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 human immunodeficiency virus type 1 Nef protein was expressed in Escherichia coli as a C-terminal fusion with glutathione S-transferase (GST). The ability of GST-Nef to act as a substrate for cellular kinases in vitro was examined by incubation of purified GST-Nef fusion proteins, immobilized on glutathione-agarose beads, with cytoplasmic extracts from a number of human cell lines. In the presence of [gamma32P]ATP, phosphorylation of Nef occurred predominantly on serine residues. Studies with protein kinase inhibitors suggested that protein kinase C (PKC) was involved in Nef phosphorylation. This was supported further by the demonstration that purified PKC was also able to phosphorylate Nef in the absence of cell extract. Serine/threonine phosphorylation of Nef was also observed in vivo when Nef was expressed with a C-terminal GST or 6-histidine tag in Spodoptera frugiperda insect cells by recombinant baculoviruses. In extracts from Jurkat T cells and U937 monocyte/macrophages Nef also associated with a 57 kDa cellular protein that was itself phosphorylated in vitro. Phosphorylation of this Nef-associated protein was inhibited by heparin and is thus likely to be mediated by casein kinase II. The observation that PKC can phosphorylate Nef in vitro raises the possibility that PKC might play a role in regulating both Nef function and the physical interactions between Nef and cellular components.
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PMID:The human immunodeficiency virus type 1 Nef protein functions as a protein kinase C substrate in vitro. 904 29

We have cloned a novel serine/threonine protein kinase (PK428) which is highly related (65%) within the kinase domain to the myotonic dystrophy protein kinase (DM-PK), as well as the cyclic AMP-dependent protein kinase (33%). Northern blots demonstrate that PK428 mRNA is distributed widely among tissues and is expressed at the highest levels in pancreas, heart, and skeletal muscle, with lower levels in liver and lung. Two PK428 mRNAs 10 and 3.8 kilobase pairs in size are seen in a number of cell lines, including hematopoietic and breast cancer cells. An antibody generated to a glutathione S-transferase-PK428 fusion protein detects a 65-kDa protein in these cell lines, and a similarly sized protein when the cloned cDNA is transiently expressed in Cos 7 cells. Immunoprecipitation of the transiently expressed PK428 protein and incubation with [gamma-32P]ATP demonstrate that it is capable of autophosphorylation. In addition, immunoprecipitates of the PK428 protein kinase also phosphorylated histone H1 and a peptide encoding a cyclic AMP-dependent protein kinase substrate. The gene corresponding to the 3.8-kb PK428 mRNA, and its corresponding 65-kDa protein, was isolated by polymerase chain reaction screening of a P1 phage human genomic library. Using this P1 phage clone as a probe, the PK428 gene was located on 1q41-42, a possible location for a human senescence gene, a gene associated with Rippling muscle disease, as well as a region associated with genetically acquired mental retardation.
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PMID:Cloning and chromosomal location of a novel member of the myotonic dystrophy family of protein kinases. 909 43

Full-length human nuclear DNA helicase II (NDH II) was cloned and overexpressed in a baculovirus-derived expression system. Recombinant NDH II unwound both DNA and RNA. Limited tryptic digestion produced active helicases with molecular masses of 130 and 100 kDa. The 130-kDa helicase missed a glycine-rich domain (RGG-box) at the carboxyl terminus, while the 100-kDa form missed both its double-stranded RNA binding domains (dsRBDs) at the amino terminus and its RGG-box. Hence, the dsRBDs and the RGG-box were dispensable for unwinding. On the other hand, the isolated DEXH core alone could neither hydrolyze ATP nor unwind nucleic acids. These enzymatic activities were not regained by fusing a complete COOH or NH2 terminus to the helicase core. Hence, an active helicase required part of the NH2 terminus, the DEXH core, and a C-terminal extension of the core. Both dsRBDs and the RGG-box were bacterially expressed as glutathione S-transferase fusion proteins. The two dsRBDs had a strong affinity to double-stranded RNA and cooperated upon RNA binding, while the RGG-box bound preferentially to single-stranded DNA. A model is suggested in which the flanking domains influence and regulate the unwinding properties of NDH II.
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PMID:Domain structure of human nuclear DNA helicase II (RNA helicase A). 911 Oct 62

