyr449 or Tyr473 abolished the insulin-induced tyrosine phosphorylation of SHPS-1 and its association with SHP-2. Surface plasmon resonance analysis showed that glutathione S-transferase fusion proteins containing the NH2-terminal or COOH-terminal SH2 domains of SHP-2 bound preferentially to phosphotyrosyl peptides corresponding to the sequences surrounding Tyr449 or Tyr473, respectively, of SHPS-1. Furthermore, phosphotyrosyl peptides containing Tyr449 or Tyr473 were effective substrates for the phosphatase activity of recombinant SHP-2 in vitro. Together, these results suggest that insulin may induce phosphorylation of SHPS-1 at Tyr449 and Tyr473, to which SHP-2 then binds through its NH2-terminal and COOH-terminal SH2 domains, respectively. SHPS-1 may play a crucial role both in the recruitment of SHP-2 from the cytosol to a site near the plasma membrane and in increasing its catalytic activity, thereby positively regulating the RAS-mitogen-activated protein kinase signaling cascade in response to insulin.
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PMID:Roles of the complex formation of SHPS-1 with SHP-2 in insulin-stimulated mitogen-activated protein kinase activation. 953 15

Insulin stimulation of Chinese hamster ovary cells expressing the human insulin receptor resulted in a time-dependent decrease in the amount of GTP bound to Rap1. The inactivation of Rap1 was associated with an insulin-stimulated decrease in the amount of Rap1 that was bound to Raf1. In parallel with the dissociation of Raf1 from Rap1, there was an increased association of Raf1 with Ras. Concomitant with the inactivation of Rap1 and decrease in Rap1-Raf1 binding, we observed a rapid insulin-stimulated dissociation of the CrkII-C3G complex which occurred in a Ras-independent manner. The dissociation of the CrkII-C3G was recapitulated in vitro using a GST-C3G fusion protein to precipitate CrkII from whole cell detergent extracts. The association of GST-C3G with CrkII was also dose dependent and demonstrated that insulin reduced the affinity of CrkII for C3G without any effect on CrkII protein levels. Furthermore, the reduction in CrkII binding affinity was reversible by tyrosine dephosphorylation with PTP1B and by mutation of Tyr221 to phenylalanine. Together, these data demonstrate that insulin treatment results in the de-repression of Rap1 inhibitory function on the Raf1 kinase concomitant with Ras activation and stimulation of the downstream Raf1/MEK/ERK cascade.
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PMID:Insulin regulates the dynamic balance between Ras and Rap1 signaling by coordinating the assembly states of the Grb2-SOS and CrkII-C3G complexes. 956 38

Phospholipase C-gamma (PLCgamma) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. In this paper, we present evidence for the association of the insulin receptor (IR) with PLCgamma. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLCgamma and coimmunoprecipitation of the IR and PLCgamma were observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLCgamma and the IR, we used a specific inhibitor of PLC, U73122, or microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLCgamma to block insulin-stimulated GLUT4 translocation. We demonstrate inhibition of 2-deoxyglucose uptake in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. We conclude that PLCgamma is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes.
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PMID:Association of the insulin receptor with phospholipase C-gamma (PLCgamma) in 3T3-L1 adipocytes suggests a role for PLCgamma in metabolic signaling by insulin. 959 25

In adipocytes, insulin stimulates the translocation of the glucose transporter, GLUT4, from an intracellular storage compartment to the cell surface. Substantial evidence exists to suggest that in the basal state GLUT4 resides in discrete storage vesicles. A direct interaction of GLUT4 storage vesicles with the plasma membrane has been implicated because the v-SNARE, vesicle-associated membrane protein-2 (VAMP2), appears to be a specific component of these vesicles. In the present study we sought to identify the cognate target SNAREs for VAMP2 in mouse 3T3-L1 adipocytes. Membrane fractions were isolated from adipocytes and probed by far Western blotting with the cytosolic portion of VAMP2 fused to glutathione S-transferase. Two plasma membrane-enriched proteins, p25 and p35, were specifically labeled with this probe. By using a combination of immunoblotting, detergent extraction, and anion exchange chromatography, we identified p35 as Syntaxin-4 and p25 as the recently identified murine SNAP-25 homologue, Syndet (mSNAP-23). By using surface plasmon resonance we show that VAMP2, Syntaxin-4, and Syndet form a ternary SDS-resistant SNARE complex. Microinjection of anti-Syndet antibodies into 3T3-L1 adipocytes, or incubation of permeabilized adipocytes with a synthetic peptide comprising the C-terminal 24 amino acids of Syndet, inhibited insulin-stimulated GLUT4 translocation to the cell surface by approximately 40%. GLUT1 trafficking remained unaffected by the presence of the peptide. Our data suggest that Syntaxin-4 and Syndet are important cell-surface target SNAREs within adipocytes that regulate docking and fusion of GLUT-4-containing vesicles with the plasma membrane in response to insulin.
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PMID:Syndet, an adipocyte target SNARE involved in the insulin-induced translocation of GLUT4 to the cell surface. 966 52

