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 ras-related protein, CDC42Hs, is a 22-kDa GTP-binding protein which is the human homolog of a Saccharomyces cerevisiae yeast-cell-division cycle protein. In attempting to isolate and biochemically characterize mammalian proteins capable of regulating various activities of CDC42Hs, we have identified an activity in bovine brain cytosol which effectively inhibits the dissociation of [3H]GDP from the platelet- or the Spodoptera frugiperda-expressed CDC42Hs protein. The purification of this activity was achieved by a series of steps which included ammonium sulfate fractionation, DEAE-Sephacel, Mono-Q, and Mono-S chromatographies. The purified CDC42Hs regulatory protein has an apparent molecular weight of 28,000, and cyanogen bromide-generated peptide sequences of this protein were identical to sequences from the carboxyl-terminal portion of rho-GDP-dissociation inhibitor (rho-GDI) (Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H., Araki, S., Ueda, T., Kikuchi, A., and Takai, Y. (1990) Oncogene 5, 1321-1328). In addition, an Escherichia coli-expressed, glutathione S-transferase-rho-GDI fusion protein fully substitutes for the GDI which we have purified from bovine brain in its ability to inhibit GDP dissociation from CDC42Hs. These findings suggest either that a common regulatory protein (GDI) is capable of inhibiting GDP dissociation from the rho and CDC42Hs proteins or that these two GTP-binding proteins interact with GDI proteins of very similar structure. The purified brain GDI protein shows little ability to inhibit GDP dissociation from the E. coli-expressed CDC42Hs and is capable of only a very weak inhibition of the dissociation of [35S]guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) from the Spodoptera frugiperda-expressed CDC42. However, brain GDI very effectively inhibits the ability of the human dbl oncogene product to catalyze GDP dissociation from CDC42Hs. In addition to influencing guanine nucleotide association with CDC42Hs, the purified brain GDI protein also appears to catalyze the dissociation of CDC42Hs from the plasma membranes of human placenta and human epidermoid carcinoma (A431) cells. This effect by the GDI protein is observed whether the membrane-associated CDC42Hs is preincubated with GDP, GTP gamma S, or no guanine nucleotides, and occurs over a similar concentration range as that necessary for the inhibition of the intrinsic GDP dissociation.
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PMID:The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein. 142 34

Cellular mechanisms for controlling membrane trafficking appear to involve small GTP-binding proteins such as the Rab proteins. Rab function is regulated by GDP dissociation inhibitor (GDI), which releases Rab proteins from membranes and inhibits GDP dissociation. Here we report the isolation of a full-length cDNA encoding a novel GDI isoform of 445 amino acids (GDI-2) with a deduced molecular weight of 50,649 from mouse skeletal muscle. Full-length and partial cDNA clones encoding a previously reported GDI protein (GDI-1) were also isolated from cDNA libraries prepared from rat brain and mouse skeletal muscle, respectively. The degree of deduced amino acid sequence identity between mouse GDI-2 and our mouse GDI-1 cDNA clone is 86%. Northern (RNA blot) analysis revealed that in human tissues, both GDI-1 and GDI-2 transcripts were abundant in brain, skeletal muscle, and pancreas but were weakly expressed in heart and liver. GDI-1 mRNA was expressed in kidney, whereas GDI-2 was almost absent, while in lung the relative amounts of these mRNA species were reversed. Specific antibodies against mouse GDI-1 and GDI-2 based on unique peptide sequences in the proteins were raised. Differentiation of 3T3-L1 fibroblasts into highly insulin-responsive adipocytes was accompanied by large increases in both mRNA and protein levels of GDI-1 and GDI-2. GDI-1 and GDI-2 expressed as glutathione S-transferase fusion proteins were both able to solubilize the membrane-bound forms of Rab4 and Rab5 in a GDP/GTP-dependent manner. Taken together, these data demonstrate that the protein products of at least two genes regulate the membrane dynamics of Rab proteins in mice.
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PMID:Cloning, characterization, and expression of a novel GDP dissociation inhibitor isoform from skeletal muscle. 751 52

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL. 749 7

This work describes the biochemical characterization of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein (GAP) regulating the RAS gene products. A fragment of 383 residues (amino acids 1644-2026) was produced in Escherichia coli as glutathione S-transferase fusion protein (GST-Ira2p-383) and highly purified (> 90%) by affinity chromatography. The affinity of Ras2p for the GST-fused Ira2p-383 was 18 microM and the maximal stimulation of the Ras2p GTPase activity 6,000 times. The Ira2p activity was confirmed to be strictly specific for Ras2p, no stimulatory effect on human c-H-ras p21 GTPase being detectable. Comparison with the GAP-like domain of mammalian p120-GAP and neurofibromin using yeast Ras2p as substrate showed that Ira2p-383 has an affinity and turnover intermediary between GAP-334 and NF1-414. The activity of Ira2p-383 was strongly inhibited by monovalent and divalent salts. The simultaneous presence of the catalytic domains of Ira2p and the yeast GDP/GTP exchange factor Cdc25p induced on Ras2p a multiple-round reaction of GTP hydrolysis and GDP/GTP exchange, showing that it is possible to reconstitute in vitro a S. cerevisiae system suitable for the study of the regulation of the Ras2p GDP/GTP cycle. The tubulin partially inhibited (25%) the GAP activity of the Ira2p-383. A larger Ira2p catalytic fragment, Ira2p-505 (amino acids 1549-2053), that showed the same Km for Ras2p as Ira2p-383, was also inhibited by tubulin to the same extent but with a higher affinity than Ira2p-383.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Properties and regulation of the catalytic domain of Ira2p, a Saccharomyces cerevisiae GTPase-activating protein of Ras2p. 757 70