Gal4p-mediated activation of galactose gene expression in Saccharomyces cerevisiae normally requires both galactose and the activity of Gal3p. Recent evidence suggests that in cells exposed to galactose, Gal3p binds to and inhibits Ga180p, an inhibitor of the transcriptional activator Gal4p. Here, we report on the isolation and characterization of novel mutant forms of Gal3p that can induce Gal4p activity independently of galactose. Five mutant GAL3(c) alleles were isolated by using a selection demanding constitutive expression of a GAL1 promoter-driven HIS3 gene. This constitutive effect is not due to overproduction of Gal3p. The level of constitutive GAL gene expression in cells bearing different GAL3(c) alleles varies over more than a fourfold range and increases in response to galactose. Utilizing glutathione S-transferase-Gal3p fusions, we determined that the mutant Gal3p proteins show altered Gal80p-binding characteristics. The Gal3p mutant proteins differ in their requirements for galactose and ATP for their Gal80p-binding ability. The behavior of the novel Gal3p proteins provides strong support for a model wherein galactose causes an alteration in Gal3p that increases either its ability to bind to Gal80p or its access to Gal80p. With the Gal3p-Gal80p interaction being a critical step in the induction process, the Gal3p proteins constitute an important new reagent for studying the induction mechanism through both in vivo and in vitro methods.
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PMID:Novel Gal3 proteins showing altered Gal80p binding cause constitutive transcription of Gal4p-activated genes in Saccharomyces cerevisiae. 911 26

We studied the molecular nature of the interaction between the integral membrane protein Sec63p and the lumenal Hsp70 BiP to elucidate their role in the process of precursor transit into the ER of Saccharomyces cerevisiae. A lumenal stretch of Sec63p with homology to the Escherichia coli protein DnaJ is the likely region of interface between Sec63p and BiP. This domain, purified as a fusion protein (63Jp) with glutathione S-transferase (GST), mediated a stable ATP-dependent binding interaction between 63Jp and BiP and stimulated the ATPase activity of BiP. The interaction was highly selective because only BiP was retained on immobilized 63Jp when detergent-solubilized microsomes were mixed with ATP and the fusion protein. GST alone was inactive in these assays. Additionally, a GST fusion containing a point mutation in the lumenal domain of Sec63p did not interact with BiP. Finally, we found that the soluble Sec63p lumenal domain inhibited efficient precursor import into proteoliposomes reconstituted so as to incorporate both BiP and the fusion protein. We conclude that the lumenal domain of Sec63p is sufficient to mediate enzymatic interaction with BiP and that this interaction positioned at the translocation apparatus or translocon at the lumenal face of the ER is vital for protein translocation into the ER.
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PMID:The lumenal domain of Sec63p stimulates the ATPase activity of BiP and mediates BiP recruitment to the translocon in Saccharomyces cerevisiae. 919 65

Tom20 is part of a multiple component, dynamic complex that functions to import specific cytosolic proteins into or through the outer membrane of the mitochondrion. To analyze the contribution of Tom20 to precursor protein recognition, the cytosolic domain of the human mitochondrial import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase and conditions established to measure specific interactions of the receptor component with precursor proteins in vitro. Reconstitution of receptor binding from purified components revealed that a prototypic matrix-destined precursor protein, pODHFR, interacts with Tom20 by a mechanism that is dependent on an active matrix targeting signal but does not require cytosolic components or ATP. Binding was influenced by both salt concentration and detergent. The effect of salt or detergent, however, varied for different precursor proteins. In particular, detergent selectively enhanced binding of pODHFR to receptor, possibly because of induced changes in the structure of the signal sequence. Finally, mutations were introduced into hTom20 which had a dramatic effect on binding of some precursor proteins but not on others. Taken together, the results suggest that hTom20 recognizes and physically interacts with precursor proteins bearing a diverse array of topogenic sequences and that such pleiotropic specificity for these precursor proteins may involve different domains within the receptor molecule.
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PMID:Interactions of the human mitochondrial protein import receptor, hTom20, with precursor proteins in vitro reveal pleiotropic specificities and different receptor domain requirements. 921 31

