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)

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

The plethora of extracellular stimuli modulating the status of a cell results in the engagement of relatively few pathways responsible for transducing signals to the interior of the cell. One such pathway is the activation of phosphatidylinositol 3-kinase (PI-3K), which results in the generation of a membrane-restricted second messenger, polyphosphatidylinositide 3'-phosphate. Among the enzymes activated by 3'-phosphorylated inositol lipids is Akt/protein kinase B (PKB). Here we describe a protocol for the expression and one-step purification of human recombinant GST-PKB in Sf9 cells. This scheme allows generating large amounts of homogeneously purified GST-PKB with high specific activity to be employed in high-throughput screening or structural studies.
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PMID:Homogeneous purification of human recombinant GST-Akt/PKB from Sf9 cells. 1049 72

The breast cancer susceptibility gene BRCA1 encodes a nuclear phosphoprotein that acts as a tumor suppressor. Phosphorylation of BRCA1 has been implicated in altering its function, however, the pathway(s) that leads to the phosphorylation of BRCA1 has not been described. Here, a signaling pathway by which heregulin induces cell cycle-independent phosphorylation of BRCA1 was delineated. We showed that heregulin stimulation induced the phosphorylation of BRCA1 and concomitant activation of the serine/threonine kinase AKT in T47D human breast cancer cells. Heregulin-induced phosphorylation of BRCA1 was abrogated by phosphatidylinositol 3-kinase (PI3K) inhibitors and by a dominant-negative AKT. In the absence of heregulin, the ectopic expression of the constitutively active p110 subunit of PI3K was sufficient to induce BRCA1 phosphorylation. Furthermore, the purified glutathione S-transferase/AKT kinase phosphorylated BRCA1 in vitro. We have also shown that the phosphorylation of BRCA1 by AKT occurs on the residue Thr-509, which is located in the nuclear localization signal. These results reveal a novel signaling pathway that links extracellular signals to the phosphorylation of BRCA1 in breast cancer cells.
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PMID:Heregulin induces phosphorylation of BRCA1 through phosphatidylinositol 3-Kinase/AKT in breast cancer cells. 1054 66

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn(2+), but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg(2+), DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH(2)-terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione S-transferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK.
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PMID:Substrate specificities and identification of putative substrates of ATM kinase family members. 1060 6

Protein tyrosine phosphorylation is an integral part of cytokine-induced proliferation and differentiation of hematopoietic cells. The authors previously reported cloning and characterization of the receptor tyrosine kinase Tif, also termed Tyro3. Using the yeast 2-hybrid technology, they recently identified that the p85 subunit of phosphatidylinositol 3-kinase (PI3 kinase) interacted with the cytoplasmic domain of Tyro3. On treatment with epidermal growth factor (EGF), NIH3T3 cells expressed EGFR/Tyro3 (a fusion receptor with the extracellular domain from epidermal growth factor receptor and the transmembrane and cytoplasmic domains from Tyro3), and EGFR/Tyro3 was rapidly phosphorylated on tyrosine residues. The interaction between Tyro3 and p85 was also confirmed by glutathione S-transferase (GST) pull-down experiments. Co-immunoprecipitation followed by Western blot analysis revealed that PI3 kinase was associated with and phosphorylated by the activated Tyro3. Tyro3-associated PI3 kinase exhibited an enhanced kinase activity. In addition, EGF treatment of EGFR/Tyro3-expressing cells led to enhanced phosphorylation of Akt, a downstream component of PI3 kinase. Treatment of NIH3T3 cells expressing a full length of rat Tyro-3, but not NIH3T3 cells, with protein S also resulted in phosphorylation of Akt. Soft agar colony assays showed that the addition of EGF to EGFR/Tyro3-transfected cells, but not to the parental NIH3T3 cells, resulted in a concentration-dependent increase in the formation of anchorage-independent colonies. Tyro3-mediated transformation of NIH3T3 cells was significantly blocked by wortmannin, a PI3 kinase-specific inhibitor. Results of these combined studies strongly suggested that the oncogenic transforming ability of Tyro3 was mediated at least in part by the PI3 kinase pathway. (Blood. 2000;95:633-638)
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PMID:Transforming activity of receptor tyrosine kinase tyro3 is mediated, at least in part, by the PI3 kinase-signaling pathway. 1062 73

