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

---

Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

p120 GTPase-activating protein (GAP) is a negative regulator of Ras that functions at a key relay point in signal transduction pathways that control cell proliferation. Among other proteins, p120 GAP associates with p190, a GAP for the Ras-related protein, Rho. To characterize the p120.p190 interaction further, we used bacterially expressed glutathione S-transferase fusion polypeptides to map the regions of p120 necessary for its interactions with p190. Our results show that both the N-terminal and the C-terminal SH2 domains of p120 are individually capable of binding p190 expressed in a baculovirus/insect cell system. Moreover, the two SH2 domains together on one polypeptide bind synergistically to p190, and this interaction is dependent on tyrosine phosphorylation of p190. In addition, mutation of the highly conserved Arg residues in the critical FLVR sequences of both SH2 domains of full-length p120 reduces binding to tyrosine-phosphorylated p190. The dependence on p190 phosphorylation for complex formation with p120 SH2 domains observed in vitro is consistent with analysis of the native p120.p190 complexes formed in vivo. These findings suggest that SH2-phosphotyrosine interaction is one mechanism by which the cell regulates p120.p190 association and thus may be a means for coordinating the Ras- and Rho-mediated signaling pathways.
...
PMID:Two SH2 domains of p120 Ras GTPase-activating protein bind synergistically to tyrosine phosphorylated p190 Rho GTPase-activating protein. 762 1

p21 Ras has been implicated in vulval differentiation in Caenorhabditis elegans. We now describe the characteristics during nematode development of the related p21 RhoA which has been ascribed a morphological role in mammals. The CeRhoA cDNA isolated in this study encodes a sequence of 192 amino acids residues with 87.6% identity to human RhoA. Genomic Southern analysis indicates the presence of a single Rho gene in C. elegans. Its 2-kilobase mRNA is expressed at the highest levels during embryogenesis and decreases gradually thereafter. However, the level of the 24-kDa protein detected by the anti-CeRhoA antibody is high at the larval stages but low in embryos. The glutathione S-transferase/CeRhoA fusion protein expressed in Escherichia coli displays conserved biochemical activities. Unlike its counterpart in mammalian cells which is predominantly cytosolic, most of CeRhoA is associated with the membrane and the cytoskeleton throughout development. Indirect immunofluorescence analysis indicates an ubiquitous expression of CeRhoA throughout development with a particular enrichment at larval stages in the pharyngeal nerve ring and at the tip of the head containing chemosensory and mechanosensory neurons. This suggests a stage-specific role for p21 RhoA in mediating the signaling pathway underlying the sensory circuitry in C. elegans post-embryonic development.
...
PMID:The Caenorhabditis elegans small GTP-binding protein RhoA is enriched in the nerve ring and sensory neurons during larval development. 779 39

The breakpoint cluster region gene product (Bcr) is a GTPase-activating protein (GAP) for members of the Rho family, Cdc42Hs, and Rac1, as is the brain protein n-chimaerin. At least 15 proteins have sequence identity to the GAP domain (150 amino acid residues) of Bcr. The widespread occurrence of proteins that possess sequence identity to the Bcr-related GAP domain makes it especially important to understand its structure/function relationships. Amino acid sequence alignment of these proteins reveals three blocks of conservation in the GAP domain. Here, we present a mutational analysis of this domain using n-chimaerin sequences. Ten mutations were constructed (at least two in each of the blocks of conservation), expressed as glutathione S-transferase fusion proteins in Escherichia coli, and purified. Seven of the mutants, including deletions, still possessed GAP activity for Rac1. Three of the mutants had no Rac1-GAP activity but were still able to bind Rac1. IC50 values obtained from competition experiments suggest that n-chimaerin and the mutants with no GAP activity bound Rac1 with similar apparent binding constants. Thus, this mutant analysis allows discrimination between Rac1-binding and Rac1 GTPase- activating residues.
...
PMID:Breakpoint cluster region gene product-related domain of n-chimaerin. Discrimination between Rac-binding and GTPase-activating residues by mutational analysis. 802 Dec 74