A new family of fluorescent probes has been developed for assessing the viability and metabolic activity of yeasts. This class of halogenated unsymmetric cyanine dyes is exemplified by the FUN-1 [2-chloro-4-(2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)- methylidene)-1-phenylquinolinium iodide] stain, a membrane-permeant nucleic acid-binding dye that has been found to give rise to cylindrical intravacuolar structures (CIVS) in Saccharomyces cerevisiae. Biochemical processing of the dye by active yeasts yielded CIVS that were markedly red shifted in fluorescence emission and therefore spectrally distinct from the nucleic acid-bound form of the dye. The formation of CIVS occurred under both aerobic and anaerobic conditions and was highly temperature dependent. Treatment of yeasts with the nonmetabolizable glucose analog 2-deoxy-D-glucose reduced cellular ATP levels approximately 6-fold and completely inhibited CIVS formation. Under aerobic conditions, the formation of CIVS was abrogated by the cytochrome oxidase inhibitors azide and cyanide; however, the H+ transport uncoupler carbonyl cyanide m-chlorophenylhydrazone inhibited CIVS formation under both aerobic and anaerobic conditions. Depletion of cellular thiols, including glutathione, with millimolar concentrations of N-ethylmaleimide, iodoacetamide, or allyl alcohol completely inhibited CIVS production. Marked reduction in the formation of CIVS by ethacrynic acid and sulfobromophthalein, inhibitors of glutathione S-transferase, suggested that dye processing can involve enzyme-mediated formation of glutathione conjugates. The conversion of FUN-1 by S. cerevisiae was studied quantitatively by using several techniques, including fluorometry, flow cytometry, and wide-field and confocal laser scanning fluorescence microscopy.
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PMID:Development of the FUN-1 family of fluorescent probes for vacuole labeling and viability testing of yeasts. 921 36

The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-gamma (PLC-gamma). A glutathione S-transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-gamma was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-gamma and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.
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PMID:The N-methyl-D-aspartate receptor subunits NR2A and NR2B bind to the SH2 domains of phospholipase C-gamma. 923 20

p38 has been shown to be a critical enzyme in the pro-inflammatory cytokine pathway and is a member of the mitogen-activated protein (MAP) kinase family. While the details for p38 activation and subsequent signal transduction have begun to be elucidated, little is known about the kinetic mechanism for p38. In this study, we have determined the kinetic mechanism for p38 MAP kinase. Data from initial velocity patterns in the presence and absence of a dead-end inhibitor and two triarylimidazole p38 inhibitors were consistent with an ordered sequential mechanism for p38 with protein substrate, glutathione S-transferase-activating transcription factor 2 (GST-ATF2), binding before ATP. The ATP analog, adenylyl methylenediphosphonate (AMP-PCP), and two triarylimidazoles were competitive inhibitors versus ATP and uncompetitive inhibitors versus GST-ATF2. Equilibrium binding studies utilizing a tritiated ATP-competitive inhibitor were also consistent with this mechanism and suggest an inability of ATP to bind to p38 in the absence of protein substrate. Moreover, the Michaelis constant for GST-ATF2 was 12-fold greater than the dissociation constant, indicating that the binding of ATP affected the binding of GST-ATF2. An ordered sequential mechanism with protein substrate binding first is unique to p38 compared to cyclic AMP-dependent protein kinase (cAPK) and most tyrosine kinases and helps to explain the interaction between enzyme, substrates, and inhibitors.
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PMID:Kinetic mechanism for p38 MAP kinase. 926 22

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.
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PMID:Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa. 926 59


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