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

---

Query: EC:2.5.1.18 (glutathione S-transferase)
22,582 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.
...
PMID:Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. 750 91

The insulin receptor is known to interact with the SH2 domain proteins p85 (the regulatory subunit of phosphatidylinositol 3-kinase), Syp (a tyrosine phosphatase), and GAP (GTPase-activating protein). In this study, we mapped the insulin receptor binding sites for each of these proteins by examining the ability of phosphopeptides, corresponding to insulin receptor phosphorylation sites, and mutant insulin receptors to inhibit an insulin receptor-SH2 domain interaction. Precipitation of partially purified insulin receptors by glutathione S-transferase fusion proteins containing the N-terminal SH2 domains of p85 and GAP and both SH2 domains of Syp was demonstrated. The effect of the addition of each phosphopeptide on insulin receptor precipitation was tested. pY1322, the C-terminal insulin receptor peptide, inhibited insulin receptor precipitation by both p85- and Syp-GST. The NPXY internalization domain peptide inhibited insulin receptor precipitation by GAP-GST. These data were confirmed by mutant insulin receptor experiments. The insulin receptor C-terminal mutants, delta CT and Y/F2, were not precipitated by p85- or Syp-GST and the NPXY mutant insulin receptors, delta Ex16 and HI delta NPEY, were not precipitated by GAP-GST. Therefore, we conclude that p85 and Syp bind to the insulin receptor C terminus at tyrosine 1322 and GAP binds to the insulin receptor NPXY domain at tyrosine 960.
...
PMID:Localization of the insulin receptor binding sites for the SH2 domain proteins p85, Syp, and GAP. 752 47

The CD45 transmembrane protein-tyrosine phosphatase (PTPase, EC 3.1.3.48) plays an essential role in T-cell activation by activating the Lck and/or Fyn protein-tyrosine kinases. However, numerous experiments have indicated that CD45 may have both stimulatory and inhibitory roles in T-cell activation. Thus, it is unlikely that the two kinases are the sole substrates of the CD45 PTPase. Furthermore, the complex regulation of the alternative splicing of the extracellular domain in various leukocyte lineages also suggests additional roles for the CD45 PTPase. To identify such functions, it is necessary to identify physiologically relevant substrates of the CD45 PTPase other than the two protein-tyrosine kinases. To this end, we searched for high-affinity substrates of the CD45 PTPase among the tyrosine-phosphorylated T-cell proteins by using purified glutathione S-transferase-CD45 fusion molecules. The enzymatically inactive CD45 C828S mutant protein, in which the cysteine residue at the catalytic center was changed to a serine residue, bound tightly to the phosphorylated CD3 zeta chain. This binding was specific to CD45 PTPase, as neither the leukocyte common antigen-related molecule (LAR) PTPase nor the CD45-LAR hybrid PTPases bound the phosphorylated CD3 zeta chain. Furthermore, phosphorylated CD3 zeta chain was preferentially dephosphorylated by the wild-type CD45 PTPase under conditions that did not significantly dephosphorylate other cellular proteins. Thus, the phosphorylated CD3 zeta chain is a specific and high-affinity substrate of the CD45 PTPase. These results suggest that CD45 is involved in the termination of the T-cell response via dephosphorylation of CD3 zeta chain.
...
PMID:Specific interaction of the CD45 protein-tyrosine phosphatase with tyrosine-phosphorylated CD3 zeta chain. 752 85

Vav is a recently described proto-oncogene expressed only in hematopoietic cells which contains an SH2 and two SH3 domains and shares homology with the Dbl GDP-GTP exchange factor and BCR. p95Vav is phosphorylated on tyrosine residues in response to stimulation of the T cell antigen receptor, cross-linking of IgE or IgM receptors and stimulation of immature hematopoietic cells by Steel factor. Monoclonal antibodies to human Vav were generated and used to examine the events which regulate tyrosine phosphorylation of p95Vav in myeloid cells. In the factor-dependent MO7e cell line, p95Vav was rapidly phosphorylated on tyrosine residues in a dose- and time-dependent manner by GM-CSF, IL-3 and Steel factor. Introduction of the BCR/ABL oncogene into this cell line resulted in factor-independent proliferation and constitutive phosphorylation of p95Vav. Tyrosine phosphorylation of p95Vav was also substantially increased by treatment of cytokine-deprived cells with the tyrosine phosphatase inhibitor sodium vanadate. Since many of the cytokines known to induce tyrosine phosphorylation of p95Vav are also known to activate JAK family tyrosine kinases, we looked for an interaction of p95Vav with JAK kinases. p95Vav co-precipitated with JAK2 in MO7e cells stimulated with GM-CSF, but not in unstimulated cells. Also, JAK2 was found to be constitutively associated with p95Vav in vivo when expressed at high levels in insect cells using baculovirus vectors. A fusion protein consisting of glutathione-S-transferase and the SH2 domain of p95Vav (GST-Vav-SH2) precipitated JAK2, suggesting that this interaction is mediated by the SH2 domain of p95Vav.(ABSTRACT TRUNCATED AT 250 WORDS)
...
PMID:Tyrosine phosphorylation of p95Vav in myeloid cells is regulated by GM-CSF, IL-3 and steel factor and is constitutively increased by p210BCR/ABL. 749 7