The Ras-like GTPase Cdc42 is essential for cell polarity and bud site assembly in Saccharomyces cerevisiae by regulating cell cycle-dependent reorganization of cortical cytoskeletal elements. However, its role in mammalian cells is unknown. To identify potential effectors of Cdc42Hs, we incubated lysates from NIH 3T3 fibroblasts or PC12 cells with immobilized glutathione S-transferase (GST)-Cdc42Hs fusion proteins bound to different guanine nucleotides and observed a specific association between the 85-kDa subunit (p85) of phosphatidylinositol 3-kinase (PI 3-kinase) and GTP gamma S (guanosine 5'-3-O-(thio)triphosphate)-bound GST-Cdc42Hs. Recombinant p85 formed a complex with GTP gamma S-bound GST-Cdc42Hs and with a GTPase-defective GTP-bound GST-Cdc42Hs-Q61L mutant, but not with a GTP gamma S-bound, effector domain GST-Cdc42HsT35A mutant. Both the Rho-GAP homology domain of p85 and the Cdc42Hs-GAP competitively inhibited the binding of recombinant p85 to Cdc42Hs. In addition, PI 3-kinase activity immunoprecipitated from cell lysates with anti-p85 antibody was stimulated 2-4-fold by GST-Cdc42-GTP gamma S. Similar interactions were observed between p85 and GST-Rac1-GTP gamma S but not between p85 and GST-RhoA-GTP gamma S. These findings suggest that PI 3-kinase, through the Rho-GAP homology domain of p85, can couple to the effector domain of Cdc42Hs and that p85 may be a target for the GTP-bound forms of Cdc42Hs and Rac1.
...
PMID:Activation of phosphoinositide 3-kinase activity by Cdc42Hs binding to p85. 803 24

Clostridium difficile toxin B exhibits cytotoxic activity that is characterized by the disruption of the microfilamental cytoskeleton. Here we studied whether the GTP-binding Rho protein, which reportedly participates in the regulation of the actin cytoskeleton, is involved in the toxin action. Toxin B treatment of Chinese hamster ovary cells reveals a time- and concentration-dependent decrease in the ADP-ribosylation of Rho by Clostridium botulinum C3 exoenzyme in the cell lysate. Disruption of the microfilament system induced by C. botulinum C2 toxin or cytochalasin D does not cause impaired ADP-ribosylation of Rho. Toxin B exhibits its effects on Rho not only in intact cells but also when added to cell lysates. Besides endogenous Rho, RhoA-glutathione S-transferase (Rho-GST) fusion protein added to cell lysate showed decreased ADP-ribosylation after toxin B treatment. Immunoblot analysis reveals identical amounts of Rho-GST and no change in molecular mass after toxin B treatment compared with controls. ADP-ribosylation of Rho-GST purified from toxin B-treated cell lysate is inhibited, indicating a modification of Rho itself. Finally, transfection of rhoA DNA under the control of a strong promoter into cells protects them from the activity of toxin B. Altogether, the data indicate that C. difficile toxin B acts directly or indirectly on Rho proteins to inhibit ADP-ribosylation and suggest that the cytotoxic effect of toxin B involves Rho.
...
PMID:Clostridium difficile toxin B acts on the GTP-binding protein Rho. 814 60

