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 carboxyl-terminal domain of the zeta 1 subunit of the mouse NMDA receptor channel produced as a fusion protein with GST was phosphorylated in vitro by PKC. A mutant of the zeta 1 subunit without serine or threonine residues in the carboxyl-terminal domain (zeta 1-2-NST) was constructed and was expressed alone or together with the epsilon 2 subunit in Xenopus oocytes. Current responses of the zeta 1-2-NST homomeric and epsilon 2/zeta 1-2-NST heteromeric NMDA receptor channels were enhanced by treatment with TPA, a PKC activator, and the extents of potentiation were comparable with the corresponding wild-type channels. These results suggest that the phosphorylation of the carboxyl-terminal domain of the zeta 1 subunit is not responsible for potentiation of NMDA receptor channels by the TPA treatment.
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PMID:Phosphorylation of the carboxyl-terminal domain of the zeta 1 subunit is not responsible for potentiation by TPA of the NMDA receptor channel. 750 80

Antigenic cross-linking of the high affinity IgE receptor (Fc epsilon R1) on mast cells results in protein tyrosine kinase activation. The object of the present study was to explore the regulation of the SH2 and SH3 domain containing adapter molecule Grb2 by Fc epsilon R1-stimulated PTK signal transduction pathways. Affinity purification of in vivo Grb2 complexes together with in vitro experiments with Grb2 glutathione S-transferase fusion proteins were used to analyze Grb2 complexes in the mast cell line RBL2H3. The data show that in RBL2H3 cells several different proteins are complexed to the SH3 domains of Grb2. These include the p21ras guanine nucleotide exchange factor Sos, two basally tyrosine-phosphorylated 110- and 120-kDa molecules, and a 75-kDa protein that is a substrate for Fc epsilon R1-activated PTKs. By analogy with Sos, p75, p110 and p120 are candidates for Grb2 effector proteins which suggests that Grb2 may be a pleiotropic adapter. Two Grb2 SH2-binding proteins were also characterized in RBL2H3 cells; the adapter Shc and a 33-kDa molecule. Shc is constitutively tyrosine phosphorylated in unstimulated cells and Fc epsilon R1 ligation induces no changes in its phosphorylation or binding to Grb2. In contrast, p33 is a substrate for Fc epsilon R1-activated PTKs and binds to Grb2 SH2 domains in Fc epsilon R1 activated but not quiescent cells. The beta subunit of the Fc epsilon R1 is a 33-kDa tyrosine phosphoprotein, but the p33 Grb2-binding protein described in the present report is not the Fc epsilon R1 beta chain and its identity is unknown. The present report thus demonstrates that there are multiple Grb2 containing protein complexes in mast cells of which a subset are Fc epsilon R1-regulated. Two other of the Grb2-binding proteins described herein are tyrosine phosphorylated in response to Fc epsilon R1 ligation: the 75-kDa protein which binds to Grb2 SH3 domains and the 33-kDa protein that associates with the Grb2 SH2 domain. We propose that protein complex formation by Grb2 is an important consequence of Fc epsilon R1 cross-linking and that this may be a signal transduction pathway which acts synergistically with calcium/PKC signals to bring about optimal mast cell end function.
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PMID:Regulation of the adapter molecule Grb2 by the Fc epsilon R1 in the mast cell line RBL2H3. 772 78

The regulatory domain of protein kinase C gamma (PKC gamma) contains the following functional elements which can interact with lipids: the pseudosubstrate motif within the first variable region (V1), cysteine-rich domains, Cys1 and Cys2 which contain zinc and bind phorbol dibutyrate (PDBu)/diacylglycerol, and the calcium-dependent lipid binding domain (CaLB). The function of individual or combined segments of the regulatory domain was investigated, using glutathione S-transferase (GST) fusion proteins and mixed micellar or liposomal assays. GST-Cys1 and GST-Cys2 bound PDBu with comparable affinity (Kd = 14-17 nM). GST-Cys1Cys2 yielded a protein with a PDBu binding affinity of 3.4 nM, in the presence of calcium, similar to that of intact PKC gamma (Kd = 2.6 nM). The phosphatidylserine (PS) dependence of PDBu binding was highly cooperative for all fusion proteins tested with Hill numbers (n) lying in the range of 3.5-4.8, similar to values obtained for intact PKC gamma. While Hill numbers were similar under all conditions, the PS concentration necessary for half-maximal PDBu binding was dependent upon the nature and presence of divalent cations. The PS requirement was lowest in the presence of calcium for GST-Cys1, GST-Cys2, and GST-Cys1Cys2 (Km for PS = 11, 14, and 12 mol %, respectively) but still significantly above the value for intact PKC gamma (5.4 mol %). The data establish Cys1 and Cys2 as independent PDBu binding domains that are modulated by divalent cations. While PDBu binding affinity to a GST-V1Cys1 fusion protein (Kd = 36 nM) was comparable to that of GST-Cys1, the CaLB domain dramatically reduced PDBu binding affinity of GST-Cys2CaLB (Kd = 912 nM). This effect of the CaLB domain on PDBu binding to Cys2 suggests that PDBu/diacylglycerol binding to native PKC gamma may occur at Cys1 and that the Cys2 domain may serve another regulatory function.
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PMID:The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations. 805 Oct 84

