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)

Several systems have been developed to allow for rapid and efficient purification of recombinant proteins expressed in bacteria. The expression of polypeptides in frame with glutathione S-transferase (GST) allows for purification of the fusion proteins from crude bacterial extracts under nondenaturing conditions by affinity chromatography on glutathione agarose (D. B. Smith and K. S. Johnson, 1988, Gene 67, 31-40). This vector expression system has also incorporated specific protease cleavage sites to facilitate proteolysis of the bacterial fusion proteins. In our hands, the cleavage of these fusion proteins at a thrombin cleavage site proceeded slowly. To facilitate the cleavage of fusion proteins, we have introduced a glycine-rich linker (glycine kinker) containing the sequence P.G.I.S.G.G.G.G.G located immediately following the thrombin cleavage site. This glycine kinker greatly increases the thrombin cleavage efficiency of several fusion proteins. The introduction of the glycine kinker into fusion proteins allows for the cleavage of the fusion proteins while they are attached to the affinity resin resulting in a single step purification of the recombinant protein. More than 2 mg of the highly purified protein was obtained from the equivalent of 100 ml of bacterial culture within a few hours when a protein tyrosine phosphatase was employed as a test protein. The vector, pGEX-KG, has also been modified to facilitate cloning of a variety of cDNAs in all reading frames and has been successfully used to express several eukaryotic proteins.
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PMID:Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. 185 37

A cDNA which encodes a protein tyrosine phosphatase with two src homology 2 (SH2) domains was isolated from a rat brain cDNA library. This phosphatase appears to be a rat homologue of PTP1D based on its amino acid sequence. The gene is expressed in a variety of tissues, and its mRNA is enriched in the brain, skeletal muscle, and lung. An RNA splice variant (PTP1Di) was also isolated which has four additional amino acid residues (Ala-Leu-Leu-Gln) in the catalytic domain. The catalytic domains of PTP1D and PTP1Di were expressed in Escherichia coli as glutathione S-transferase fusion proteins and purified to near homogeneity. Whereas both PTP1D and PTP1Di had catalytic activity, the Vmax of PTP1Di relative to that of PTP1D was 8-fold lower for para-nitrophenylphosphate, 20-fold lower for nicotinic acetylcholine receptor, and 14-fold lower for myelin basic protein. The Km values of PTP1Di were lower than those of PTP1D for both nicotinic acetylcholine receptor and myelin basic protein, suggesting a higher affinity of PTP1Di for a protein substrate. These two forms also differed in optimum pH for para-nitrophenylphosphate and sensitivity to the inhibitory effects of vanadate, molybdate, and spermidine. In order to see if this insert would affect the catalytic activity of other related phosphatases, the 4-amino acids were inserted in the corresponding region of the catalytic domain of PTP1C. Whereas both the wild type and PTP1Ci which contained the 4-amino acid insert dephosphorylated para-nitrophenylphosphate, nicotinic receptor, and myelin basic protein, the enzyme activity of PTP1Ci was only 11-24% of that of PTP1C wild type. These results demonstrate that the 4-amino acid insert in the catalytic domains of PTP1D down-regulates its phosphatase activity and suggests that RNA splicing may serve as a regulatory mechanism of protein tyrosine phosphatase activity.
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PMID:RNA splicing regulates the activity of a SH2 domain-containing protein tyrosine phosphatase. 751 64

