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)

One of the immediate cellular responses to stimulation by various growth factors is the activation of a phosphatidylinositol (PI) 3-kinase. We recently cloned the 85-kDa subunit of PI 3-kinase (p85) from a lambda gt11 expression library, using the tyrosine-phosphorylated carboxy terminus of the epidermal growth factor (EGF) receptor as a probe (E. Y. Skolnik, B. Margolis, M. Mohammadi, E. Lowenstein, R. Fischer, A. Drepps, A. Ullrich, and J. Schlessinger, Cell 65:83-90, 1991). In this study, we have examined the association of p85 with EGF and platelet-derived growth factor (PDGF) receptors and the tyrosine phosphorylation of p85 in 3T3 (HER14) cells in response to EGF and PDGF treatment. Treatment of cells with EGF or PDGF markedly increased the amount of p85 associated with EGF and PDGF receptors. Binding assays with glutathione S-transferase (GST) fusion proteins demonstrated that either Src homology region 2 (SH2) domain of p85 is sufficient for binding to EGF and PDGF receptors and that receptor tyrosine autophosphorylation is required for binding. Binding of a GST fusion protein expressing the N-terminal SH2 domain of p85 (GST-N-SH2) to EGF and PDGF receptors was half-maximally inhibited by 2 and 24 mM phosphotyrosine (P-Tyr), respectively, suggesting that the N-SH2 domain interacts more stably with PDGF receptors than with EGF receptors. The amount of receptor-p85 complex detected in HER14 cells treated with EGF or PDGF. Growth factor treatment also increased the amount of p85 found in anti-PDGF-treated HER14 cells, suggesting that the vast majority of p85 in the anti-P-Tyr fraction is receptor associated but not phosphorylated on tyrosine residues. Only upon transient overexpression of p85 and PDGF receptor did p85 become tyrosine phosphorylated. These are consistent with the hypothesis that p85 functions as an adaptor molecule that targets PI 3-kinase to activated growth factor receptors.
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PMID:Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors. 137 91

The molecular interactions of the Src homology 2 (SH2) domain and the N-terminal proline-rich sequence motifs (pro-1 to pro-5) of the SH2 protein Shb with other components were presently characterised. Using a degenerate phosphopeptide library the preferred binding site for the Shb SH2 domain was determined to pTyr-Thr/Val/Ile-X-Leu at positions +1 to +3 relative the phosphotyrosine residue. Experiments with competing peptides and platelet-derived growth factor (PDGF) beta-receptor mutants with Y to F substitutions in autophosphorylation sites revealed multiple binding sites for the Shb SH2 domain in the receptor. The Shb SH2 domain also binds to in vitro phosphorylated fibroblast growth factor receptor-1 (FGFR-1) mainly through position Y776. The receptor experiments suggest that other residues besides the +1 to +3 positions may also be of significance for Shb binding. The pro-4/pro-5 motif of Shb binds in vitro particularly well to the Src, p85 alpha PI3-kinase and Eps8 SH3 domains expressed as GST fusion proteins. However, the GST-SH3 domain fusion proteins tested bind in vitro to peptides corresponding to the pro-1 to pro-5 motifs of Shb with low affinity and selectivity, suggesting that sequences outside the core proline motif may also be important for Shb-SH3 domain interactions. In vivo association between Shb-SH3 domain proteins v-Src and Eps8 was detected by coimmunoprecipitation. PDGF treatment did not affect the association between Eps8 and Shb. The data suggest that Shb is an adaptor protein linking SH3 domain proteins to tyrosine kinases or other tyrosine phosphorylated proteins.
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PMID:Molecular interactions of the Src homology 2 domain protein Shb with phosphotyrosine residues, tyrosine kinase receptors and Src homology 3 domain proteins. 753 62