Specific [32P]ADP-ribosylation by Clostridium botulinum exoenzyme C3 was used to study the involvement of phosphorylation in the regulation of the low-molecular-mass GTP-binding protein Rho. Dephosphorylation of CHO cell extracts by alkaline phosphatase treatment resulted in a 80-90% reduction in the C3-catalysed [32P]ADP-ribosylation of Rho proteins in both cytosolic and membrane fractions. Similar results were obtained after dephosphorylation with protein phosphatase type-1 from bovine retina, whereas type-2B and type-2C phosphatases had no effect on the level of subsequent [32P]ADP-ribosylation of Rho by C3. Incubation of CHO cell lysate under phosphorylation conditions increased the subsequent C3-mediated [32P]ADP-ribosylation of Rho proteins. The protein kinase inhibitors H7 and H9 had no effect on [32P]ADP-ribosylation at concentrations which are specific for inhibition of protein kinase A or C. Recombinant glutathione S-transferase-RhoA fusion protein (GST-RhoA) was phosphorylated by protein kinase A; however, the phosphorylation had no stimulatory effect on the ADP-ribosylation of GST-RhoA by C3. An approx. 48 kDa phosphoprotein was identified which bound specifically to recombinant GST-RhoA fusion protein. By gel-permeation chromatography, Rho-containing complexes of approx. 50 kDa and 130-170 kDa were detected. The ADP-ribosylation of Rho in the 130-170 kDa complex was reduced by alkaline phosphatase pretreatment. The data suggest that Rho activity is influenced by phosphorylation of Rho-associated regulatory factors. Phosphorylation/dephosphorylation of these Rho-regulating factors appears to alter the ability of Rho to serve as a substrate for C3-induced [32P]ADP-ribosylation.
...
PMID:ADP-ribosylation of Rho proteins by Clostridium botulinum exoenzyme C3 is influenced by phosphorylation of Rho-associated factors. 819 24

R-Ras, belonging to the Ras small GTP-binding protein superfamily, has been implicated in regulation of various cell functions such as gene expression, cell proliferation, and apoptotic cell death. In the present study, we purified an R-Ras-interacting protein with molecular mass of about 98 kDa (p98) from bovine brain cytosol by glutathione S-transferase (GST)-R-Ras affinity column chromatography. This protein bound to GTP gamma S (guanosine 5'-(3-O-thio)triphosphate, a nonhydrolyzable GTP analog).R-Ras but not to GDP.R-Ras, GTP gamma S.R-Ras with a mutation in the effector domain (R-RasA64), GTP gamma S.Ha-Ras, or GTP gamma S.RalA. We obtained a cDNA encoding p98 on the basis of its partial amino acid sequences. The predicted protein consists of 834 amino acids whose calculated mass, 95,384 Da, is close to the apparent molecular mass of p98. The amino acid sequence shows a high degree of sequence similarity to the entire sequence of Gap1m, one of the GTPase-activating proteins (GAP) for Ha-Ras. A recombinant protein consisting of the GAP-related domain of p98 fused to maltose-binding protein stimulated GTPase activity of R-Ras, and showed a weak effect on that of Ha-Ras but not that of Rap1 or Rho. These results clearly indicate that p98 is a novel GAP for R-Ras. Thus, we designated this protein as R-Ras GAP.
...
PMID:A novel GTPase-activating protein for R-Ras. 853 Apr 88

The Escherichia coli transcription factor NusA and the bacteriophage lambda antiterminator Q proteins were expressed as inducible glutathione S-transferase (GST) fusion proteins. The fusion proteins were purified under nondenaturing conditions by affinity chromatography on glutathione agarose. Thrombin cleavage of the glutathione agarose-bound fusion proteins yielded homogeneously pure NusAN+15 (5 mg/g cells) and almost homogeneously pure QN+13 protein (0.7 mg/g cells), where N+x indicates the presence of x additional amino acids at the N-terminus of the protein. The purified NusAN+15 exhibited the same activities as wildtype NusA in enhancement of transcriptional pausing, enhancement of termination at Rho-independent terminators, and enhancement of Q-mediated antitermination in vitro. The QN+13 protein exhibited both anti-pausing and antitermination activities in Q-mediated transcription antitermination. However, the antitermination activity of QN+13 was lost gradually during storage if the thrombin used for cleavage of the GST fusion protein was not removed. This was due to cleavage by thrombin after Arg22 within the Q protein itself, at a noncanonical thrombin cleavage site, so the truncated protein (QN+22) lacked the first 22 amino acids at the N-terminus of Q. The expression vectors described here can be used to rapidly produce large quantities of these proteins, and the truncated Q protein can be used to evaluate the requirement for the N-terminus of Q in antitermination, anti-pausing, interactions with the DNA template (qut site), and interaction with RNA polymerase itself.
...
PMID:Expression and functional characterization of Escherichia coli NusA and lambda Q as glutathione S-transferase fusion proteins. 853 55

