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 BCR gene is involved in the formation of the BCR-ABL oncogene responsible for the pathogenesis of Philadelphia chromosome-positive human leukemias. We have previously shown that P210 BCR-ABL binds to the xeroderma pigmentosum group B protein (XPB) through the portion of BCR that is homologous to the catalytic domain of GDP-GTP exchangers such as yeast CDC24 and Dbl. In the baculovirus overexpression system which facilitates binding of coexpressed proteins, we now show that XPB binds to the intact BCR protein efficiently but not to CDC24 or Dbl, suggesting specificity of this interaction. The binding of endogenous BCR and XPB proteins was also detected in Hela cells, and this was inhibited by a blocking peptide. Full-length (1-782) XPB and its truncated form (203-782), which does not contain the nuclear localization signal, were tagged with glutathione S-transferase (GST) and were expressed in Rat1 fibroblasts. GST-XPB(203-782) was localized predominantly in the cytoplasm and bound to BCR but not to p62, one of the other components in TFIIH. GST-XPB(1-782) was largely in the nucleus and bound to p62 and BCR. Although the biological significance of the binding remains to be uncovered, BCR binds to the XPB/p62 complex.
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PMID:BCR binds to the xeroderma pigmentosum group B protein. 1040 66

IQGAP1, a target of Cdc42 and Rac1 small GTPases, directly interacts with beta-catenin and negatively regulates E-cadherin-mediated cell-cell adhesion by dissociating alpha-catenin from the cadherin-catenin complex in vivo (Kuroda, S., Fukata, M., Nakagawa, M., Fujii, K., Nakamura, T., Ookubo, T., Izawa, I., Nagase, T., Nomura, N., Tani, H., Shoji, I., Matsuura, Y., Yonehara, S., and Kaibuchi, K. (1998) Science 281, 832-835). Here we investigated how Cdc42 and Rac1 regulate the IQGAP1 function. IQGAP1 interacted with the amino-terminal region (amino acids 1-183) of beta-catenin, which contains the alpha-catenin-binding domain. IQGAP1 dissociated alpha-catenin from the beta-catenin-alpha-catenin complex in a dose-dependent manner in vitro. Guanosine 5'-(3-O-thio)triphosphate (GTPgammaS).glutathione S-transferase (GST)-Cdc42 and GTPgammaS. GST-Rac1 inhibited the binding of IQGAP1 to beta-catenin in a dose-dependent manner in vitro, whereas neither GDP.GST-Cdc42, GDP. GST-Rac1, nor GTPgammaS.GST-RhoA did. The coexpression of dominant active Cdc42 with IQGAP1 suppressed the dissociation of alpha-catenin from the cadherin-catenin complex induced by the overexpression of IQGAP1 in L cells expressing E-cadherin (EL cells). Consistent with this, the overexpression of either dominant negative Cdc42 or Rac1 resulted in the reduction of E-cadherin-mediated cell adhesive activity in EL cells. These results indicate that Cdc42 and Rac1 negatively regulate the IQGAP1 function by inhibiting the interaction of IQGAP1 with beta-catenin, leading to stabilization of the cadherin-catenin complex.
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PMID:Cdc42 and Rac1 regulate the interaction of IQGAP1 with beta-catenin. 1047 51

By using the mRNA differential display approach to isolate defense signaling genes active at the early stage of fungal infection two cDNA fragments with high sequence homology to rab-specific GDP-dissociation inhibitors (GDIs) were identified in rice (Oryza sativa L.) suspension cells. Using polymerase-chain-reaction products as probes, two full-length cDNA clones were isolated from a cDNA library of fungal-elicitor-treated rice, and designated as OsGDI1 and OsGDI2. The deduced amino acid sequences of the isolated cDNAs exhibited substantial homology to Arabidopsis rab-GDIs. Northern analysis revealed that transcripts detected with the 3'-gene-specific DNA probes accumulated to high levels within 30 min after treatment with a fungal elicitor derived from Magnaporthe grisea. The functionality of the OsGDIs was demonstrated by their ability to rescue the Sec19 mutant of Saccharomyces cerevisiae which is defective in vesicle transport. The proteins, expressed in Escherchia coli, cross-reacted with a polyclonal antibody prepared against bovine rab-GDI. Like bovine rab-GDI, the OsGDI proteins efficiently dissociated rab3A from bovine synaptic membranes. Using the two-hybrid system, it was shown that the OsGDIs specifically interact with the small GTP-binding proteins belonging to the rab subfamily. The specific interaction was also demonstrated in vitro by glutathione S-transferase resin pull-down assay.
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PMID:Characterization of two fungal-elicitor-induced rice cDNAs encoding functional homologues of the rab-specific GDP-dissociation inhibitor. 1059 42

