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)

Growth hormone (GH) signaling requires activation of the GH receptor (GHR)-associated tyrosine kinase, JAK2. JAK2 activation by GH is believed to facilitate initiation of various pathways including the Ras, mitogen-activated protein kinase, STAT, insulin receptor substrate (IRS), and phosphatidylinositol 3-kinase systems. In the present study, we explore the biochemical and functional involvement of the Src homology 2 (SH2)-containing protein-tyrosine phosphatase, SHP-2, in GH signaling. GH stimulation of murine NIH 3T3-F442A fibroblasts, cells that homologously express GHRs, resulted in tyrosine phosphorylation of SHP-2. As assessed specifically by anti-SHP-2 coimmunoprecipitation and by affinity precipitation with a glutathione S-transferase fusion protein incorporating the SH2 domains of SHP-2, GH induced formation of a complex of tyrosine phosphoproteins including SHP-2, GHR, JAK2, and a glycoprotein with properties consistent with being a SIRP-alpha-like molecule. A reciprocal binding assay using IM-9 cells as a source of SHP-1 and SHP-2 revealed specific association of SHP-2 (but not SHP-1) with a glutathione S-transferase fusion incorporating GHR cytoplasmic domain residues 485-620, but only if the fusion was first rendered tyrosine-phosphorylated. GH-dependent tyrosine phosphorylation of SHP-2 was also observed in murine 32D cells (which lack IRS-1 and -2) stably transfected with the GHR. Further, GH-dependent anti-SHP-2 coimmunoprecipitation of the Grb2 adapter protein was detected in both 3T3-F442A and 32D-rGHR cells, indicating that biochemical involvement of SHP-2 in GH signaling may not require IRS-1 or -2. Finally, GH-induced transactivation of a c-Fos enhancer-driven luciferase reporter in GHR- and JAK2-transfected COS-7 cells was significantly reduced when a catalytically inactive SHP-2 mutant (but not wild-type SHP-2) was coexpressed; in contrast, expression of a catalytically inactive SHP-1 mutant allowed modestly enhanced GH-induced transactivation of the reporter in comparison with that found with expression of wild-type SHP-1. Collectively, these biochemical and functional data imply a positive role for SHP-2 in GH signaling.
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PMID:Involvement of the Src homology 2-containing tyrosine phosphatase SHP-2 in growth hormone signaling. 944 80

Cell adhesion kinase beta (CAKbeta) is a protein tyrosine kinase closely related to focal adhesion kinase (FAK) in structure. CAKbeta contains two proline-rich sequences within its C-terminal region. Since proline-rich sequences present in the corresponding region of FAK are known to mediate protein-protein interactions by binding to SH3 domains, we investigated binding of CAKbeta to a panel of SH3 domains. Affinity precipitation from rat brain lysate revealed selective interactions of CAKbeta with glutathione S-transferase (GST)-fused SH3 domains of p130(Cas)(Cas)-related proteins and Graf. Mutational analysis indicated that the proline-rich sequences of CAKbeta mediate this interaction. Each of the two proline-rich sequences fused to GST bound directly to these SH3 domains in dot blot analysis. A competitive binding assay revealed that the first proline-rich sequence of CAKbeta preferentially associated with the SH3 domain of Cas. The second proline-rich sequence of CAKbeta bound to the SH3 domain of Graf with higher specificity than the corresponding proline-rich sequence of FAK. Finally, we showed co-immunoprecipitation of CAKbeta with Graf from rat brain lysate. These results indicate that CAKbeta associates in vivo with Graf through its SH3 domain.
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PMID:Interaction of two proline-rich sequences of cell adhesion kinase beta with SH3 domains of p130Cas-related proteins and a GTPase-activating protein, Graf. 949 93