This study demonstrates that the isolated regulatory (R) domain (amino acids 1-270) of human protein kinase C alpha (PKC alpha) is a potent inhibitor of PKC beta-I activity in a yeast expression system. The PKC alpha R domain fused to glutathione-S-transferase competitively inhibited the activity of yeast-expressed rat PKC beta-I in vitro (Ki = 0.2 microns) and was 400-fold more potent than a synthetic pseudosubstrate peptide corresponding to amino acids 19-36 from PKC alpha. In contrast, the fusion protein did not affect the activity of the purified catalytic subunit of cAMP-dependent protein kinase. The PKC alpha R domain (without glutathione-S-transferase [GST]) also was tested for its ability to inhibit PKC beta-I activity in vivo, in a yeast strain expressing rat PKC beta-I. Upon treatment with a PKC-activating phorbol ester, yeast cells expressing rat PKC beta-I were growth-inhibited and a fraction of the cells appeared as long chains. Coexpression of the R domain with rat PKC beta-I blocked the phorbol ester-induced inhibition of yeast cell growth and the phorbol ester-dependent alterations in yeast cell morphology. These results indicate that the R domain of PKC alpha acts as a dominant inhibitor of PKC activity in vivo and thus provides a useful genetic tool to assess the roles of PKC in various signal transduction processes.
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PMID:Regulatory domain of human protein kinase C alpha dominantly inhibits protein kinase C beta-I-regulated growth and morphology in Saccharomyces cerevisiae. 860 Jan 65

The regulatory (R) domain of PKC alpha fused to glutathione-S-transferase (GST-R alpha) competitively inhibited PKC activity associated with extracts of Y1 mouse adrenocortical tumor cells and the activities of several specific PKC isozymes. GST-R alpha did not inhibit the activities of cAMP-dependent protein kinase, cGMP-dependent protein kinase or calmodulin-dependent myosin light chain kinase. GST-R alpha inhibited PKC activities 20 times more potently than did a synthetic peptide corresponding to the pseudosubstrate sequence of PKC alpha. In intact yeast cells, the R domain prevented PKC beta-1-induced inhibition of growth and cytokinesis. These results indicate that the R domain of PKC alpha acts as a specific, dominant inhibitor of PKC activity, and suggest that the PKC alpha R domain may provide a useful genetic tool to assess the roles of PKC in various signal transduction processes.
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PMID:Molecular strategies for the dominant inhibition of protein kinase C. 896 21

In this investigation, untreated non-B-type acute lymphoblastic leukemia (ALL) of 104 children was analyzed using immunocytochemistry for expression of protein kinase C, proto-oncogene products (Fos, Jun, Ras) and resistance-related proteins (topoisomerase II, P-glycoprotein, glutathione S-transferase-pi, metallothionein, dihydrofolate-reductase, thymidylate-synthase). The aim of the analysis was to find out whether combining those factors with the most important clinical prognostic factor (blast cell count) can improve the prognostic value (relapse-free interval). Univariate analysis shows that protein kinase D (PKC), Fos, P-glycoprotein (P-170) and glutathione S-transferase-pi (GST-pi) are significant prognostic factors independent of blast cell count (PBC) for the relapse-free intervals of children with ALL. The presence of the proteins Fos, PKC, P-170 and GST-pi was not independent within the patient population. The multivariate analysis showed that in combination with PBC and PKC, both P-170 and GST-pi have only limited prognostic influence. Combining the factors PKC, Fos and GST-pi as a categorical variable showed that this variable is a strong prognostic factor in addition to PBC.
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PMID:Prognostic value of protein kinase C, proto-oncogene products and resistance-related proteins in newly diagnosed childhood acute lymphoblastic leukemia. 898 47