Based on the previous experiments with the N17 mutant of CDC42, it has been speculated, but not proved as yet, that CDC42 is required for Ras-induced malignant transformation of fibroblasts. However, since this inhibitor could sequester many GDP-dissociation stimulators (GDSs), such as DBL, OST and Tiam-1 which activate not only CDC42, but also Rho or Rac, in fact it is not a specific inhibitor that inactivates only CDC42. Thus, we have taken the minimum CDC42-binding domain (residues 504 - 545, called ACK42) of the Tyr-kinase ACK-1 that binds only CDC42 in the GTP-bound form, and thereby blocking the interactions of CDC42-GTP with its downstream effectors such as ACKs, PAKs and N-WASP. First of all, using the ACK42-GST fusion protein as a specific ligand for the GTP-CDC42 complex, we have revealed that CDC42 is activated by oncogenic Ras mutants such as v-Ha-Ras in NIH3T3 fibroblasts, and similarly in PC12 cells by both NGF (Nerve Growth Factor) and EGF (Epidermal Growth Factor) which activate the endogenous normal Ras, providing the first direct evidence that CDC42 acts downstream of Ras and NGF/EGF. Furthermore, over-expression of ACK42 completely reversed Ras-induced malignant phenotypes such as focus formation and anchorage/serum-independent growth of the fibroblasts, and a cell-permeable derivative of ACK42 called WR-ACK42 strongly inhibited the growth of Ras transformants, with little effect on the parental normal cell growth, and also abolished Ras-induced filopodium/microspike formation of the fibroblasts which is CDC42-dependent. These observations unambiguously proved for the first time that the RAS-induced activation of CDC42 is indeed essential for Ras to transform the fibroblasts, and furthermore suggest that ACK42 or its peptidomimetics are potentially useful for genotherapy or chemotherapy of Ras-associated cancer.
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PMID:The CDC42-specific inhibitor derived from ACK-1 blocks v-Ha-Ras-induced transformation. 1061 19

A cDNA clone encoding a small GTP binding protein (Brho) was isolated from an embryonic cDNA library of Bombyx mori that encoded a polypeptide with 202 amino acids sharing 60-80% similarity with the Rho1 family of GTP binding proteins. The effector site and one of the guanine nucleotide binding sites differed from other members of the Rho family. To characterize the biochemical properties of Brho, the clone was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. The recombinant protein was purified to homogeneity with glutathione S-Sepharose. The fusion protein bound [(35)S] GTPgammaS and [(3)H] GDP with association constants of 11x10(6) M(-1) and 6.2x10(6) M(-1), respectively. The binding of [(35)S] GTPgammaS was inhibited by GTP and GDP, but by no other nucleotides. The calculated GTP-hydrolysis activity was 89.6 m mol/min/mol of Brho. Bound [(35)S] GTPgammaS and [(3)H] GDP were exchanged with GTPgammaS most efficiently in the presence of 6 mM MgCl(2). These results suggest that Brho has a higher affinity for GTP than GDP, converts from the GTP-bound state into the GDP-bound state by intrinsic GTP hydrolytic activity, and returns to the GTP-bound state with the exchange of GDP with GTP. Arch.
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PMID:Molecular cloning of a cDNA for a small GTP binding protein, BRho, from the embryo of Bombyx mori and its characterization after expression and purification. 1073 20