The cytoplasmic insulin receptor substrate-1 (IRS-1), which is multiply phosphorylated in vivo on tyrosine residues, is a known binding protein for the tandem src homology 2 (SH2) domain-containing protein tyrosine phosphatase, SH-PTP2. Eleven phosphotyrosyl (pY) peptides from IRS-1 were screened for allosteric activation of SH-PTP2 phosphatase activity toward phosphorylated, reduced, carboxyamidomethylated, and maleylated-lysozyme. Peptides IRS-1pY895, IRS-1pY1172, and IRS-1pY1222 showed up to 50-fold acceleration of dephosphorylation. Analyses of Arg to Lys mutants in either or both SH2 domains indicate that both the N-terminal (N-SH2) and C-terminal (C-SH2) domains function in allosteric activation. Direct determination by surface plasmon resonance of the dissociation constants between pY peptides and glutathione S-transferase fusions to N-SH2 and C-SH2 domains reveals a 240-fold preference of the N-SH2 domain (compared with the C-SH2 domain) for IRS-1pY1172. The N-SH2 domain prefers IRS-1pY1172 > IRS-1pY895 > IRS-1pY1222, whereas C-SH2 domain prefers IRS-1pY1222 > IRS-1pY895 > IRS-1pY1172. These data suggest that each SH2 domain can bind to a distinct pY sequence of multiply phosphorylated protein substrates such as IRS-1, while activating hydrolysis at a third pY sequence bound in the SH-PTP2 active site. In addition, proteolysis and truncation studies reveal an autoregulatory function for the C-terminal region of SH-PTP2. Limited tryptic cleavage within the C-terminus results in 27-fold activation of protein tyrosine phosphatase activity. The activated tryptic fragment cannot be further activated by pY peptide binding to the SH2 domains indicating that autoregulatory functions of the SH2 domains are dependent on the C-terminal region. These data suggest that multiple levels for control of SH-PTP2 enzymatic activity may exist in vitro and in vivo.
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PMID:Activation of the SH2-containing protein tyrosine phosphatase, SH-PTP2, by phosphotyrosine-containing peptides derived from insulin receptor substrate-1. 751 3

A novel cDNA encoding PTP (protein tyrosine phosphatase) was cloned from PC12h cells and designated as PCPTP1 (gene encoding PC12 protein Tyr phosphatase). The longest open reading frame (ORF) of this clone encodes a 656-amino-acid (aa) protein with a single PTP catalytic domain. Western blot analysis using a polyclonal Ab (antibody) raised against the cytoplasmic region of PCPTP1 detected two products, a major 65-kDa and minor 42-kDa protein, designated PCPTP1-MFI and PCPTP1-MVQ, respectively, in PC12h cells. These two proteins correspond to the products translated from the second and fifth methionine of PCPTP1, respectively. The bacterially expressed GST::PCPTP1-MVQ fusion protein had phosphatase activity with pNPP (p-nitrophenyl phosphate) as a substrate. Alignment of the aa sequence of PCPTP1-MVQ with those of other PTP showed the highest similarity to STEP and LC-PTP/HePTP, with 54 and 51% identity, respectively. Northern blot analysis showed only one 3.9-kb transcript in PC12h cells, indicating that PCPTP1 corresponds to this 3.9-kb transcript. The 3.9-kb PCPTP1 mRNA was detected in the brain and adrenal gland, but not in other non-neuronal tissues in adult rats. Two other transcripts of 3.3 and 1.7 kb were also detected in brain. NGF (nerve growth factor) and glucocorticoid are known to bimodally regulate the cell fate decision of sympathoadrenal precursors like PC12 cells, with NGF promoting the neuronal phenotype and glucocorticoid promoting the chromaffin phenotype. Still, both agents decreased the level of PCPTP1 mRNA in PC12h cells. Therefore, it is likely that the decrease in the level of PCPTP1 mRNA might be associated or correlated with cell differentiation in PC12h cells.
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PMID:Cloning and expression of PCPTP1 encoding protein tyrosine phosphatase. 755 44

Protein tyrosine phosphorylation and thus dephosphorylation are part of the interleukin (IL)-11 response in mouse 3T3-L1 cells. We report here for the first time the involvement and interactions of the SH2-containing protein tyrosine phosphatase Syp in the IL-11 signal transduction pathway. Addition of IL-11 to 3T3-L1 cells resulted in an increase in the tyrosine phosphorylation of Syp. When cell lysates were precipitated with glutathione S-transferase fusion products of Syp, the C-terminal SH2 domain of Syp was shown to precipitate several proteins of 70, 130, 150, and 200 kDa that were tyrosine phosphorylated in response to IL-11. Reciprocal immunoprecipitation experiments showed that Syp was inducibly associated with both gp130 and Janus kinase 2 (JAK2). A phosphopeptide containing the sequence for a potential Syp binding site (YXXV) was used to compete with the associations of Syp with gp130 and JAK2. The phosphopeptide reduced the Syp association with both gp130 and JAK2. To summarize, Syp has multiple interactions in IL-11 signal transduction. In addition to the IL-11-induced tyrosine phosphorylation of Syp, Syp coprecipitated with gp130, JAK2, and other tyrosine-phosphorylated proteins in response to IL-11. These findings may have extensive significance to IL-11 and related cytokine signal transduction, suggesting new pathways and mechanisms.
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PMID:Syp associates with gp130 and Janus kinase 2 in response to interleukin-11 in 3T3-L1 mouse preadipocytes. 755 3

