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)

Recent studies from our laboratory have shown that insulin induces relaxation of vascular smooth muscle cells (VSMCs) via stimulation of myosin phosphatase and inhibition of Rho kinase activity. In this study, we examined the mechanism whereby insulin inhibits Rho signaling and its impact on actin cytoskeleton organization. Incubation of confluent serum-starved VSMCs with thrombin or phenylephrine (PE) caused a rapid increase in glutathione S-transferase-Rhotekin-Rho binding domain-associated RhoA, Rho kinase activation, and actin cytoskeleton organization, which was blocked by preincubation with insulin. Preexposure to N(G)-monomethyl L-arginine acetate (L-NMMA), a nitric oxide synthase inhibitor, and Rp-8 CPT-cyclic guanosine monophosphate (RpcGMP), a cyclic guanosine monophosphate (cGMP) antagonist, attenuated the inhibitory effect of insulin on RhoA activation and restored thrombin-induced Rho kinase activation, and site-specific phosphorylation of the myosin-bound regulatory subunit (MBS(Thr695)) of myosin-bound phosphatase (MBP), and caused actin fiber reorganization. In contrast, 8-bromo-cGMP, a cGMP agonist, mimicked the inhibitory effects of insulin and abolished thrombin-mediated Rho activation. Insulin inactivation of RhoA was accompanied by inhibition of isoprenylation via reductions in geranylgeranyl transferase-1 activity as well as increased RhoA phosphorylation, which was reversed by pretreatment with RpcGMP and L-NMMA. We conclude that insulin may inhibit Rho signaling by affecting posttranslational modification of RhoA via nitric oxide/cGMP signaling pathway to cause MBP activation, actin cytoskeletal disorganization, and vasodilation.
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PMID:Negative regulation of rho signaling by insulin and its impact on actin cytoskeleton organization in vascular smooth muscle cells: role of nitric oxide and cyclic guanosine monophosphate signaling pathways. 1208 58

The male-germ-cell Rac GTPase-activating protein gene (MgcRacGAP) was initially described as a human RhoGAP gene highly expressed in male germ cells at spermatocyte stage, but exhibits significant levels of expression in most cell types. In somatic cells, MgcRacGAP protein was found to both concentrate in the midzone/midbody and be required for cytokinesis. As a RhoGAP, MgcRacGAP has been proposed to down-regulate RhoA, which is localized to the cleavage furrow and midbody during cytokinesis. Due to embryonic lethality in MgcRacGAP -null mutant mice and to the lack of an in vitro model of spermatogenesis, nothing is known regarding the role and mode of action of MgcRacGAP in male germ cells. We have analysed the expression, subcellular localization and molecular interactions of MgcRacGAP in male germ cells. Whereas MgcRacGAP was found only in spermatocytes and early spermatids, the widespread RhoGTPases RhoA, Rac1 and Cdc42 (which are, to various extents, in vitro substrates for MgcRacGAP activity) were, surprisingly, not detected at these stages. In contrast, Rnd2, a Rho family GTPase-deficient G-protein was found to be co-expressed with MgcRacGAP in spermatocytes and spermatids. MgcRacGAP was detected in the midzone of meiotic cells, but also, unexpectedly, in the Golgi-derived pro-acrosomal vesicle, co-localizing with Rnd2. In addition, a stable Rnd2-MgcRacGAP molecular complex could be evidenced by glutathione S-transferase pull-down and co-immunoprecipitation experiments. We conclude that Rnd2 is a probable physiological partner of MgcRacGAP in male germ cells and we propose that MgcRacGAP, and, quite possibly, other RhoGAPs, may participate in signalling pathways involving Rnd family proteins.
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PMID:Rho family GTPase Rnd2 interacts and co-localizes with MgcRacGAP in male germ cells. 1259 Jun 51