beta gamma Subunits of heterotrimeric GTP-binding proteins (G beta gamma) activate the inwardly rectifying muscarinic K+ channel, GIRK1. The significant role for the carboxyl (C) terminus of GIRK1 in this interaction has been suggested. However, it is still unknown whether G beta gamma directly interacts with GIRK1. To elucidate the molecular basis of G beta gamma-activation of GIRK1, we examined the binding properties of G beta gamma to the C terminus of GIRK1 cloned from mouse brain cDNA library (MB-GIRK1). The C terminus of MB-GIRK1 fused with glutathione S-transferase directly bound to purified G beta gamma. Incubation of the C terminus with Gi pretreated with GTP gamma S, but not with GDP, resulted in the binding of Gi beta gamma to the protein. Purified G alpha-GDP, but not G alpha-GTP gamma S, inhibited the binding of G beta gamma to the fusion protein. These results indicate that G beta gamma dissociated from G alpha may directly bind to the C terminus of GIRK1.
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PMID:G beta gamma directly binds to the carboxyl terminus of the G protein-gated muscarinic K+ channel, GIRK1. 762 88

We previously purified a protein factor, named REKS (Ras-dependent Extracellular Signal-regulated Kinase (ERK)/mitogen-activated protein kinase Kinase (MEK) Stimulator), from Xenopus eggs by use of a cell-free assay system in which recombinant GTP gamma S (guanosine 5'-(3-O-thio)triphosphate)-Ki-Ras activates recombinant MEK. By use of this assay system, we purified here bovine REKS to near homogeneity from the cytosol fraction of bovine brain by successive chromatographies of Mono S, Mono Q, GTP gamma S-glutathione S-transferase-Ha-Ras-coupled glutathione-agarose, and Mono Q columns. It was composed of three proteins with masses of about 95, 32, and 30 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 95-, 32-, and 30-kDa proteins were identified by immunoblot analysis to be B-Raf protein kinase, 14-3-3 protein, and 14-3-3 protein, respectively. Moreover, the REKS activity was specifically immunoprecipitated by an anti-B-Raf antibody. Bovine REKS was activated by lipid-modified GTP gamma S-Ki-Ras far more effectively than by a lipid-unmodified one. Lipid-modified GDP-Ki-Ras was inactive. Exogenous addition of 14-3-3 proteins stimulated further the REKS activity both in the presence and absence of GTP gamma S-Ki-Ras. These results indicate that at least one of the direct targets of Ras is B-Raf complexed with 14-3-3 proteins in bovine brain.
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PMID:Purification of a Ras-dependent mitogen-activated protein kinase kinase kinase from bovine brain cytosol and its identification as a complex of B-Raf and 14-3-3 proteins. 774 15

The GST (glutathione S-transferase)-NDK (nucleoside diphosphate kinase) fusion protein was expressed in Escherichia coli. The GST-NDK protein was capable of transferring gamma-phosphate from ATP to nucleoside diphosphates such as GDP, CDP, TDP and UDP. Western blot analysis using anti-NDK antibody indicated that NDK in endosperm gradually decreased during 36 h of imbibition. On the contrary, NDK in embryo increased during the same period. NDK activities in both tissues were in accord with these observations. Whereas the NDK protein in roots of rice seedlings during 7 days of imbibition remained constant, in shoots it declined after 5 days of imbibition. Thus, NDK may play a significant role in the cellular event modulated by adenylate energy charge level.
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PMID:Expression of functional proteins of cDNA encoding rice nucleoside diphosphate kinase (NDK) in Escherichia coli and organ-related alteration of NDK activities during rice seed germination (Oryza sativa L.). 776 75