Potential signaling substrates for the insulin-like growth factor I (IGF-I) receptor are SH2 domain proteins including the p85 subunit of phosphatidylinositol 3-kinase, the tyrosine phosphatase Syp, GTPase activating protein (GAP), and phospholipase C-gamma (PLC-gamma). In this study, we demonstrate an association between the IGF-I receptor and p85, Syp, and GAP, but not with PLC-gamma in lysates of cells overexpressing the human IGF-I receptor. We further investigated these interactions using glutathione S-transferase (GST) fusion proteins containing the amino-terminal SH2 domains of p85 or GAP, or both SH2 domains of Syp or PLC-gamma to precipitate the IGF-I receptor from purified receptor preparations and from whole cell lysates. p85-, Syp-, and GAP-GSTs precipitated the IGF-I receptor, whereas the PLC-gamma-GST did not. Using phosphopeptides corresponding to IGF-I receptor phosphorylation sites, we determined that the p85- and Syp-GST association with the IGF-I receptor could be inhibited by a carboxyl-terminal peptide containing pY1316 and that the GAP-GST association could be inhibited by a NPXY domain peptide. The GAP-GST binding site was confirmed by showing that a mutant IGF-I receptor with a deletion of the NPXY domain including tyrosine 950 was poorly precipitated by the GAP-GST. We conclude that p85 and Syp may bind directly to the IGF-I receptor at tyrosine 1316, and that GAP may bind to the IGF-I receptor at and PLC-gamma was not evident. p85, Syp, and GAP are potential modulators of IGF-I receptor signal transduction.
...
PMID:Localization of the insulin-like growth factor I receptor binding sites for the SH2 domain proteins p85, Syp, and GTPase activating protein. 764 82

Insulin activates hexose transport via at least two mechanisms: a p21ras-dependent pathway, leading to an increase in the amount of cell surface GLUT1; and a metabolic, p21ras-independent pathway, leading to translocation of the insulin-responsive transporter GLUT4 to the cell surface. Following insulin stimulation, SHPTP2, a non-transmembrane protein-tyrosine phosphatase, associates with insulin receptor substrate 1 via its Src homology 2 (SH2) domains. Microinjection of a glutathione S-transferase fusion protein encoding the N- and C-terminal SH2 domains of SHPTP2 (GST-NC-SH2) or anti-SHPTP2 antibodies into NIH-3T3 fibroblasts overexpressing the insulin receptor blocks insulin-induced DNA synthesis. Microinjection of either GST-NC-SH2 or anti-SHPTP2 antibodies into 3T3-L1 adipocytes inhibited the insulin-stimulated increase in expression of GLUT1. In contrast, translocation of GLUT4 to the cell surface was unaffected by either GST-NC-SH2 or anti-SHPTP2 antibodies. These data confirm a role for SHPTP2 in insulin-stimulated mitogenesis and indicate that whereas SHPTP2 is necessary for insulin-stimulated expression of GLUT1, it is not required for activation of the metabolic pathway leading to GLUT4 translocation.
...
PMID:Different signaling roles of SHPTP2 in insulin-induced GLUT1 expression and GLUT4 translocation. 776 84

Sequence analysis of the genomes of the Leporipoxviruses myxoma virus and Shope fibroma virus (SFV) led to the discovery of open reading frames homologous to the vaccinia H1L gene encoding a soluble protein phosphatase with dual tyrosine/serine specificity. These viral phosphatase genes were subsequently localized to the myxoma BamHI-I fragment and the SFV BamHI-M fragment, and the resulting encoded proteins were designated I1L and M1L, respectively. The localization and orientation of the myxoma I1L and SFV M1L open reading frames within the well conserved central core of the viral genomes closely mirror that of the Orthopoxviruses vaccinia virus and variola virus. The myxoma I1L and SFV M1L phosphatases each contain the conserved tyrosine phosphatase signature sequence motif, (I/V)HCXAGXXR(S/T)G, including the active site cysteine, found previously to be essential for phosphotyrosine dephosphorylation. The vaccinia H1L phosphatase was originally shown to have the ability to dephosphorylate phosphotyrosyl and phosphoseryl residues in vitro. To assess whether this is a common feature of poxvirus phosphatases, myxoma I1L was expressed as a GST-fusion protein, purified, and shown to dephosphorylate substrates containing tyrosine and serine phosphorylated residues, in a similar fashion to vaccinia H1L. A myxoma I1L variant, in which the active site cysteine 110 was mutated to serine, was expressed in a parallel fashion to the wild-type I1L protein and found to be completely deficient in its ability to dephosphorylate both phosphotyrosine and phosphoserine amino acids. In an attempt to ascertain the biological requirement for the myxoma I1L phosphatase, we constructed a recombinant myxoma virus containing a disrupted I1L open reading frame. This I1L mutant virus was able to successfully propagate in tissue culture only in the presence of a wild-type complementing gene, and pure virus clones containing only the disrupted allele were not viable. Thus, we conclude that the myxoma I1L dual specificity phosphatase is an essential factor for virus viability.
...
PMID:Myxoma virus and Shope fibroma virus encode dual-specificity tyrosine/serine phosphatases which are essential for virus viability. 783 13

