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)

Protein tyrosine phosphatases all contain a conserved cysteine that forms an intermediate thiophosphate ester bond during tyrosine phosphate hydrolysis. A bacterial glutathione S-transferase fusion protein containing rat brain phosphatase PTP1b was constructed in which this conserved cysteine was mutated to serine. The resulting catalytically inactive enzyme was labeled in vivo to high specific activity with 35S, and the binding of this labeled fusion protein to the immunoprecipitated epidermal growth factor (EGF) receptor was evaluated. The binding was ligand-dependent, and saturation analysis revealed a nonlinear Scatchard plot, with a Kd for high affinity binding of approximately 100 nM. A number of glutathione S-transferase fusion proteins containing src homology 2 (SH2) domains attenuated phosphatase binding in a concentration-dependent manner. Phospholipase C (PLC) gamma and the GTPase-activating protein of ras were the most potent inhibitors. Tyrosine-phosphorylated EGF receptor peptide fragments were evaluated for specific inhibition of PTP1b and PLC gamma SH2 binding to the activated receptor. One such peptide, modeled on EGF receptor tyrosine 992, blocked the binding of both fusion proteins. Another phosphopeptide, modeled on tyrosine 1148, inhibited the binding of PTP1b but not the PLC gamma fusion protein. This site specificity was confirmed by analysis of equilibrium binding of the fusion proteins to EGF receptors mutated in each of these phosphorylation sites. The results revealed clear sequence specificity in the binding of proteins involved in the regulation of intracellular signaling by receptor tyrosine kinases.
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PMID:Sequence specificity in recognition of the epidermal growth factor receptor by protein tyrosine phosphatase 1B. 769 94

The neoplastic transformation of cultured rat liver epithelial cells by various means has consistently been associated with the development of resistance to the mito-inhibitory effect of transforming growth factor beta (TGF-beta), suggesting that such phenotype plays a mechanistic role during the transformation of these cells. We have studied the induction of the "TGF-beta-resistant" phenotype in a clonal strain of early passage WB-F344 normal cultured rat liver epithelial cells, the proliferation of which was markedly inhibited by TGF-beta. The control WB cells in continuous culture slowly developed TGF-beta resistance. However, when the same cells were exposed to step-wise increases of TGF-beta concentration in their culture medium, the development of TGF-beta resistance was accelerated. Cells which had been grown in medium containing 1 ng/ml TGF-beta developed colony-forming capacity in soft agar containing epidermal growth factor. Cells which were grown in media containing 5 and 10 ng/ml TGF-beta demonstrated a low level of colony-forming efficiency in soft agar medium without added epidermal growth factor and tumorigenicity in isogeneic rats. These TGF-beta-resistant cells also exhibited progressively increasing levels of expression of the c-fos and and myc mRNA, and increased resistance to the cytotoxicity of Adriamycin and melphalan. The latter phenomenon was accompanied by an increase in the mdr-1 mRNA expression, cellular glutathione level, and glutathione S-transferase activity. The results suggest that chronic exposure to high concentration of TGF-beta promotes the spontaneous neoplastic transformation of cultured rat liver epithelial cells, and that this process may represent one of the mechanisms of cellular adaptation for induction of the multidrug-resistant phenotype during the carcinogenesis of epithelial cells.
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PMID:Transforming growth factor beta 1 promotes spontaneous transformation of cultured rat liver epithelial cells. 795 58

