Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.5.1.18 (glutathione S-transferase)
 22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Steroidogenic acute regulatory protein (StAR) is required for efficient adrenal cortical and gonadal but not trophoblast steroid hormone synthesis. StAR gene expression in gonadal cells is stimulated by tropic hormones acting through the intermediacy of cAMP. DNA sequence analysis of the human StAR gene promoter revealed two motifs resembling binding sites for steroidogenic factor 1 (SF-1), a member of the orphan nuclear receptor transcription factor family that controls expression of steroidogenic hydroxylases. The 5'-most sequence (distal site) is a consensus SF-1 binding site. The proximal site is a consensus estrogen receptor binding half-site. The StAR gene promoter is not active in BeWo choriocarcinoma cells, COS-1 cells, HeLa cells, or SK-OV-3 ovarian adenocarcinoma cells, all of which do not express significant levels of SF-1 mRNA. Introduction of SF-1 into these cells stimulated StAR promoter activity, particularly in response to cAMP. Two orphan nuclear transcription factors that bind to sequences similar to SF-1 sites, NGFI-B/Nur77 and RNR-1, did not support cAMP-stimulated StAR promoter activity in BeWo cells. Mutation of the distal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity in BeWo cells by 82% and 71%, respectively. Mutation of the proximal putative SF-1 binding site reduced basal and cAMP-stimulated promoter activity by 89% and 96%, respectively. Mutations in both sites reduced basal promoter activity to 7% of wild type promoter activity and cAMP-stimulated promoter activity to less than 5% of the wild type. Deletion analyses of promoter activity were consistent with the mutation studies. Electrophoretic mobility shift assays (EMSAs) demonstrated that the distal site binds to SF-1 expressed in COS-1 cells and to an SF-1-GST fusion protein with high affinity, but that the mutated distal sequence does not. An anti-SF-1 antibody ablated the characteristic SF-1-DNA complex with the distal sequence. The proximal site formed a number of protein-DNA complexes with COS-1 cell extracts, but appeared to have at best only very modest affinity for SF-1. Collectively, our findings demonstrate that SF-1 plays a key role in controlling the basal and cAMP-stimulated expression of the StAR gene. SF-1 can function at two distinct sites in the human StAR gene promoter, apparently by two different types of interaction, to control transcription.
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PMID:Steroidogenic factor 1-dependent promoter activity of the human steroidogenic acute regulatory protein (StAR) gene. 870 8

The PDE4A (type IV) cAMP-specific, rolipram-inhibited phosphodiesterase RPDE-6 (RNPDE4A5), when transiently expressed in COS7 cells, could be complexed with the v-Src-SH3 domain expressed as a glutathione S-transferase (GST) fusion protein. RPDE-6 did not interact with GST itself. This complex was not disrupted by treatment with high NaCl concentration together with Triton X-100. Interaction was apparently determined by the N-terminal splice region of RPDE-6, as the PDE4A splice variant RPDE-39, which differs from RPDE-6 at the extreme N-terminus, failed to associate with v-Src-SH3; met26RD1 (where RD1 is rat 'dunc-like' PDE), which has the N-terminal splice region deleted, failed to associate with v-Src-SH3, and the association of RPDE-6 and v-Src-SH3 was blocked by a fusion protein formed from the N-terminal splice region. RDPE-6 showed binding to GST fusion proteins of both the intact Src kinase and an SH2-SH3 construct but did not bind to the Src-SH2 domain or to the adaptor protein Grb-2. RPDE-6 could be co-immunoprecipitated from cytosol extracts of transfected cells by using anti-Src antiserum. RPDE-6 exhibited selectivity in binding to the SH3 domains of c-Abl, Crk, Csk, Lck, Lyn, Fyn and v-Src, with binding to the SH3 regions of the Src-related tyrosyl kinases Lyn and Fyn being the most effective. The binding of RPDE-6 to the SH3 domains of Crk, Csk and Lck led to a marked reduction in PDE activity, but no change was apparent in complexes with other species. Endogenous RPDE-6 from brain, but not endogenous RPDE-39 from testis, bound to the Src-SH3 domain. We suggest that the PDE4A splice variant RPDE-6 has a propensity for interaction with selective SH3 domains, in particular those from Src and the Src-related tyrosyl kinases Lyn and Fyn. This interaction seems to be governed by alternative splicing of the PDE4A gene, because RPDE-39, a splice variant that lacks the proline-rich N-terminal splice region of RPDE-6, does not interact with these SH3 domains. It is proposed that the binding site on RPDE-6 for SH3 domains lies within the unique first 102 residues of its N-terminal splice domain, where two motifs representing Class I SH3 binding sites with selectivity for Src kinase SH3 domains can be identified and one motif for a putative Class II SH3 binding site.
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PMID:The SH3 domain of Src tyrosyl protein kinase interacts with the N-terminal splice region of the PDE4A cAMP-specific phosphodiesterase RPDE-6 (RNPDE4A5). 876 80