Chemoattractant-stimulated polymorphonuclear leukocytes (PMNs) that are adherent to extracellular matrix proteins exhibit a massive, sustained respiratory burst that requires cell spreading. However, the signaling pathways culminating in PMN spreading are not well characterized. Studies showing that protein tyrosine phosphorylation increases with PMN spreading suggest that phosphorylation is critical for this process. In the present study, we observed increased tyrosine phosphorylation of both focal adhesion kinase and Syk in FMLP-activated PMNs that had been plated onto fibrinogen; an increase in Syk activity, but not focal adhesion kinase activity, was apparent. The time course of Syk phosphorylation correlated with the initiation of cell spreading and H2O2 release. Pretreatment of PMNs with piceatannol, a Syk-selective inhibitor, blocked Syk activity, cell spreading, and H2O2 release, indicating that Syk activity was required for the activation of adherent PMNs. Paxillin is a cytoskeletally associated protein that is also tyrosine phosphorylated during PMN spreading and H2O2 release. Paxillin phosphorylation is kinetically slower than Syk phosphorylation and is inhibited with piceatannol, suggesting that paxillin is a substrate for Syk. An analysis of Syk immunoprecipitates indicated that Syk and paxillin associate during PMN spreading. This interaction is not mediated by the src kinases Lyn and Fgr, since neither kinase coprecipitated with Syk. Syk from FMLP-activated, adherent PMNs phosphorylated paxillin-glutathione S-transferase, suggesting that paxillin is a substrate for Syk in vivo. These results indicate that PMN spreading and H2O2 release require a Syk-dependent signaling pathway leading to paxillin phosphorylation.
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PMID:Syk activation is required for spreading and H2O2 release in adherent human neutrophils. 959 Feb 68

Platelets are anuclear, membrane-bounded fragments derived from megakaryocytes which, upon stimulation, assemble an actin skeleton including stress fibres and focal contacts. The focal contacts resemble those of tissue culture cells. However, they lack paxillin, a conspicuous component of these organelles. We found that instead of paxillin, platelets contain a related protein with a molecular mass of 55 kDa that crossreacts with a monoclonal antibody against paxillin. The gene for the 55 kDa protein was cloned from a bone marrow cDNA library and turned out to be identical to a recently discovered gene encoding hic-5. Like paxillin, hic-5 is a cytoskeletal protein containing four carboxy-terminal LIM domains and LD motifs in the amino-terminal half. The LIM domains of both hic-5 and paxillin are capable of targetting green fluorescent protein to focal contacts. In addition, GST-hic-5 precipitates the focal adhesion kinase pp125(FAK) and talin from platelet extracts. Only trace amounts of hic-5 occur in DAMI cells, a megakaryocytic cell line, and in megakaryocytes cultured from CD34+ cells obtained from umbilical cord blood. However, RT-polymerase chain reactions performed with RNA obtained from platelets gave a positive result when primers specific for hic-5 were used, but were negative with paxillin-specific primers, indicating that a switch from paxillin expression to hic-5 expression must occur late in the maturation of megakaryocytes into platelets.
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PMID:Recruitment of the LIM protein hic-5 to focal contacts of human platelets. 966 39

We have previously reported that non-activated platelets can be induced by morphological changes from the recombinant fusion protein of GST-rhodostomin [GST-RHO(RGD)], a member of disintegrin with an arginine-glycine-aspartic acid (RGD) motif. In this study, we further characterized the factors involved in platelet shape changes induced by rhodostomin. From less to full-spreading, four cell spreading indexes, p1, p2, s1 and s2, were designated to the platelet shape based on the scanning electron micrographs. Results of peptide competition and antibody blocking confirmed that interaction between the RGD of rhodostomin and the alpha(IIb)beta3 integrins of platelets was required for induction of a higher percentage of s2 cells. When platelets were pretreated with calphostin C, herbimycin A and cytochalasin B, respectively, the percentage of p1 and p2 cells on rhodostomin-coated plates was increased and, concomitantly, the percentage of s1 and s2 cells was decreased. Biochemical analyses indicated that the focal adhesion kinase (FAK or pp125FAK) in platelets that adhered to GST-RHO(RGD) was phosphorylated in contrast to little or no phosphorylation of FAK in cells adhered to fibrinogen or non-activated cells. Furthermore, the degree of FAK phosphorylation was consistently correlated with morphological changes in platelets treated with various drugs. Taking all the results together, we suggested that rhodostomin could directly bind to integrins of platelets and then trigger signal transduction leading to FAK phosphorylation and actin polymerization and finally resulting in platelet full-spreading.
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PMID:Full-spreading platelets induced by the recombinant rhodostomin are via binding to integrins and correlated with FAK phosphorylation. 969 Jul 77