The Galpha subunits of the G(12) family of heterotrimeric G proteins, defined by Galpha(12) and Galpha(13), have many cellular functions in common, such as stress fiber formation and neurite retraction. However, a variety of G protein-coupled receptors appear to couple selectively to Galpha(12) and Galpha(13). For example, thrombin and lysophosphatidic acid (LPA) have been shown to induce stress fiber formation via Galpha(12) and Galpha(13), respectively. We recently showed that active forms of Galpha(12) and Galpha(13) interact with Ser/Thr phosphatase type 5 through its tetratricopeptide repeat domain. Here we developed a novel assay to measure the activities of Galpha(12) and Galpha(13) by using glutathione S-transferase-fused tetratricopeptide repeat domain of Ser/Thr phosphatase type 5, taking advantage of the property that tetratricopeptide repeat domain strongly interacts with active forms of Galpha(12) and Galpha(13). By using this assay, we identified that thrombin and LPA selectively activate Galpha(12) and Galpha(13), respectively. Galpha(12) and Galpha(13) show a high amino acid sequence homology except for their N-terminal short sequences. Then we generated chimeric G proteins Galpha(12N/13C) and Galpha(13N/12C), in which the N-terminal short sequences are replaced by each other, and showed that thrombin and LPA selectively activate Galpha(12N/13C) and Galpha(13N/12C), respectively. Moreover, thrombin and LPA stimulate RhoA activity through Galpha(12) and Galpha(13), respectively, in a Galpha(12) family N-terminal sequence-dependent manner. Thus, N-terminal short sequences of the G(12) family determine the selective couplings of thrombin and LPA receptors to the Galpha(12) family.
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PMID:N-terminal short sequences of alpha subunits of the G12 family determine selective coupling to receptors. 1259 20

Rho GTPases are critical for actin cytoskeletal regulation, and alterations in their activity may contribute to altered cytoskeletal organization that characterizes many pathological conditions, including ischemia. G protein activity is a function of the ratio of GTP-bound (active) to GDP-bound (inactive) protein, but the effect of altered energy metabolism on Rho protein activity has not been determined. We used antimycin A and substrate depletion to induce depletion of intracellular ATP and GTP in the kidney proximal tubule cell line LLC-PK10 and measured the activity of RhoA, Rac1, and Cdc42 with GTPase effector binding domains fused to glutathione S-transferase. RhoA activity decreased in parallel with the concentration of ATP and GTP during depletion, so that by 60 min there was no detectable RhoA-GTP, and recovered rapidly when cells were returned to normal culture conditions. Dissociation of the membrane-actin linker ezrin, a target of RhoA signaling, from the cytoskeletal fraction paralleled the decrease in RhoA activity and was augmented by treatment with the Rho kinase inhibitor Y27632. The activity of Cdc42 did not decrease significantly during depletion or recovery. Rac1 activity decreased moderately to a minimum at 30 min of depletion but then increased from 30 to 90 min of depletion, even as ATP and GTP levels continued to fall. Our data are consistent with a principal role for RhoA in cytoskeletal reorganization during ischemia and demonstrate that the activity of Rho GTPases can be maintained even at low GTP concentrations.
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PMID:Rho GTPases show differential sensitivity to nucleotide triphosphate depletion in a model of ischemic cell injury. 1262 Aug 11

A yeast two-hybrid screen was utilized to identify novel Smad 3 binding proteins expressed in developing mouse orofacial tissue. Three proteins (Erbin, Par-3, and Dishevelled) were identified that share several similar structural and functional characteristics. Each contains at least one PDZ domain and all have been demonstrated to play a role in the establishment and maintenance of cell polarity. In GST (glutathione S-transferase) pull-down assays, Erbin, Par-3, and Dishevelled bound strongly to the isolated MH2 domain of Smad 3, with weaker binding to a full-length Smad 3 protein. Failure of Erbin, Par-3, and Dishevelled to bind to a Smad 3 mutant protein that was missing the MH2 domain confirms that the binding site resides within the MH2 domain. Erbin, Par-3, and Dishevelled also interacted with the MH2 domains of other Smads, suggesting broad Smad binding specificity. Dishevelled and Erbin mutant proteins, in which the PDZ domain was removed, still retained their ability to bind Smad 3, albeit with lower affinity. While transforming growth factor beta (TGFbeta) has been suggested to alter cell polarity through a Smad-independent mechanism involving activation of members of the RhoA family of GTP binding proteins, the observation that Smads can directly interact with proteins involved in cell polarity, as shown in the present report, suggests an additional means by which TGFbeta could alter cell polarity via a Smad-dependent signaling mechanism.
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PMID:Identification of three novel Smad binding proteins involved in cell polarity. 1265 Sep 46