The catalytic domain of the Saccharomyces cerevisiae SDC25 gene product, including the last 550 C-terminal residues (Sdc25p-C), was produced as an Escherichia coli recombinant protein fused with glutathione S-transferase. The highly purified (greater than 95%) stable fusion protein, obtained by affinity chromatography, was very active in enhancing the dissociation rate or the GDP/GTP exchange of the GDP complex of Ras2p or human H-ras p21. This activity was further increased (three times) by glutathione S-transferase cleavage with thrombin. The stimulation of the guanine nucleotide release by Sdc25p-C was stronger for Ras2p.GDP than Ras2p.GTP, an effect that was less pronounced in the case of the p21 complexes. The association rate of the Ras2p.GDP (GTP) complex was also enhanced by Sdc25p-C. Monovalent and divalent salts inhibit the nucleotide-releasing activity of Sdc25p-C. Retention phenomena occurring on gel-filtration chromatography hindered the use of highly purified Sdc25p-C to study the formation of stable complexes with Ras2p. For this purpose, Sdc25p-C was produced as a non-glutathione-S-transferase fusion protein via pTTQ19. Upon partial purification, this product yielded a 54-kDa truncated form of Sdc25p-C (truncated Sdc25p-C) showing the same specific activity as the 64-kDa Sdc25p-C protein. On gel filtration, truncated Sdc25p-C and nucleotide-free Ras2p (or p21) formed a stable 1:1 stoichiometric complex that was dissociated by increasing concentrations of GDP. The properties of this complex were analyzed by using the mutant [S24N]Ras2p, the homologue of [S17N]p21 known to induce a dominant negative phenotype, [R80D, N81D]Ras2p, a recessive negative mutant insensitive to the truncated form of Sdc25p-C in vitro. The complex with [S24N]Ras2p was greater than 100-fold less sensitive to the dissociating effect of GDP, whereas [R80D, N81D]Ras2p was unable to form a stable complex with truncated Sdc25p-C. These results strongly suggest that the residues R80 and N81 are situated in or closely associated with the Ras2p specific site binding Sdc25p.
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PMID:Properties of the catalytic domain of sdc25p, a yeast GDP/GTP exchange factor of Ras proteins. Complexation with wild-type Ras2p, [S24N]Ras2p and [R80D, N81D]Ras2p. 785 34

We have previously identified a protein factor, named REKS (Ras-dependent Extracellular signal-regulated kinase/Mitogen-activated protein kinase kinase (MEK) Stimulator), which is necessary for Ras-dependent MEK activation. In this study, we attempted to highly purify and characterize REKS. We have highly purified REKS by successive column chromatographies using a cell-free assay system in which REKS activates recombinant extracellular signal-regulated kinase 2 through recombinant MEK in a guanosine 5'-O-(thiotriphosphate) (GTP gamma S)-Ki-Ras-dependent manner. REKS formed a stable complex with GTP gamma S-Ras; REKS was coimmunoprecipitated with GTP gamma S-Ki-Ras or GTP gamma S-Ha-Ras, but not with GDP-Ki-Ras or GDP-Ha-Ras by an anti-Ras antibody. REKS was absorbed to a GTP gamma S-glutathione S-transferase (GST)-Ha-Ras-coupled glutathione-agarose column but not to a GDP-GST-Ha-Ras-coupled glutathione-agarose column and was coeluted with GTP gamma S-GST-Ha-Ras by reduced glutathione. The minimum molecular mass of REKS was estimated to be about 98 kDa on SDS-polyacrylamide gel electrophoresis. REKS phosphorylated this 98-kDa protein as well as recombinant MEK. REKS was not recognized by any of the anti-c-Raf-1, anti-Mos, and anti-mSte11 antibodies. These results indicate that REKS is a Ras-dependent MEK kinase.
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PMID:Purification and characterization of REKS from Xenopus eggs. Identification of REKS as a Ras-dependent mitogen-activated protein kinase kinase kinase. 785 6

Different domains of the serine/threonine kinase, raf-1, were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and purified to near homogeneity by affinity chromatography. A cysteine-rich domain of raf-1 was found to contain 2 mol of zinc (molar basis), similar to analogous cysteine-rich domains of protein kinase C. GST-fusion proteins, containing the cysteine-rich domain of raf-1, bound to liposomes in a phosphatidylserine-dependent manner. In contrast to protein kinase C, the translocation of raf-1 was not dependent upon diacylglycerol, phorbol ester, or calcium, nor did raf-1 bind phorbol esters. A GST-fusion protein encoding residues 1-147 of raf-1 bound to normal GTP-ras with high affinity, but not to mutant GTP-Ala35 ras; no binding was detected to GDP-ras. The binding of a smaller fusion protein (residues 1-130 of raf-1) was about 10-fold weaker, inferring that a 17-amino acid sequence represents a critical binding determinant in intact raf-1. These residues are adjacent to the amino-terminal end of, and partially extend into, the cysteine-rich domain (amino acids 139-184). A synthetic peptide corresponding to this 17-amino acid sequence blocked the interaction of raf-1 with ras. The function of the cysteine-rich region of raf-1 homologous to protein kinase C is to promote translocation of raf-1 kinase to membranes and to form part of the high affinity binding site for GTP-ras.
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PMID:The cysteine-rich region of raf-1 kinase contains zinc, translocates to liposomes, and is adjacent to a segment that binds GTP-ras. 814 97


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