To identify serum-inducible genes in the insulin-producing cell line beta TC-1, a library subtraction screening procedure was performed on serum-deprived (G0) and serum-restimulated (G1) insulin-producing beta TC-1 cells. A cDNA containing a motif with strong homology to Src homology 2 (SH2) domains was found using this procedure and called Shb. The Shb cDNA contains two methionine codons in its N-terminus and thus may code for two proteins of 67 and 56 kDa, each with one SH2 domain in its C-terminus. No other structural similarity to proteins with catalytic activity could be detected, suggesting that Shb is a so called adaptor. Shb contains the proline-rich sequence PPPGPGR between the two proposed initiator methionines which resembles a sequence for binding to Src homology 3 (SH3) domains. A second proline-rich sequence was detected after the second methionine codon. The Shb cDNA hybridized to a similar or identical mRNA of 3.1 kb expressed in mouse brain, liver, kidney, heart, NIH3T3 fibroblasts and beta TC-1 cells. Western blot analysis of the same tissues using an antiserum directed against a synthetic peptide corresponding to a part of the SH2 domain of Shb, revealed reactivity with two proteins of 56 and 67 kDa. In addition, a third reactive component of 40 kDa was detected in most tissues. Transfection and transient expression of the Shb cDNA in COS-1 cells yielded increased expression of the 67, 56 and 40 kDa proteins. Transfection and stable expression of the Shb cDNA in pig aortic endothelial cells showed increased expression primarily of the 67 kDa protein. A fusion protein consisting of the SH2 domain of Shb linked to glutathione S-transferase showed increased binding to glycoproteins of cells stimulated with platelet-derived growth factor (PDGF-BB). Furthermore, the autophosphorylated PDGF beta-receptor but not the autophosphorylated epidermal growth factor (EGF) receptor bound specifically to immobilized fusion protein. It is concluded that Shb is a novel SH2-containing protein with proline-rich domains and therefore probably involved in the signal-transduction of some ligand-activated tyrosine kinase receptors.
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PMID:Shb is a ubiquitously expressed Src homology 2 protein. 830 79

The interaction between SH2 domains and phosphotyrosine-containing sequences was examined by real-time measurements of kinetic parameters. The SH2 domains of the p85 subunit of the phosphatidylinositol 3-kinase as well as of other signaling molecules were expressed in bacteria as glutathione S-transferase fusion proteins. Phosphotyrosine-containing peptides, corresponding to two autophosphorylation sites on the human platelet-derived growth factor beta-receptor that are responsible for phosphatidylinositol 3-kinase binding, were synthesized and used as capturing molecules, immobilized on a biosensor surface. The association and dissociation rate constants for binding to both sites were determined for intact p85 and the recombinant SH2 domains. High association rates were found to be coupled to very fast dissociation rates for all interactions studied. A binding specificity was observed for the two SH2 domains of p85, with the N-terminal SH2 binding with high affinity to the Tyr-751 site but not to the Tyr-740 site, and the C-terminal SH2 interacting strongly with both sites. This approach should be generally applicable to the study of the specificity inherent in the assembly of signaling complexes by activated protein-tyrosine kinase receptors.
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PMID:Interactions between SH2 domains and tyrosine-phosphorylated platelet-derived growth factor beta-receptor sequences: analysis of kinetic parameters by a novel biosensor-based approach. 838 38

Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable.
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PMID:Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression. 862 63

Screening of a human breast epithelial cell cDNA library with the tyrosine-phosphorylated C terminus of the epidermal growth factor receptor identified a novel member of the GRB7 gene family, designated GRB14. In addition to a pleckstrin homology domain-containing central region homologous to the Caenorhabditis elegans protein F10E9.6/mig 10 and a C-terminal Src homology 2 (SH2) domain, a conserved N-terminal motif, P(S/A)IPNPFPEL, can now be included as a hallmark of this family. GRB14 mRNA was expressed at high levels in the liver, kidney, pancreas, testis, ovary, heart, and skeletal muscle. Anti-Grb14 antibodies recognized a protein of approximately 58 kDa in a restricted range of human cell lines. Among those of breast cancer origin, GRB14 expression strongly correlated with estrogen receptor positivity, and differential expression was also observed among human prostate cancer cell lines. A GST-Grb14 SH2 domain fusion protein exhibited strong binding to activated platelet-derived growth factor (PDGF) receptors (PDGFRs) in vitro, but association between Grb14 and beta-PDGFRs could not be detected in vivo. In serum-starved cells, Grb14 was phosphorylated on serine residues, which increased with PDGF, but not EGF, treatment. Grb14 is therefore a target for a PDGF-regulated serine kinase, an interaction that does not require PDGFR-Grb14 association.
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PMID:Cloning and characterization of GRB14, a novel member of the GRB7 gene family. 864 58