We recently reported the cloning of full-length cDNAs corresponding to mRNAs of three GST-pi genes, hGSTP1*A, hGSTP1*B and hGSTP1*C, as well as, the isolation of the full-length hGSTP1*C, of the human glutathione S-transferase-pi (GST-pi) gene that is characterized by a A-->G transition at +1404 in exon 5 and a C-->T transition at +2294 in exon 6. Although the promoter of the isolated gene was identical to that of the previously described GST-pi gene isolated from the MCF 7 and the HPB-ALL cell lines, both of which were hGSTP1*A, a number of structural differences were observed, including, nucleotide transitions, transversions, deletions and insertions, some of which created new restriction enzyme cleavage sites. A guanine insertion in the insulin response element, IRE, in intron 1 created an additional site for 5'-cytosine methylation. Seven repeat retinoic acid response element (RARE) consensus half sites, A(G)GG(T)TC(G)A at +1521 to +1644 were identified in the cloned hGSTP1*C. Five of the RARE half-sites had the minimal spacer nucleotide requirement for functionality and DNA mobility shift analysis with different pairs of the RARE half-sites and supershift studies using antibodies against RAR-beta showed significant binding of nuclear protein complexes from RA-treated cells to these RAREs. GST-pi gene expression was increased significantly in cells transfected with the GST-pi gene and treated with all-trans RA. These results contrast with those in a previous report in which RA was shown to suppress the GST-pi promoter, and indicate a complex mechanism of RA-mediated GST-pi gene regulation in tumor cells.
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PMID:Structure of the human allelic glutathione S-transferase-pi gene variant, hGSTP1 C, cloned from a glioblastoma multiforme cell line. 967 46

pp120, a substrate of the insulin receptor tyrosine kinase, is a plasma membrane glycoprotein that is expressed in the hepatocyte as two spliced isoforms differing by the presence (full-length) or absence (truncated) of most of the intracellular domain including all phosphorylation sites. Co-expression of full-length pp120, but not its phosphorylation-defective isoforms, increased receptor-mediated insulin endocytosis and degradation in NIH 3T3 fibroblasts. We, herein, examined whether internalization of pp120 is required to mediate its effect on insulin endocytosis. The amount of full-length pp120 expressed at the cell surface membrane, as measured by biotin labeling, markedly decreased in response to insulin only when insulin receptors were co-expressed. In contrast, when phosphorylation-defective pp120 mutants were co-expressed, the amount of pp120 expressed at the cell surface did not decrease in response to insulin. Indirect immunofluorescence analysis revealed that upon insulin treatment of cells co-expressing insulin receptors, full-length, but not truncated, pp120 co-localized with alpha-adaptin in the adaptor protein complex that anchors endocytosed proteins to clathrin-coated pits. This suggests that full-length pp120 is part of a complex of proteins required for receptor-mediated insulin endocytosis and that formation of this complex is regulated by insulin-induced pp120 phosphorylation by the receptor tyrosine kinase. In vitro GST binding assays and co-immunoprecipitation experiments in intact cells further revealed that pp120 did not bind directly to the insulin receptor and that its association with the receptor may be mediated by other cellular proteins.
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PMID:Insulin stimulates pp120 endocytosis in cells co-expressing insulin receptors. 971 32

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

To investigate the difference in signaling between insulin and insulin-like growth factor I (IGF-I), we studied the effects of these hormones on the phosphorylation state of Crk-associated substrate (Cas) in cells expressing human insulin receptor (HIRc). In the basal state, Cas was heavily tyrosine-phosphorylated, and insulin dephosphorylated Cas in a time- and dose-dependent manner. On the other hand, IGF-I phosphorylated rather than dephosphorylated Cas in HIRc cells. In HIRY/F2 cells expressing a mutant insulin receptor lacking a binding site of SHP-2, a protein-tyrosine phosphatase containing src homology 2 (SH2) regions, insulin accelerated phosphorylation of Cas, as did IGF-I. In HIRc cells expressing a mutant SHP-2 lacking a PTPase domain (DeltaPTP), which interfered with SHP-2 function, insulin failed to dephosphorylate Cas. In whole cell lysate obtained in the basal state, Cas bound to a glutathione-S transferase fusion protein containing SH2 domains of SHP-2 and dissociated from this GST protein in response to insulin. These results indicate that the opposite regulation of Cas phosphorylation by insulin and IGF-I may be mediated through different properties of their receptors, and that the interaction of the insulin receptor with SHP-2 may play an important role in determining the tyrosine-phosphorylation state of Cas.
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PMID:Opposite regulation of tyrosine-phosphorylation of p130(Cas) by insulin and insulin-like growth factor I. 983 15

Phosphatidylinositol (PI) 3-kinase plays an important role in transducing the signals of various growth factor receptors. However, the regulatory mechanism of PI3-kinase activity by these growth factor receptors is not completely understood. Therefore, we attempted to clarify the regulatory mechanism of PI3-kinase using insulin and 3T3 L1 fibroblasts. Our results showed that insulin stimulated PI3-kinase activity seven-fold and concomitantly phosphorylated a p85 subunit at the tyrosine residue. However, this tyrosine phosphorylation was not significant in the activation of PI3-kinase as the PI3-kinase pulled down by the overexpressed GST-p85 fusion protein showed as high an activity as the immunoprecipitated one. The p110 subunit was phosphorylated at both serine and tyrosine residues without insulin treatment. Since the phosphorylation state was not changed by insulin. The results suggested that phosphorylation of the p110 subunit does not control PI3-kinase activity. Finally, it was shown that the insulin receptor substrate-1 (IRS-1) binding to PI3-kinase was not sufficient for full activation because the amount of IRS-1 pulled down by the GST-p85 fusion protein reached almost maximum, after incubation with insulin-treated cell lysates for 20 min, whereas PI3-kinase activity reached its maximum only after incubation for 5 h. All results suggest that the phosphorylation of p85 subunit at tyrosine residues and phosphorylation of p110 subunit at tyrosine or serine residues are not functionally significant in the regulation of PI3-kinase activity. They also suggest that P13-kinase is needed to bind to other protein(s) as well as the insulin receptor substrate-1 for full activation.
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PMID:The regulatory mechanism of phosphatidylinositol 3-kinase by insulin in 3T3 L1 fibroblasts: phosphorylation-independent activation of phosphatidylinositol 3-kinase. 989 59


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