A mutational analysis of the Ras-binding domain (RBD) of c-Raf-1 identified three amino acid positions (Asn(64), Ala(85), and Val(88)) where amino acid substitution with basic residues increases the binding of RBD to recombinant v-Ha-Ras. The greatest increase in binding (6-9-fold) was observed with the A85K-RBD mutant. The elevated binding for the A85K-RBD and V88R-RBD mutants was also detected with Ras expressed in cultured mammalian cells, namely NIH-3T3 and BAF cells. None of the wild type residues in RBD positions Asn(64), Ala(85), and Val(88) have been previously implicated in the interaction with Ras (Block, C., Janknecht, R., Herrmann, C., Nassar, N., and Wittinghofer, A. (1996) Nat. Struct. Biol. 3, 244-251; Nassar, N., Horn, G., Herrmann, C., Scherer, A., McCormick, F., and Wittinghofer, A. (1995) Nature 375, 554-560). The discovery of elevated binding among the mutants in these positions implies that additional RBD residues can be used to generate the Ras. RBD complex. These findings are of particular significance in the design of Ras antagonists based on the RBD prototype. The A85K-RBD mutant can be used to develop an assay for measuring the level of activated Ras in cultured cells; Sepharose-linked A85K-RBD.GST fusion protein served as an activation-specific probe to precipitate Ras.GTP but not Ras.GDP from epidermal growth factor-stimulated cells. A85K-RBD precipitates up to 5-fold more Ras.GTP from mammalian cells than wild type RBD.
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PMID:Point mutants of c-raf-1 RBD with elevated binding to v-Ha-Ras. 1088 84

GTPases of the Rho family control a wide variety of cellular processes such as cell morphology, motility, proliferation, differentiation, and apoptosis. We report here the characterization of a new Rho member, which shares 85% and 78% amino acid similarity to TC10 and Cdc42, respectively. This GTPase, termed as TC10-like (TCL) is encoded by an unexpectedly large locus, made of five exons spanning over 85 kilobases on human chromosome 14. TCL mRNA is 2.5 kilobases long and is mainly expressed in heart. In vitro, TCL shows rapid GDP/GTP exchange and displays higher GTP dissociation and hydolysis rates than TC10. Using the yeast two-hybrid system and GST pull-down assays, we show that GTP-bound but not GDP-bound TCL protein directly interacts with Cdc42/Rac interacting binding domains, such as those found in PAK and WASP. Despite its overall similarity to TC10 and Cdc42, the constitutively active TCL mutant displays distinct morphogenic activity in REF-52 fibroblasts, producing large and dynamic F-actin-rich ruffles on the dorsal cell membrane. Interestingly, TCL morphogenic activity is blocked by dominant negative Rac1 and Cdc42 mutants, suggesting a cross-talk between these three Rho GTPases.
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PMID:Characterization of TCL, a new GTPase of the rho family related to TC10 andCcdc42. 1096 94

Both protein kinase C and protein tyrosine kinases have been shown to be involved in phospholipase D (PLD) activation in intact rat myometrium [Le Stunff, Dokhac and Harbon (2000) J. Pharmacol. Exp. Ther. 292, 629-637]. In this study we assessed the involvement of monomeric G-proteins in PLD activation in a cell-free system derived from myometrial tissue. Both the PLD1 and PLD2 isoforms were detected. Two forms of PLD activity, essentially membrane-bound, were found in myometrial preparations. One form was stimulated by oleate and insensitive to guanosine 5'-[gamma-thio] triphosphate (GTP[S]). The second required ammonium sulphate to be detected and was stimulated by GTP[S]. ADP-ribosylation factors (ARF1 and ARF6) and RhoA were immunodetected in myometrial preparations. ARF1 and RhoA were present in the membrane and cytosolic fractions whereas ARF6 was detected exclusively in the membrane fraction. A synthetic myristoylated peptide corresponding to the N-terminal domain of ARF6 [myrARF6((2-13))] totally abolished PLD activation in the presence of ammonium sulphate and GTP[S], whereas myrARF1((2-17)) and the inhibitory GDP/GTP-exchange factor, Rho GDI, did not. These data are consistent with a membrane-bound ARF6-regulated PLD activity. Finally, the stimulation of PLD by ARF6 was inhibited by AlF(-)(4) and this inhibition was counteracted by the fusion protein glutathione S-transferase-beta-adrenergic receptor kinase 1 (495-689) and by the QEHA peptide (from adenylate cyclase ACII), which act as G-protein betagamma-subunit scavengers. It is concluded that G-protein subunits betagamma are involved in a pathway modulating PLD activation by ARF6, illustrating cross-talk between heterotrimeric and monomeric G-proteins.
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PMID:Phospholipase D in rat myometrium: occurrence of a membrane-bound ARF6 (ADP-ribosylation factor 6)-regulated activity controlled by betagamma subunits of heterotrimeric G-proteins. 1108 43