We reported that ecto-NAD+ glycohydrolase activity induced upon differentiation of HL-60 cells with retinoic acid is localized on the extracellular domain of CD38 and that CD38 ligation by a specific monoclonal antibody, HB-7, is followed by rapid tyrosine phosphorylation of cellular proteins including a proto-oncogene product, Cbl. In the present study, we investigated intracellular signaling linked to the HB-7-induced Cbl phosphorylation in dibutyryl cAMP-treated THP-1 cells. The 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase was immunoprecipitated with anti-Cbl antibody in a manner dependent on the tyrosine phosphorylation of Cbl. PI 3-kinase activity was also observed in the immunoprecipitated fractions containing tyrosine-phosphorylated Cbl. The phosphorylated form of Cbl, which had been separated from the CD38-stimulated cells, was capable of directly binding to a recombinant p85 fused to glutathione S-transferase. Thus, the direct association of tyrosine-phosphorylated Cbl with PI 3-kinase, possibly leading to the kinase activation, appeared to be involved in intracellular signaling caused by the CD38 ligation.
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PMID:Association of phosphatidylinositol 3-kinase with the proto-oncogene product Cbl upon CD38 ligation by a specific monoclonal antibody in THP-1 cells. 894 25

Many fungal pathogens invade plants using specialized infection structures called appressoria that differentiate from the tips of fungal hyphae contacting the plant surface. We demonstrate a role for a MAP kinase that is essential for appressorium formation and infectious growth in Magnaporthe grisea, the fungal pathogen responsible for rice blast disease. The PMK1 gene of M. grisea is homologous to the Saccharomyces cerevisiae MAP kinases FUS3/KSS1, and a GST-Pmk1 fusion protein has kinase activity in vitro. pmk1 mutants of M. grisea fail to form appressoria and fail to grow invasively in rice plants. pmk1 mutants are still responsive to cAMP for early stages of appressorium formation, which suggests Pmk1 acts downstream of a cAMP signal for infection structure formation. PMK1 is nonessential for vegetative growth and sexual and asexual reproduction in culture. Surprisingly, when expressed behind the GAL1 promoter in yeast, PMK1 can rescue the mating defect in a fus3 kss1 double mutant. These results demonstrate that PMK1 is part of a highly conserved MAP kinase signal transduction pathway that acts cooperatively with a cAMP signaling pathway for fungal pathogenesis.
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PMID:MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. 894 11

A potential role for cAMP in regulating the differentiation of myoblasts has led us to examine the components of the cAMP signaling system, including the type IV, cAMP-specific phosphodiesterases. The full coding sequence of the phosphodiesterase PDE4D1 was inserted in the bacterial expression vector pGEX-KG. N- and C-terminal truncations were also placed in the same vector, allowing the expression and purification of glutathione S-transferase (GST)-PDE fusion proteins using glutathione-Sepharose. The purified PDE was active [V(max) = 318 +/- 18 nmol min(-1)(mg of protein)(-1)] and inhibited by RO 20-1724, rolipram, and MIX (IC50 values of 2, 0.4, and 40 microM, respectively). The requirement of PDE4D1 for a divalent cation was also examined. It was able to use Mg2+, Co2+, and Mn2+, but not Zn2+, suggesting that it is not a zinc hydrolase as has been proposed for other PDE types. Deletion of both C- and N-terminal regions affected the apparent native size of the enzyme. The C-terminal region was involved in dimer formation, whereas an N-terminal region was responsible for larger aggregates. Removal of the last 35 amino acids of an N-terminal 80-residue highly conserved region (UCR2) resulted in a 6-fold increase in PDE activity, providing evidence that this part of the molecule acts as an intramolecular inhibitor. The availability of a highly purified, enzymatically active protein in substantial quantities has allowed us to directly examine PDE4D1 for the first time.
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PMID:Recombinant expression of a type IV, cAMP-specific phosphodiesterase: characterization and structure-function studies of deletion mutants. 906 27