The genome of avian sarcoma virus CT10 encodes a fusion protein in which viral Gag sequences are fused to cellular Crk sequences containing primarily Src homology 2 (SH2) and Src homology 3 (SH3) domains. Transformation of chicken embryo fibroblasts (CEF) with the Gag-Crk fusion protein results in the elevation of tyrosine phosphorylation on specific cellular proteins with molecular weights of 130,000, 110,000, and 70,000 (p130, p110, and p70, respectively), an event which has been correlated with cell transformation. In this study, we have identified the 70-kDa tyrosine-phosphorylated protein in CT10-transformed CEF (CT10-CEF) as paxillin, a cytoskeletal protein suggested to be important for organizing the focal adhesion. Tyrosine-phosphorylated paxillin was found to be complexed with v-Crk in vivo as evident from coimmunoprecipitation studies. Moreover, a bacterially expressed recombinant glutathione S-transferase (GST)-CrkSH2 fragment bound paxillin in vitro with a subnanomolar affinity, suggesting that the SH2 domain of v-Crk is sufficient for binding. Mapping of the sequence specificity of a GST-CrkSH2 fusion protein with a partially degenerate phosphopeptide library determined a motif consisting of pYDXP, and in competitive coprecipitation studies, an acetylated A(p)YDAPA hexapeptide was able to quantitatively inhibit the binding of GST-CrkSH2 to paxillin and p130, suggesting that it meets the minimal structural requirements necessary for the interaction of CrkSH2 with physiological targets. To investigate the mechanism by which v-Crk elevates the tyrosine phosphorylation of paxillin in vivo, we have treated normal CEF and CT10-CEF with sodium vanadate to inhibit protein tyrosine phosphatase activity. These data suggest that paxillin is involved in a highly dynamic kinase-phosphatase interplay in normal CEF and that v-Crk binding may interrupt this balance to increase the steady-state level of tyrosine phosphorylation. By contrast, the 130-kDa protein was not tyrosine phosphorylated upon vanadate treatment of normal CEF and only weakly affected in the CT10-CEF, suggesting that a different mechanism may be involved in its phosphorylation.
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PMID:Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. 768 42

In addition to their role in bacterial killing, reactive oxygen intermediates (ROI) produced by the NADPH oxidase may participate in the regulation of intracellular pathways. We have recently demonstrated that ROI produced by the oxidase regulate tyrosine phosphorylation in neutrophils, possibly by alterations in the cellular redox state. The purpose of the present study was to characterize the identities of certain of the redox-sensitive tyrosine-phosphorylated substrates and the significance of the increased phosphorylation. As a prominent 42-44-kDa phosphorylated band was noted in oxidant-treated cells, we investigated the possible phosphorylation and activation of mitogen-activated protein (MAP) kinase under these conditions. Immunoprecipitation of MAP kinase followed by immunoblotting with anti-phosphotyrosine antibodies indicated that a 42-44-kDa polypeptide was tyrosine-phosphorylated in response to treatment of cells, either with the oxidizing agent diamide or with H2O2 in cells where catalase was inhibited. Using an in vitro renaturation assay with myelin basic protein as the substrate, oxidant-induced stimulation of kinase activity of a 42-44-kDa band was observed in both whole cell extracts and in MAP kinase immunoprecipitates. The mechanism of redox-sensitive activation of MAP kinase was examined. First, exposure of cells to oxidants caused a significant increase in the activity of MEK (the putative activator of MAP kinase), as determined by an in vitro kinase assay using recombinant catalytically inactive glutathione S-transferase-MAP kinase as the substrate. Additionally, oxidant treatment of cells resulted in inhibition of the activity of CD45, a protein tyrosine phosphatase known to dephosphorylate and inactivate MAP kinase. We conclude that oxidant treatment of neutrophils can activate MAP kinase by stimulating its tyrosine and (presumably) threonine phosphorylation via MEK activation, a response that may be potentiated by inhibition of MAP kinase dephosphorylation by phosphatases such as CD45.
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PMID:Activation of the mitogen-activated protein kinase signaling pathway in neutrophils. Role of oxidants. 798 67