In yeast, the Ypt1 GTPase is required for ER-to-cis-Golgi and cis-to-medial-Golgi protein transport, while Ypt31/32 are a functional pair of GTPases essential for exit from the trans-Golgi. We have previously identified a Ypt1 guanine nucleotide exchange factor (GEF) activity and characterized it as a large membrane-associated protein complex that localizes to the Golgi and can be extracted from the membrane by salt, but not by detergent. TRAPP is a large protein complex that is required for ER-to-Golgi transport and that has properties similar to those of Ypt1 GEF. Here we show that TRAPP has Ypt1 GEF activity. GST-tagged Bet3p or Bet5p, two of the TRAPP subunits, were expressed in yeast cells and were precipitated by glutathione-agarose (GA) beads. The resulting precipitates can stimulate both GDP release and GTP uptake by Ypt1p. The majority of the Ypt1 GEF activity associated with the GST-Bet3p precipitate has an apparent molecular weight of > 670 kDa, indicating that the GEF activity resides in the TRAPP complex. Surprisingly, TRAPP can also stimulate nucleotide exchange on the Ypt31/32 GTPases, but not on Sec4p, a Ypt-family GTPase required for the last step of the exocytic pathway. Like the previously characterized Ypt1 GEF, the TRAPP Ypt1-GEF activity can be inhibited by the nucleotide-free Ypt1-D124N mutant protein. This mutant protein also inhibits the Ypt32 GEF activity of TRAPP. Coprecipitation and overexpression studies suggest that TRAPP can act as a GEF for Ypt1 and Ypt31/32 in vivo. These data suggest the exciting possibility that a GEF complex common to Ypt1 and Ypt31/32 might coordinate the function of these GTPases in entry into and exit from the Golgi.
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PMID:The TRAPP complex is a nucleotide exchanger for Ypt1 and Ypt31/32. 1110 33

AGS3, a 650-amino acid protein encoded by an approximately 4-kilobase (kb) mRNA enriched in rat brain, is a Galpha(i)/Galpha(t)-binding protein that competes with Gbetagamma for interaction with Galpha(GDP) and acts as a guanine nucleotide dissociation inhibitor for heterotrimeric G-proteins. An approximately 2-kb AGS3 mRNA (AGS3-SHORT) is enriched in rat and human heart. We characterized the heart-enriched mRNA, identified the encoded protein, and determined its ability to interact with and regulate the guanine nucleotide-binding properties of G-proteins. Screening of a rat heart cDNA library, 5'-rapid amplification of cDNA ends, and RNase protection assays identified two populations of cDNAs (1979 and 2134 nucleotides plus the polyadenylation site) that diverged from the larger 4-kb mRNA (AGS3-LONG) in the middle of the protein coding region. Transfection of COS-7 cells with AGS3-SHORT cDNAs resulted in the expression of a major immunoreactive AGS3 polypeptide (M(r) approximately 23,000) with a translational start site at Met(495) of AGS3-LONG. Immunoblots indicated the expression of the M(r) approximately 23,000 polypeptide in rat heart. Glutathione S-transferase-AGS3-SHORT selectively interacted with the GDP-bound versus guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS)-bound conformation of Galpha(i2) and inhibited GTPgammaS binding to Galpha(i2). Protein interaction assays with glutathione S-transferase-AGS3-SHORT and heart lysates indicated interaction of AGS3-SHORT with Galpha(i1/2) and Galpha(i3), but not Galpha(s) or Galpha(q). Immunofluorescent imaging and subcellular fractionation following transient expression of AGS3-SHORT and AGS3-LONG in COS-7 and Chinese hamster ovary cells indicated distinct subcellular distributions of the two forms of AGS3. Thus, AGS3 exists as a short and long form, both of which apparently stabilize the GDP-bound conformation of Galpha(i), but which differ in their tissue distribution and trafficking within the cell.
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PMID:Identification of a truncated form of the G-protein regulator AGS3 in heart that lacks the tetratricopeptide repeat domains. 1127 52


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