Protein phosphorylation is one of several representative post-translational modifications. Cyclic AMP-dependent protein kinase (PKA) plays the crucial and varying role of signal transduction. On the other hand, ras proteins plays an important role in cell proliferation and growth. Although a previous report showed that H-ras protein was phosphorylated by PKA, the stoichiometry was not determined, so we investigated the stoichiometry of phosphorylation of the protein by PKA. H-ras cDNA inserted into a pGEX-2T expressing vector produced high levels of recombinant H-ras (rH-ras) in a fusion protein with glutathione S-transferase. rH-ras was obtained after cleavage by thrombin. Phosphorylation of ras protein by the catalytic subunit of PKA was performed, and the radioactivity was counted after SDS-PAGE and autoradiography. The results indicate that less than 0.1 mol of phosphate was incorporated per mol of H-ras protein, and suggest that H-ras protein could not be a physiologically meaningful substrate for PKA.
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PMID:Phosphorylation of H-ras proteins by protein kinase A. 906 27

All eukaryotic protein kinases share a conserved catalytic core. In the catalytic (C) subunit of cAMP-dependent protein kinase (cAPK) this core is preceded by a myristylation motif followed by a long helix with Trp 30 at the end of this A-helix filling a hydrophobic cavity between the two lobes of the core. To understand the importance of the A-helix, the myristylation motif (delta 1-14) as well as the entire N-terminal segment (delta 1 -39) were deleted. In addition, Trp 30 was replaced with both Tyr and Ala. All proteins were overexpressed in E. coli and purified to homogeneity. rC(delta 1-14), rC(W30Y), and rC(W30A) all had reduced thermostability, but were catalytically indistinguishable from wild-type C. Based on Surface Plasmon Resonance, all three also formed stable holoenzyme complexes with the RI-subunit, although the appKds were reduced by more than 10-fold due to decrease in the association rate. Surprisingly, however, the holoenzymes were even more thermostable than wild-type holoenzyme. To obtain active enzyme, it was necessary to purify rC(delta 1-39) as a fusion protein with glutathione-S-transferase (GST-rC(delta 1-39), although its thermostability (Tm) was decreased by 12.5 degrees C, was catalytically similar to wild-type C and was inhibited by both the type I and II R-subunits and the heat-stable protein kinase inhibitor (PKI). The Tm for holoenzyme II formed with GST-rC(delta 1-39) was 16.5 degrees C greater than the Tm for free GST-rC(delta 1-39), and the Ka(cAMP) was increased nearly 10-fold. These mutants point out striking and unanticipated differences in how the RI and RII subunits associate with the C-subunit to form a stable holoenzyme and indicate, furthermore, that this N-terminal segment, far from the active site cleft, influences those interactions. The importance of the A-helix and Trp 30 for stability correlates with its location at the cleft interface where it orients the C-helix in the small lobe and the activation loop in the large so that these subdomains are aligned in a way that allows for correct configuration of residues at the active site. This extensive network of contacts that links the A-helix directly to the active site in cAPK is compared to other kinases whose crystal structures have been solved.
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PMID:Importance of the A-helix of the catalytic subunit of cAMP-dependent protein kinase for stability and for orienting subdomains at the cleft interface. 907 Apr 39