We recently identified SH2-Bbeta as a JAK2-binding protein and substrate involved in the signaling of receptors for growth hormone and interferon-gamma. In this work, we report that SH2-Bbeta also functions as a signaling molecule for platelet-derived growth factor (PDGF). SH2-Bbeta fused to glutathione S-transferase (GST) bound PDGF receptor (PDGFR) from PDGF-treated but not control cells. GST fusion protein containing only the SH2 domain of SH2-Bbeta also bound PDGFR from PDGF-treated cells. An Arg to Glu mutation within the FLVRQS motif in the SH2 domain of SH2-Bbeta inhibited GST-SH2-Bbeta binding to tyrosyl-phosphorylated PDGFR. The N-terminal truncated SH2-Bbeta containing the entire SH2 domain interacted directly with tyrosyl-phosphorylated PDGFR from PDGF-treated cells but not unphosphorylated PDGFR from control cells in a Far Western assay. These results suggest that the SH2 domain of SH2-Bbeta is necessary and sufficient to mediate the interaction between SH2-Bbeta and PDGFR. PDGF stimulated coimmunoprecipitation of endogenous SH2-Bbeta with endogenous PDGFR in both 3T3-F442A and NIH3T3 cells. PDGF stimulated the rapid and transient phosphorylation of SH2-Bbeta on tyrosines and most likely on serines and/or threonines. Similarly, epidermal growth factor stimulated the phosphorylation of SH2-Bbeta; however, phosphorylation appears to be predominantly on serines and/or threonines. In response to PDGF, SH2-Bbeta associated with multiple tyrosyl-phosphorylated proteins, at least one of which (designated p84) does not bind to PDGFR. Taken together, these data strongly argue that, in response to PDGF, SH2-Bbeta directly interacts with PDGFR and is phosphorylated on tyrosine and most likely on serines and/or threonines, and acts as a signaling protein for PDGFR.
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PMID:Platelet-derived growth factor (PDGF) stimulates the association of SH2-Bbeta with PDGF receptor and phosphorylation of SH2-Bbeta. 969 82

The Src homology 3 (SH3) domains are a modular structure of about 60 amino acid residues found in many proteins important in signal transduction. Each SH3 domain has a binding specificity to sequences containing a PXXP motif in ligand proteins. We found that a focal adhesion kinase (FAK)-related protein, cell adhesion kinase beta (CAKbeta), was bound in vitro by the SH3 domain of embryonal Fyn-associated substrate (Efs), a docking protein structurally related to p130Cas (Cas) and HEF1. Here, we employed a dot far-Western blotting technique to evaluate the affinity and specificity of the binding by the SH3 domains of Efs and its related proteins. The SH3 domains and their ligands were prepared as glutathione S-transferase fusion proteins, and one of the binding components was immobilized on membranes while the other was labeled with 32P to use as a probe. The amount of the bound probe was determined by autoradiography using an imaging plate and a bioimaging analyzer. A competitive binding assay showed that Efs, compared with Cas and HEF1, had a SH3 domain with a lower relative affinity to CAKbeta and FAK and with a preference to interact with FAK rather than CAKbeta. Our assay based on dot far-Western blotting is a simple and sensitive method to evaluate fine differences in the binding affinity of SH3-mediated interactions.
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PMID:Dot far-western blot analysis of relative binding affinities of the Src homology 3 domains of Efs and its related proteins. 975 Jan 31