A protein serine/threonine kinase, p160(ROCK), has been identified as a putative Rho target protein that is activated when bound to the GTP-bound form of the small GTPase Rho (Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatu, A., Fujita, A., Watanabe, N. Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S. (1996) EMBO J. 15, 1885-1893). p160(ROCK) has a serine/threonine kinase domain in its NH2-terminal region, followed by an approximately 600-amino acid-long alpha-helix, a cysteine-rich zinc finger-like motif, and a pleckstrin homology region in the COOH terminus. To identify the Rho binding domain of this protein, we divided p160 into five fragments, expressed each as a His-tagged recombinant protein, and performed a ligand overlay assay using [35S]guanosine-5'-3-O-(thio)triphosphate (GTPgammaS)-bound glutathione S-transferase-RhoA. Specific GTPgammaS-Rho binding was observed only in the fragment M2, which covered most of the carboxyl half of the alpha-helix between amino acids 727 and 1021. This fragment was further subdivided into several fragments, and the ligand overlay assay as well as the yeast two hybrid system was carried out to identify the Rho-binding region. These studies localized the minimum Rho binding region to amino acids 934-1015. To identify critical amino acids for Rho binding, we analyzed the Rho binding activity of the subfragment with various point mutations. This analysis revealed that K934M, L941A, and E1008A mutations significantly weakened Rho binding and an I1009A mutation abolished Rho binding. The amino acid sequence in this region had no significant homology with Rho effector motif class 1, which is shared by putative Rho targets, PKN, rhophilin, and rhotekin, (Reid, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S. (1996) J. Biol. Chem. 271, 13556-13560) and may define a distinct class of Rho effector motif.
...
PMID:Identification of the Rho-binding domain of p160ROCK, a Rho-associated coiled-coil containing protein kinase. 879 90

A genetic screen for GTPase-activating proteins (GAPs) or other negative regulators of the Rac/Rho family GTPase Cdc42p in Saccharomyces cerevisiae identified ZDS1, a gene encoding a protein of 915 amino acids. Sequence from the yeast genome project identified a homolog, ZDS2, whose predicted product of 942 amino acids is 38% identical in sequence to Zds1p. Zds1p and Zds2p have no detectable homology to known Rho-GAPs or to other known proteins. However, by several assays, it appears that overexpression of either Zds1p or Zds2p decreases the level of Cdc42p activity. Deletion analysis also suggests that Zds1p and Zds2p are at least partially overlapping in function. Deletion of ZDS2 produced no obvious phenotype, and deletion of ZDS1 produced no obvious phenotype other than a mild effect on cell shape. However, the zds1 zds2 double mutant grew slowly with an apparent mitotic delay and produced elongated cells and buds with other evidence of abnormal morphogenesis. A glutathione S-transferase-Zds1p fusion protein that fully complemented the double mutant localized to presumptive bud sites and the tips of small buds. The similarity of this localization to that of Cdc42p suggests that Zds1p may interact directly with Cdc42p. As ZDS1 and ZDS2 have recently been identified also by numerous other groups studying a wide range of biological phenomena, the roles of Cdc42p in intracellular signaling may be more diverse than has previously been appreciated.
...
PMID:ZDS1 and ZDS2, genes whose products may regulate Cdc42p in Saccharomyces cerevisiae. 881 39


1 2 3 4 5 6 7 8 9 10 Next >>

---