Recently, it was shown that Yersinia outer protein T (YopT) belongs to a new family of cysteine proteases containing invariant C, H, and D residues that are crucial for its activity. YopT cleaves RhoA, Rac, and Cdc42 at their C termini, thereby releasing them from the membrane. Moreover, YopT inhibits the Rho-rhotekin and Rho-guanine nucleotide dissociation inhibitor interactions. To characterize the active domain of YopT, we constructed N- and C-terminal truncations and expressed them as glutathione S-transferase fusion proteins in Escherichia coli. The toxin fragments were tested for stability by trypsin digestion. The activity of the proteins was studied by membrane release assay, rhotekin pulldown experiments, and microinjection. Whereas deletion of the first 74 N-terminal amino acids did not influence the activity of YopT, deletion of 8 amino acids from the C terminus led to complete loss of activity. N-terminal deletion of 100 amino acids led to an inactive protein, although it still contained the amino acids C139, H258, and D274, which are essential for catalysis. Loss of activity of the N-terminal deletions corresponded to the block of interaction with RhoA, indicating that residues 75 to 100 of YopT are essential for binding to the GTPase. By contrast, when up to 15 amino acids of the C terminus were deleted, the protein had no activity but was still able to interact with RhoA, suggesting a role for the C terminus in the enzyme activity of YopT.
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PMID:The C terminus of YopT is crucial for activity and the N terminus is crucial for substrate binding. 1287 42

Unique among the phospholipase C isozymes, the recently identified phospholipase C-epsilon (PLC-epsilon) contains an amino-terminal CDC25 domain capable of catalyzing nucleotide exchange on Ras family GTPases as well as a tandem array of Ras-associating (RA) domains near its carboxyl terminus that are effector binding sites for activated H-Ras and Rap. To determine whether other small GTPases activate PLC-epsilon, we measured inositol phosphate accumulation in COS-7 cells expressing a broad range of GTPase-deficient mutants of Ras superfamily proteins. RhoA, RhoB, and RhoC all markedly stimulated inositol phosphate accumulation in PLC-epsilon-expressing cells. This stimulation matched or exceeded phospholipase activation promoted by co-expression of PLC-epsilon with the known regulators Ras, Galpha12/13, or Gbeta1gamma2. In contrast, little effect was observed with the other Rho family members Rac1, Rac2, Rac3, and Cdc42. Truncation of the two carboxyl-terminal RA domains caused loss of responsiveness to H-Ras but not to Rho. Truncation of PLC-epsilon to remove the CDC25 and pleckstrin homology (PH) domains also did not cause loss of responsiveness to Rho, Galpha12/13, or Gbeta1gamma2. Comparative sequence analysis of mammalian phospholipase C isozymes revealed a unique approximately 65 amino acid insert within the catalytic core of PLC-epsilon not present in PLC-beta, gamma, delta, or zeta. A PLC-epsilon construct lacking this region was no longer activated by Rho or Galpha12/13 but retained regulation by Gbetagamma and H-Ras. GTP-dependent interaction of Rho with PLC-epsilon was illustrated in pull-down experiments with GST-Rho, and this interaction was retained in the PLC-epsilon construct lacking the unique insert within the catalytic core. These results are consistent with the conclusion that Rho family GTPases directly interact with PLC-epsilon by a mechanism independent of the CDC25 or RA domains. A unique insert within the catalytic core of PLC-epsilon imparts responsiveness to Rho, which may signal downstream of Galpha12/13 in the regulation of PLC-epsilon, because activation by both Rho and Galpha12/13 is lost in the absence of this sequence.
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PMID:Direct activation of phospholipase C-epsilon by Rho. 1290 Apr 2

Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis. Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation. The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546). The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation. Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol. Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells. The toxin fragment (approximately 65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B. The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells. After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells. Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol. Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane. The data indicate that toxin B is proteolytically processed during its cellular uptake process.
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PMID:Cellular uptake of Clostridium difficile toxin B. Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells. 1294 36

Reorganization of the cytoskeleton and association of contractile proteins are important steps in modulating smooth muscle contraction. Heat shock protein (HSP) 27 has significant effects on actin cytoskeletal reorganization during smooth muscle contraction. We investigated the role of phosphorylated HSP27 in modulating acetylcholine-induced sustained contraction of smooth muscle cells from the rabbit colon by transfecting smooth muscle cells with phosphomimic (3D) or nonphosphomimic (3G) HSP27. In 3G cells, the initial peak contractile response at 30 s was inhibited by 25% (24.0 +/- 4.5% decrease in cell length, n = 4). The sustained contraction was greatly inhibited by 75% [9.3 +/-.9% decreases in cell length (n = 4)]. Furthermore, in 3D cells, translocation of both PKCalpha and of RhoA was greatly enhanced and resulted in a greater association of PKCalpha-RhoA in the membrane fraction. In 3G transfected cells, PKCalpha and RhoA failed to translocate in response to stimulation with acetylcholine, resulting in an inhibition of association of PKCalpha-RhoA in the membrane fraction. Studies using GST-RhoA fusion protein indicate that there is a direct association of RhoA with PKCalpha and with HSP27. The results suggest that phosphorylated HSP27 plays a crucial role in the maintenance of association of PKCalpha-RhoA in the membrane fraction and in the maintenance of acetylcholine-induced sustained contraction.
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PMID:Phosphorylated HSP27 essential for acetylcholine-induced association of RhoA with PKCalpha. 1459 45

We have characterized the cDNA for a Rho GTPase activating protein (GAP) mapping to chromosome 13q12. The cDNA was characterized by determining the complete sequence of a 4.8 kb cDNA clone that represents the 5' untranslated region (UTR), the translated region, and the 3' UTR. The protein has a sterile alpha-motif (SAM), a distinct GAP domain, and a conserved START (StAR related lipid transfer) domain. The cDNA has 5 instability motifs (ATTTA) in the 3' UTR and one motif in the translated region between GAP and START domains. The RhoGAP transcript is truncated in some breast carcinoma cell lines and it has low expression in other breast cancer cell lines as compared to a normal breast cell line. We have previously observed the absence of RhoGAP transcript in a breast tumor specimen. A GST-fusion of the RhoGAP was tested for its specificity on RhoA, Cdc42, and Rac1. The protein was most active for RhoA. Transfection of RhoGAP into MCF7 cells significantly inhibited cell growth. The introduction of the RhoGAP construct into MDAMB231 cells that had previously been transfected with a p21 construct did not affect cell proliferation, indicating the involvement of p21 in Rho-mediated proliferation of cancer cells. NIH3T3 cells overexpressing RhoGAP showed considerable inhibition of stress fiber formation. Several cDNAs were identified as RhoGAP interactors by using the yeast two-hybrid assay system. These cDNAs correspond to SWI/SNF, alpha-tubulin, HMG CoA reductase, and TAX1 binding protein (TAX1BP1). The interaction with HMG CoA reductase may partially explain the growth inhibition of breast carcinoma cells by statin class of cholesterol lowering drugs. The biological significance of the interacting proteins is discussed in the context of their involvement in tumorigenesis. Our results indicate that loss of RhoGAP or its altered activity suppresses the growth of breast tumor cells. The presence of various motifs in RhoGAP and its interaction with several other proteins suggest that the protein may regulate Rho signaling in multiple ways and possibly function in a Rho-independent manner.
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PMID:Chromosome 13q12 encoded Rho GTPase activating protein suppresses growth of breast carcinoma cells, and yeast two-hybrid screen shows its interaction with several proteins. 1498 79


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