Activation of protein kinase B (PKB) by growth factors has been demonstrated to proceed via phosphatidylinositol 3-kinase (PI3-kinase). Here, we show that agents which raise intracellular cAMP can also stimulate PKB. However, this effect is not sensitive to wortmannin, indicating that it is PI3-kinase independent. This activation does not appear to result from direct phosphorylation by protein kinase A (PKA) since GST-PKB is not an effective PKA substrate. In addition, the activation pathway of PKB by cAMP seems to be linked to that of growth factors, albeit downstream of PI3-kinase. Evidence for this is that a constitutive active PKB, T308D, S473D, containing activating mutations in the serine and threonine residues which are phosphorylated subsequent to PI3-kinase activation, cannot be further stimulated by cAMP elevations. Hence, these data suggest that, in addition to growth factors, cAMP can also lead to activation of PKB. This cAMP stimulatory action appears to require phosphorylation of T308 and S473, and hence would indicate that cAMP modulates the phosphorylation event of these PKB regulatory sites.
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PMID:cAMP stimulates protein kinase B in a Wortmannin-insensitive manner. 920 56

Vibrio cholerae O139 has pandemic potential and it produces copious amounts of fluid secretion. The levels of various second messengers (intracellular Ca2+, cAMP, IP3, PKC) were measured to determine the cause of fluid secretion produced by this strain of V. cholerae. There was a significant increase in the levels of these second messengers in V. cholerae O139 treated ileum as compared to control ileum (enterocytes). Levels of these second messengers were also assessed in V. cholerae 569B induced fluid secretion in rabbit ileum and it was found that the levels were raised more in V. cholerae O139 treated ileum than in V. cholerae 569B treated rabbit ileum. The intestinal damage was assessed by measuring changes in the extent of lipid peroxidation of the enterocytes. Intracellular second messengers are known to raise the extent of lipid peroxidation. In V. cholerae O139 treated loops calcium ionophore A23187 enhanced the extent of lipid peroxidation whereas l-verapamil could only marginally decrease the lipid peroxidation. Dantrolene and H7 significantly decreased the extent of lipid peroxidation of enterocytes in V. cholerae O139 treated rabbit ileum. However, PMA could not enhance further the extent of lipid peroxidation in V. cholerae O139 treated rabbit ileum. So intracellular calcium and protein kinase C appear to be involved in intestinal damage caused by V. cholerae O139. Reactive oxygen species are responsible for causing tissue damage and the extent of oxidative damage depends on the balance between the pro-oxidants and the anti-oxidants. So the changes in the enterocytes' antioxidant level during V. cholerae O139 mediated intestinal infection was estimated. There was a significant decrease in the enterocyte level of the antioxidant enzymes SOD, catalase, glutathione peroxidase, glutathione reductase, glutathione transferase and glucose-6-phosphate dehydrogenase in V. cholerae O139 mediated intestinal infection. So a significant decrease in the levels of antioxidant defenses and a significant increase in the levels of second messengers appear to be important in mediating V. cholerae O139 induced lipid peroxidation which contributes to the changes in membrane permeability and thus to fluid secretion.
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PMID:Role of intracellular second messengers and reactive oxygen species in the pathophysiology of V. cholera O139 treated rabbit ileum. 963 66

There have been speculations that the regulatory (R) subunit of the cAMP-dependent protein kinase (PKA) may have other functions. A recent study has shown that the catalytic (C) subunit of PKA may be regulated in a cAMP- and R subunit-independent manner. However, evidence linking a function to the R subunit apart from inhibiting the C subunit has been elusive. In this report, interaction cloning experiments showed that the RIalpha subunit association with the cytochrome c oxidase subunit Vb (CoxVb) is cAMP-sensitive. Interaction was detected with a GST-RIalpha fusion protein as well as by coimmunoprecipitation. Transient treatment with cAMP-elevating agents inhibited cytochrome c oxidase in Chinese hamster ovary (CHO) cells with a concomitant decrease in cytochrome c levels in the mitochondria and an increase in its release into the cytosol. Furthermore, mutant cells harboring a defective RIalpha show increased cytochrome c oxidase activity and also constitutively lower levels of cytochrome c in comparison to either the wild-type cells or the C subunit mutant. These results suggest a novel mechanism of cAMP signaling through the interaction of RIalpha with CoxVb thereby regulating cytochrome c oxidase activity as well as the cytochrome c levels.
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PMID:Novel function of the regulatory subunit of protein kinase A: regulation of cytochrome c oxidase activity and cytochrome c release. 976 Feb 54


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