Phospholipase C-gamma (PLCgamma) is the isozyme of PLC phosphorylated by multiple tyrosine kinases including epidermal growth factor, platelet-derived growth factor, nerve growth factor receptors, and nonreceptor tyrosine kinases. In this paper, we present evidence for the association of the insulin receptor (IR) with PLCgamma. Precipitation of the IR with glutathione S-transferase fusion proteins derived from PLCgamma and coimmunoprecipitation of the IR and PLCgamma were observed in 3T3-L1 adipocytes. To determine the functional significance of the interaction of PLCgamma and the IR, we used a specific inhibitor of PLC, U73122, or microinjection of SH2 domain glutathione S-transferase fusion proteins derived from PLCgamma to block insulin-stimulated GLUT4 translocation. We demonstrate inhibition of 2-deoxyglucose uptake in isolated primary rat adipocytes and 3T3-L1 adipocytes pretreated with U73122. Antilipolytic effect of insulin in 3T3-L1 adipocytes is unaffected by U73122. U73122 selectively inhibits mitogen-activated protein kinase, leaving the Akt and p70 S6 kinase pathways unperturbed. We conclude that PLCgamma is an active participant in metabolic and perhaps mitogenic signaling by the insulin receptor in 3T3-L1 adipocytes.
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PMID:Association of the insulin receptor with phospholipase C-gamma (PLCgamma) in 3T3-L1 adipocytes suggests a role for PLCgamma in metabolic signaling by insulin. 959 25

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

c-Myc plays a key role in the cell cycle dependent control of the PDGF beta-receptor mRNA. The mouse platelet-derived growth factor (PDGF) beta-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF beta-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF beta-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.
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PMID:Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor. 1128 29

PtdIns(3,4) P (2), a breakdown product of the lipid second messenger PtdIns(3,4,5) P (3), is a key signalling molecule in pathways controlling various cellular events. Cellular levels of PtdIns(3,4) P (2) are elevated upon agonist stimulation, mediating downstream signalling pathways by recruiting proteins containing specialized lipid-binding modules, such as the pleckstrin homology (PH) domain. A recently identified protein, TAPP1 (tandem-PH-domain-containing protein 1), has been shown to interact in vitro with high affinity and specificity with PtdIns(3,4) P (2) through its C-terminal PH domain. In the present study, we have utilized this PH domain tagged with glutathione S-transferase (GST-TAPP1-PH) as a probe in an on-section immunoelectron microscopy labelling procedure, mapping the subcellular distribution of PtdIns(3,4) P (2). As expected, we found accumulation of PtdIns(3,4) P (2) at the plasma membrane in response to the agonists platelet-derived growth factor and hydrogen peroxide. Importantly, however, we also found agonist stimulated PtdIns(3,4) P (2) labelling of intracellular organelles, including the endoplasmic reticulum and multivesicular endosomes. Expression of the 3-phosphatase PTEN (phosphatase and tensin homologue deleted on chromosome 10) in PTEN-null U87MG cells revealed differential sensitivity of these lipid pools to the enzyme. These data suggest a role for PtdIns(3,4) P (2) in endomembrane function.
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PMID:Detection of novel intracellular agonist responsive pools of phosphatidylinositol 3,4-bisphosphate using the TAPP1 pleckstrin homology domain in immunoelectron microscopy. 1460 33


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