PG-M is a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds. We previously isolated cDNA clones encoding the core protein of PG-M (Shinomura, T., Nishida, Y., Ito, K., and Kimata, K. (1993) J. Biol. Chem. 268, 14461-14469). The amino acid sequence deduced from the cDNA analysis revealed the presence of two epidermal growth factor-like domains, a C-type lectin-like domain, and a complement regulatory protein (CRP)-like domain at the COOH terminus. The COOH-terminal portion has been expressed as a fusion protein with glutathione S-transferase in Escherichia coli to test its carbohydrate binding activity using affinity chromatography. The purified fusion protein binds to immobilized D-mannose, D-galactose, L-fucose, and N-acetyl-D-glucosamine in a calcium-dependent manner. Furthermore, the fusion protein binds to heparin- or heparan sulfate-Sepharose. To investigate roles of each COOH-terminal domain, we have made a truncated construct which lacks the CRP-like domain and determined if the CRP-like domain is involved in the binding activity. The removal of this domain resulted in the complete loss of both C-type lectin-like and heparin binding activities. The results suggest that a whole set of epidermal growth factor-, lectin-, and CRP-like domains may serve a functional structure for these bindings.
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PMID:Expression and binding activity of the carboxyl-terminal portion of the core protein of PG-M, a large chondroitin sulfate proteoglycan. 796 77

We have expressed in bacteria the C-terminal part of Plasmodium yoelii merozoite surface protein-1 (MSP1) containing the two epidermal growth factor-like domains. The protein, either alone or fused to glutathione S-transferase, was highly effective as a vaccine and protected mice against challenge infection. Reduction and alkylation abolished the protection obtained with the protein. This shows for the first time the absolute requirement of the disulphide-bonded conformation for immunogenicity. In a short term experiment, mice were protected against a massive challenge. The immunity was effective at the time of merozoite release/reinvasion. Recombinant protein based on this part of MSP1 may be suitable as a vaccine against malaria.
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PMID:Immunization against malaria with a recombinant protein. 801 56

Autophosphorylation of receptor tyrosine kinases provides binding sites for signaling proteins containing Src homology 2 (SH2) domains. We determined the binding sites of Shc, SH2-containing adaptor protein, within epidermal growth factor (EGF) receptors, using Chinese hamster ovary cells overexpressing EGF receptor mutants in which autophosphorylation sites, either alone or in combination, were replaced by phenylalanine. Binding of Shc to EGF receptor mutants lacking single tyrosine residues at 1148 or 1173 decreased by approximately 60 or approximately 15%, respectively, whereas other single point mutants bound the wild-type level of Shc. Binding of Shc markedly decreased in mutants lacking both tyrosine residues at 1148 and 1173. In peptide inhibition assay, phosphorylated nonameric peptide representing tyrosine 1148, DNPDpYQQDF, but not pentameric peptide, pYQQDF, inhibited the binding of glutathione S-transferase-Shc SH2 domain fusion protein to in vitro autophosphorylated EGF receptors, suggesting that N-terminal sequences adjacent to phosphotyrosine are necessary for the association of Shc. Based on results of peptide inhibition assays in which phosphorylated peptides representing tyrosines 992, 1148, and 1173 inhibited Shc binding to the receptor, we constructed another EGF receptor mutant in which one of these tyrosine residues was retained. The amount of Shc bound to mutant receptors retaining tyrosines 1148, 1173, or 992 was approximately 80, approximately 40, or approximately 10% of wild-type level, respectively. These results indicate that tyrosine 1148 of activated human EGF receptors is a major binding site of Shc and tyrosine 1173 is a secondary binding site in intact cells.
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PMID:Tyrosines 1148 and 1173 of activated human epidermal growth factor receptors are binding sites of Shc in intact cells. 803 16

Protein kinases share a number of highly conserved or invariant amino acid residues in their catalytic domains, suggesting that these residues are necessary for kinase activity. In p180erbB3, a receptor tyrosine kinase belonging to the epidermal growth factor (EGF) receptor subfamily, three of these residues are altered, suggesting that this protein might have an impaired protein tyrosine kinase activity. To test this hypothesis, we have expressed human EGF receptor and bovine p180erbB3 in insect cells via baculovirus infection and have compared their autophosphorylation and substrate phosphorylation activities. We have found that, while the EGF receptor readily undergoes EGF-stimulated autophosphorylation and catalyzes the incorporation of phosphate into the model substrates (E4Y1)n (random 4:1 copolymer of glutamic acid and tyrosine) and GST-p85 (glutathione S-transferase fusion protein with the 85-kDa subunit of phosphatidylinositol 3-kinase), p180erbB3 autophosphorylation and substrate phosphorylation are at least 2 orders of magnitude less efficient. However, p180erbB3 is capable of binding the ATP analog 5'-p-fluorosulfonylbenzoyladenosine, indicating that the lack of observed kinase activity is probably not due to nonfunctional or denatured receptors expressed by the insect cells. On the basis of these results, we propose that p180erbB3 possesses an impaired intrinsic tyrosine kinase activity.
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PMID:Insect cell-expressed p180erbB3 possesses an impaired tyrosine kinase activity. 805 68