We have investigated the interaction between Cbl and the Src-related tyrosine kinase Fyn. Fyn was observed to be constitutively associated with Cbl in lysates of several different cell types including the interleukin-3-dependent murine myeloid cell line 32Dcl3, and the prolactin-dependent rat thymoma cell line Nb2. Binding studies indicated that Cbl could bind to glutathione S-transferase (GST) fusion proteins encoding the unique, Src homology domain 3 (SH3), and SH2 domains of Fyn, Hck, or Lyn. Fusion proteins encoding either the SH3 or SH2 domains of Fyn bound to Cbl as effectively as the fusion protein encoding the unique, SH3, and SH2 domains of Fyn. The Fyn SH2 domain bound to both tyrosine-phosphorylated and nonphosphorylated Cbl, implying that this interaction might be phosphotyrosine-independent. Binding of the Fyn SH2 domain to Cbl was not disrupted by the addition of phosphotyrosine, phosphoserine, or phosphothreonine. A GST fusion protein encoding the proline-rich region of Cbl bound to Fyn present in a total cell lysate. Far Western blot analysis also indicated that the SH3 domain of Fyn bound preferentially to the proline-rich region of Cbl. The addition of [gamma-32P]ATP to either anti-Cbl immunoprecipitates or anti-Fyn immunoprecipitates resulted in the phosphorylation of both Cbl and Fyn as demonstrated by immunoprecipitation of the phosphorylated proteins with specific antisera. Fyn directly phosphorylated a GST fusion protein containing the C-terminal region of Cbl (GST-CBL-LZIP). In contrast, immunoprecipitated JAK2 was not able to phosphorylate this same region of Cbl. The GST-CBL-LZIP fusion protein contains a binding site for the SH2 domain of the p85 subunit of phosphatidylinositol 3-kinase, which mapped to Tyr731, which is present in the sequence YEAM. Mutation of Tyr731 in GST-CBL-LZIP eliminated binding of the p85 subunit of phosphatidylinositol 3-kinase and substantially reduced the phosphorylation of this fusion protein by Fyn, despite the presence of four other tyrosine residues in this fusion protein. These data are consistent with the hypothesis that Cbl represents a substrate for Src-like kinases that are activated in response to the engagement of cell surface receptors, and that Src-like kinases are responsible for the phosphorylation of a tyrosine residue in Cbl that may regulate activation of phosphatidylinositol 3-kinase.
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PMID:Fyn associates with Cbl and phosphorylates tyrosine 731 in Cbl, a binding site for phosphatidylinositol 3-kinase. 989 Sep 70

While cancer drug resistance has been extensively studied in cell culture, little is known about more clinically relevant in vivo resistance. The in vivo resistance of a murine mammary carcinoma EMT-6 to alkylating agents was demonstrated in the present study to be associated with multiple biochemical changes. These included an up to 1.5-fold increase in activity of phase II drug metabolizing enzymes (DMEs), such as glutathione (GSH), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPX) and aldehyde dehydrogenase (ALDH), and an up to 88% decrease of phase I DME activity [7-ethoxycumarin O-deethylase (ECOD), P450 reductase (PR)] in the resistant tumors compared with the parental tumor. Transplant of either parental or resistant tumors to mice was accompanied by a decrease of both phase I and phase II DME activity in the livers of female Balb/C mice compared with the non-tumor mice. Moreover, at the protein level, while cytochrome P450 (CYP) IIB1/2 in the liver of mouse bearing both the sensitive and the resistant tumor was significantly diminished compared to that in the liver of non-tumor control mouse in Western analysis, there was actually an increase of this protein in the liver of the host bearing either of the two resistant tumors compared to that of the sensitive tumor-bearing animal. Although this in vivo resistance phenotype is not expressed in cell culture, the profile of most of the enzyme changes in the resistant tumors remained similar in in vitro culture of the isolated tumor cells. Collectively, these results demonstrate that this in vivo alkylating agent resistance is associated with multiple changes of both phase I and phase II DMEs in the resistant tumors, and some of these, such as CYP IIB1/2 protein are further altered in the resistant tumor-bearing mouse liver, suggesting a potential role of systemic factors in this resistance phenotype.
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PMID:Biochemical characterization of in vivo alkylating agent resistance of a murine EMT-6 mammary carcinoma. Implication for systemic involvement in the resistance phenotype. 992 73

Two minisatellite (D1S80, D17S5) and 10 microsatellite (D2S1328, TPO, D3S1358, D9S926, D11S2010, THO1, VWF, FES, D16S310, and D18S848) polymorphic loci were analyzed in 5 Greek population groups (eastern Macedonia, central Macedonia, Thessaly, Epirus, and Greeks from Asia Minor) using the polymerase chain reaction. The genotypes at these loci conformed to Hardy-Weinberg equilibrium, and pairwise comparisons between them were in agreement with the expectation of independence between loci. This along with the low values of the coefficient of gene differentiation (GST) and the high heterozygosity levels of all loci allows the use of allele frequency data from the 12 hypervariable DNA markers for medicolegal casework in the Greek population groups studied. The small genetic distances indicate a genetic affinity among the 5 population samples. However, a few markers seem to allow some discrimination among the groups. No significant differences with other European populations were found for the loci studied.
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PMID:Genetic studies in 5 Greek population samples using 12 highly polymorphic DNA loci. 997 96


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