The receptor-like protein tyrosine phosphatase, PTPmu, displays structural similarity to cell-cell adhesion molecules of the immunoglobulin superfamily. We have investigated the ability of human PTPmu to function in such a capacity. Expression of PTPmu, with or without the PTPase domains, by recombinant baculovirus infection of Sf9 cells induced their aggregation. However, neither a chimeric form of PTPmu, containing the extracellular and transmembrane segments of the EGF receptor and the intracellular segment of PTPmu, nor the intracellular segment of PTPmu expressed as a soluble protein induced aggregation. PTPmu mediates aggregation via a homophilic mechanism, as judged by lack of incorporation of uninfected Sf9 cells into aggregates of PTPmu-expressing cells. Homophilic binding has been demonstrated between PTPmu-coated fluorescent beads (Covaspheres) and endogenously expressed PTPmu on MvLu cells. Additionally the PTPmu-coated beads specifically bound to a bacterially expressed glutathione-S-transferase fusion protein containing the extracellular segment of PTPmu (GST/PTPmu) adsorbed to petri dishes. Covaspheres coated with the GST/PTPmu fusion protein aggregated in vitro and also bound to PTPmu expressed endogenously on MvLu cells. These results suggest that the ligand for this transmembrane PTPase is another PTPmu molecule on an adjacent cell. Thus homophilic binding interactions may be an important component of the function of PTPmu in vivo.
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PMID:Homophilic binding of PTP mu, a receptor-type protein tyrosine phosphatase, can mediate cell-cell aggregation. 839 72

Two protein tyrosine phosphatase genes, PTP1 and PTP2, are known in Saccharomyces cerevisiae. However, the functions of these tyrosine phosphatases are unknown, because mutations in either or both phosphatase genes have no clear phenotypic effects. In this report, we demonstrate that although ptp2 has no obvious phenotype by itself, it has a profound effect on cell growth when combined with mutations in a novel protein phosphatase gene. Using a colony color sectoring assay, we isolated 25 mutants in which the expression of PTP1 or PTP2 is required for growth. Complementation tests of the mutants showed that they have a mutation in one of three genes. Cloning and sequence determination of one of these gene, PTC1, indicated that it encodes a homolog of the mammalian protein serine/threonine phosphatase 2C (PP2C). The amino acid sequence of the PTC1 product is approximately 35% identical to PP2C. Disruption of PTC1 indicated that the PTC1 function is nonessential. In contrast, ptc1 ptp2 double mutants showed a marked growth defect. To examine whether PTC1 encodes an active protein phosphatase, a glutathione S-transferase (GST)-PTC1 fusion gene was constructed and expressed in Escherichia coli. Purified GST-PTC1 fusion protein hydrolyzed a serine phosphorylated substrate in the presence of the divalent cation Mg2+ or Mn2+. GST-PTC1 also had weak (approximately 0.5% of its serine phosphatase activity) protein tyrosine phosphatase activity.
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PMID:Mutations in a protein tyrosine phosphatase gene (PTP2) and a protein serine/threonine phosphatase gene (PTC1) cause a synthetic growth defect in Saccharomyces cerevisiae. 839 5

The polymerase chain reaction was used to amplify protein tyrosine phosphatase (PTPase)-related cDNA from a template of total RNA isolated from human skeletal muscle. A novel PTPase, which we term PTP-PEST, was detected by this method. The polymerase chain reaction fragment was used to screen two different HeLa cell libraries to obtain full length cDNA clones. The cDNA predicts a protein of 510 amino acids, approximately 60 kDa, that does not contain an obvious signal sequence or transmembrane segment suggesting it is a nonreceptor type enzyme. The PTPase domain is located in the N-terminal portion of the molecule and displays approximately 35% identity to other members of this family of enzymes. The C-terminal segment is rich in Pro, Glu, Asp, Ser, and Thr residues, possessing features of PEST motifs which have previously been identified in proteins with very short intracellular half-lives. The protein was expressed in Escherichia coli as a fusion product with glutathione S-transferase. Intrinsic activity was demonstrated in vitro against a variety of phosphotyrosine-containing substrates including BIRK, the autophosphorylated cytoplasmic kinase domain of the insulin receptor beta subunit. It did not dephosphorylate phosphoseryl-phosphorylase a. PTP-PEST mRNA is broadly distributed in a variety of cell lines. Stimulation of human rhabdomyosarcoma A204 cells, a transformed muscle line, with insulin led to an approximately 4-fold induction of PTP-PEST mRNA within 36 h.
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PMID:Cloning and expression of PTP-PEST. A novel, human, nontransmembrane protein tyrosine phosphatase. 834 45


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