Syp (SH-PTP2) was recently identified as a phosphotyrosine phosphatase containing two SH2 domains within its primary structure. In response to appropriate growth factor stimulation, Syp becomes phosphorylated on tyrosine residues and associates with insulin receptor substrate 1 (IRS-1) and/or the corresponding growth factor receptor via its SH2 domains, leading to increased Syp activity. To assess the importance of Syp in mitogenic signaling, we microinjected mammalian fibroblasts with several reagents designed to interfere with Syp SH2/phosphotyrosine interaction in vivo. Insulin-, insulin-like growth factor-1-, and epidermal growth factor-stimulated DNA synthesis, indicated by bromodeoxyuridine (BrdUrd) incorporation, was dramatically decreased following microinjection of a Syp antibody (Ab) (65-85%) or a Syp GST-SH2 fusion protein (approximately 90%) in comparison with cells microinjected with control IgG or glutathione S-transferase (GST), respectively. In addition, microinjection of an IRS-1-derived phosphonopeptide, which inhibits in vitro binding of Syp-SH2 to IRS-1 with an ED50 value of approximately 23 microM, also decreased BrdUrd incorporation in vivo by approximately 50-75%. Microinjection of the Syp Ab, Syp GST-SH2 fusion protein, or the phosphonopeptide had no effect on serum-stimulated BrdUrd incorporation. In conclusion, disruption of Syp function in living cells inhibited cell cycle progression in response to growth factor stimulation, indicating that Syp is a critical positive regulator of mitogenic signal transduction.
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PMID:Syp (SH-PTP2) is a positive mediator of growth factor-stimulated mitogenic signal transduction. 806 47

Glutathione S-transferase P-form (GST-P, EC 2.5.1.18) mRNA was expressed by epidermal growth factor as well as by 3,4,5,3',4'-penta-chlorinated biphenyl (PenCB) in primary cultured rat liver parenchymal cells. The expression of GST-P was suppressed by inhibitors of protein kinase C and dexamethasone, an antagonist of AP-1 transcription factor activity, whereas expression of cytochrome P450IA2 by PenCB was not affected by these reagents. The AP-1 related transcription factor may be essential for the expression of GST-P by PenCB as also may be a protein kinase C type enzyme.
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PMID:Expression of glutathione S-transferase P-form in primary cultured rat liver parenchymal cells by coplanar polychlorinated biphenyl congeners is suppressed by protein kinase inhibitors and dexamethasone. 822 47

It has recently been shown that Ras proteins interact directly with Raf serine/threonine kinases in vitro and in the yeast two-hybrid system, leading to speculation that Raf proteins function as effectors for Ras. Here it is demonstrated that the endogenous Raf-1 protein co-immunoprecipitates with Ras from mammalian cells when the non-neutralizing anti-Ras monoclonal antibody Y13-238 is used. The formation of a Ras-Raf complex is absolutely dependent on prior treatment of the cells with a stimulus that activates Ras: phorbol ester or anti-T cell receptor antibody in the case of human peripheral blood T lymphoblasts, or epidermal growth factor in the case of Rat-1 fibroblasts. Up to 3% of cellular Raf-1 can be found in association with Ras. The association is not competed by addition of exogenous GST-Raf to the cell lysates and is therefore unlikely to be due to Ras-Raf binding after cell lysis. Specific interaction of Ras and Raf therefore occurs in intact mammalian cells in response to stimuli that cause Ras to become GTP-bound.
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PMID:Interaction of Ras and Raf in intact mammalian cells upon extracellular stimulation. 830 46

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


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