We have characterized a growth factor-inducible gene, erp, and demonstrated that it encodes a 367-amino-acid nontransmembrane tyrosine phosphatase protein with significant similarity to the vaccinia virus H1 protein. Immunoprecipitation analyses show that the erp protein, ERP, is rapidly induced following serum stimulation of quiescent fibroblasts. ERP has been expressed as a fusion protein with glutathione S-transferase and shown to have tyrosine as well as serine protein phosphatase activity. The enzymatic activity of ERP depends on the presence of reducing agents such as dithiothreitol, and its tyrosine phosphatase activity is inhibited by sodium vanadate, a potent inhibitor of protein tyrosine phosphatases. The number of stable NIH 3T3 clones obtained after transfection with a vector expressing the complete ERP protein is reduced more than 90% compared with that after transfection with a vector expressing a mutated inactive ERP protein. The remaining ERP-expressing clones present a significant increase in the proportion of bi- and multinucleated cells and a decrease in proliferation rate. Studies on the genomic structure reveal that the erp transcription unit is 2.8 kbp long and split into four exons. The erp gene maps to the 17A2-17C region of the murine genome. Our results demonstrate that the protein product of the immediate-early gene erp has a negative effect on cell proliferation.
...
PMID:Structure, mapping, and expression of erp, a growth factor-inducible gene encoding a nontransmembrane protein tyrosine phosphatase, and effect of ERP on cell growth. 835 78

The pGEX glutathione S-transferase (GST) fusion protein system is used extensively for high level expression and rapid purification of fusion proteins from bacterial and eukaryotic cell lysates. Unfortunately, many GST fusion proteins are partially or completely insoluble, and thus cannot be purified efficiently from a crude lysate. We have adapted a protocol, previously used to solubilize actin, for the purification of otherwise insoluble GST fusion proteins. Using a GST fusion of the nontransmembrane protein tyrosine phosphatase 1B, we demonstrate that tyrosine phosphatase enzymatic activity is maintained during the purification process. We provide methods for the purification of GST fusion proteins at analytical and preparative scales, and demonstrate that saturation of glutathione agarose is dependent on fusion protein molecular weight. Finally, we present strategies for eluting purified fusion proteins from glutathione agarose beads, for storing eluted protein, and for preparing covalently coupled affinity matrices.
...
PMID:Solubilization and purification of enzymatically active glutathione S-transferase (pGEX) fusion proteins. 848 15

p56lck is a potential in vivo substrate for the tyrosine-specific phosphatase, CD45. In this study, recombinant purified p56lck was found to specifically associate with recombinant CD45 cytoplasmic domain protein, but not to the cytoplasmic domain of another related tyrosine phosphatase, receptor protein-tyrosine phosphatase alpha. Under equilibrium binding conditions, the binding was saturable and occurred at a 1:1 molar stoichiometry. A fusion protein containing only the amino-terminal region of p56lck (residues 34-150) also bound to recombinant CD45, and further analysis of this region indicated that glutathione S-transferase fusion proteins of the unique amino-terminal region and the SH2 domain, but not the SH3 domain of p56lck, bound to recombinant CD45. The SH2 domain protein bound with a higher affinity than the amino-terminal region, but both were able to compete for the binding of p56lck to CD45, and when added together worked synergistically to compete for p56lck binding. The SH2 domain interaction with CD45 was specific as glutathione S-transferase-SH2 fusion proteins from p85 alpha subunit of phosphatidylinositol 3-kinase and SHC did not bind to CD45. In addition, this interaction occurred in the absence of any detectable tyrosine phosphorylation on CD45, suggesting a nonconventional SH2 domain interaction.
...
PMID:Demonstration of a direct interaction between p56lck and the cytoplasmic domain of CD45 in vitro. 857 15


1 2 3 4 Next >>

---