Five in vitro assays have been applied to screen the efficacy of potential chemopreventive agents. These assays measure a) inhibition of morphological transformation in rat tracheal epithelial (RTE) cells, b) inhibition of anchorage independence in human lung tumor (A427) cells, c) inhibition of hyperplastic alveolar nodule formation in mouse mammary organ cultures (MMOC), d) inhibition of anchorage independence in mouse JB6 epidermal cells, and e) the inhibition of calcium tolerance in human foreskin epithelial cells. The efficacy of many of these same agents in whole animal studies of lung, colon, mammary gland, skin, and urinary bladder carcinogenesis has also been measured. The aim herein is to estimate the positive and negative predictive values of these in vitro assays against whole animal chemopreventive efficacy data using the same chemicals. For three of these assays--using RTE, A427 cells and mouse mammary organ culture (MMOC)-enough data are available to allow the estimate to be made. Such extrapolations of in vitro data to the in vivo situation are difficult at best. There are many dissimilarities between the two assay systems. The in vitro assays use respiratory and mammary epithelial cells, while the in vivo assays use respiratory, mammary, colon, bladder and skin cells. The in vitro assays use the carcinogens benzo(a)pyrene (B(a)P) and 7,12-dimethylbenz(a)anthracene (DMBA), while the in vivo assays use B(a)P, DMBA, N-methyl-N-nitrosourea (MNU), N,N'-diethylnitrosamine (DEN), azoxymethane (AOM), and N-butyl-N-(4-hydroxybutyl)nitrosoamine (OH-BBN). There are vast differences in pharmacodynamics and pharmacokinetics in vitro and in vivo, yet it is possible to rapidly screen chemicals in vitro for efficacy at one-tenth the cost and complete tests in weeks instead of months. A positive in vitro assay was defined as a 20% inhibition (compared with control) for the RTE and A427 assays and a 60% inhibition for the MMOC assay at nontoxic concentrations. For in vivo assays, the criterion for a positive result was a statistically significant inhibition of incidence, multiplicity or a significant increase in latency (mean time to first tumor). For an agent to be considered negative in animals, it required negative results in at least two different organ systems and no positive results. Using the battery of three in vitro tests, the positive predictive value for having one, two, or three positive in vitro assays and at least one positive whole animal test was 76%, 80%, and 83% respectively. The negative predictive values for one, two or all three in vitro assays was 25%, 27%, and 50%. From these data it is observed that in vitro assays give valuable positive predictive values and less valuable negative predictive values. The mechanisms of chemoprevention are not well understood. Seven categories of agents were examined for their cancer preventing both in vitro and in vivo: antiinflammatories, antioxidants, arachadonic acid metabolism inhibitors, GSH inducers, GST inducers, ODC inhibitors, and PKC inhibitors. Three or even five in vitro assays cannot be all-inclusive of the many mechanisms of cancer prevention. However, three assays help to predict whole animal efficacy with reasonable positive predictive values. Much work and development remains to be done to rapidly identify new chemopreventive drugs.
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PMID:Use of in vitro assays to predict the efficacy of chemopreventive agents in whole animals. 915 67

Increased activity of the Na(+)-H+ exchanger (NHE-1 isoform) has been observed in cells and tissues from hypertensive humans and animals, including the spontaneously hypertensive rat (SHR). No mutation in NHE-1 DNA sequence or alteration in NHE-1 mRNA and protein expression has been demonstrated in hypertension, indicating that alterations in proteins that regulate NHE-1 activity are responsible for increased activity. The recent finding that NHE-1 phosphorylation in SHR vascular smooth muscle cells (VSMCs) was greater than in Wistar-Kyoto rat (WKY) VSMCs suggested that NHE-1 kinases may represent an abnormal regulatory pathway present in hypertension. To define NHE-1 kinases altered in the hypertensive phenotype. We measured NHE-1 kinase activity by an in-gel-kinase assay using a recombinant glutathione S-transferase NHE-1 fusion protein as a substrate. At least 7 NHE-1 kinases (42 to 90 kD) were present in VSMCs. We studied a 90-kD kinase because it was the major NHE-1 kinase and exhibited differences between SHR and WKY. Comparison of 90-kD kinase activity revealed that SHR VSMCs had increased activity in growth-arrested cells and in cells stimulated by angiotensin II (100 nmol/L for 5 minutes). Activation of the 90-kD kinase by angiotensin II was Ca2+ dependent, PKC independent, and partially dependent on the mitogen-activated protein kinase pathway. These findings indicate that increased activity of a 90-kD NHE-1 kinase is a characteristic of SHR VSMCs in culture and suggest that alterations in the 90-kD NHE-1 kinase and/or proteins that regulate its activity may be a pathogenic component in hypertension in the SHR.
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PMID:A 90-kD Na(+)-H+ exchanger kinase has increased activity in spontaneously hypertensive rat vascular smooth muscle cells. 918 Jun 27

Protein kinase C (PKC) is involved in cell-matrix and cell-cell adhesion, and the cytoplasmic domain of syndecan-2 contains two serines (residues 197 and 198) which lie in a consensus sequence for phosphorylation by PKC. Other serine and threonine residues are present but not in a consensus sequence. We investigated phosphorylation of syndecan-2 cytoplasmic domain by PKC, using purified GST-syndecan-2 fusion proteins and synthetic peptides corresponding to regions of the cytoplasmic domain. A synthetic peptide encompassing the entire cytoplasmic domain of syndecan-2 was phosphorylated by PKC with high affinity. Peptide mapping and substitution studies showed that both serines were phosphoacceptors, but each had slightly different affinity, with that of serine-197 being higher than serine-198. The efficiency of phosphorylation was concentration-dependent. At low concentrations, the cytoplasmic domain peptides were monomeric, with 2 mol/mol serine phosphorylation. At higher concentrations, however, the peptides formed dimers, with only 0.5 mol/mol phosphorylation. Concentration-dependent dimerization was not altered by phosphorylation. Phosphorylation is, therefore, dependent on the conformation of syndecan-2 cytoplasmic domain, but does not affect its oligomeric status.
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PMID:Serine phosphorylation of syndecan-2 proteoglycan cytoplasmic domain. 924 83

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


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