Akt activation requires phosphorylation of Thr(308) and Ser(473) by 3-phosphoinositide-dependent kinase-1 and 2 (PDK1 and PDK2), respectively. While PDK1 has been cloned and sequenced, PDK2 has yet to be identified. The present study shows that phosphatidylinositol 3-kinase-dependent p38 kinase activation regulates Akt phosphorylation and activity in human neutrophils. Inhibition of p38 kinase activity with SB203580 inhibited Akt Ser(473) phosphorylation following neutrophil stimulation with formyl-methionyl-leucyl-phenylalanine, FcgammaR cross-linking, or phosphatidylinositol 3,4,5-trisphosphate. Concentration inhibition studies showed that Ser(473) phosphorylation was inhibited by 0.3 microm SB203580, while inhibition of Thr(308) phosphorylation required 10 microm SB203580. Transient transfection of HEK293 cells with adenoviruses containing constitutively active MKK3 or MKK6 resulted in activation of both p38 kinase and Akt. Immunoprecipitation and glutathione S-transferase (GST) pull-down studies showed that Akt was associated with p38 kinase, MK2, and Hsp27 in neutrophils, and Hsp27 dissociated from the complex upon activation. Active recombinant MK2 phosphorylated recombinant Akt and Akt in anti-Akt, anti-MK2, anti-p38, and anti-Hsp27 immunoprecipitates, and this was inhibited by an MK2 inhibitory peptide. We conclude that Akt exists in a signaling complex containing p38 kinase, MK2, and Hsp27 and that p38-dependent MK2 activation functions as PDK2 in human neutrophils.
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PMID:p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. 1104 4

Hepatocyte growth factor (HGF)/scatter factor is a multifunctional cytokine that induces mitogenesis, motility, and morphogenesis in epithelial, endothelial, and neuronal cells. The receptor for HGF/scatter factor was identified as c-Met tyrosine kinase, and activation of the receptor induces multiple signaling cascades. To gain further insight into c-Met-mediated multiple events at a molecular level, we isolated several signaling molecules including a novel binding partner of c-Met, SH2 domain-containing inositol 5-phosphatase 1 (SHIP-1). Western blot analysis revealed that SHIP-1 is expressed in the epithelial cell line, Madin-Darby canine kidney (MDCK) cells. SHIP-1 binds at phosphotyrosine 1356 at the multifunctional docking site. Because a number of signaling molecules such as Grb2, phosphatidylinositol 3-kinase, and Gab1 bind to the multifunctional docking site, we further performed an in vitro competition study using glutathione S-transferase- or His-tagged signaling molecules with c-Met tyrosine kinase. Our binding study revealed that SHIP-1, Grb2, and Gab1 bound preferentially over phosphatidylinositol 3-kinase. Surprisingly, MDCK cells that overexpress SHIP-1 demonstrated branching tubulogenesis within 2 days after HGF treatment, whereas wild-type MDCK cells showed tubulogenesis only after 6 days following treatment without altering cell scattering or cell growth potency. Furthermore, overexpression of a mutant SHIP-1 lacking catalytic activity impaired HGF-mediated branching tubulogenesis.
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PMID:Src homology 2-containing inositol 5-phosphatase 1 binds to the multifunctional docking site of c-Met and potentiates hepatocyte growth factor-induced branching tubulogenesis. 1106 26

The epithelial Na+ channel (ENaC) constitutes the rate-limiting step for Na+ transport across tight epithelia and is the principal target of hormonal regulation, particularly by insulin and mineralocorticoids. Recently, the serine-threonine kinase (SGK) was identified as a rapidly mineralocorticoid-responsive gene, the product of which stimulates ENaC-mediated Na+ transport. Like its close relative, protein kinase B (also called Akt), SGK's kinase activity is dependent on phosphatidylinositol 3-kinase (PI3K), a key mediator of insulin signaling. In our study we show that PI3K is required for SGK-dependent stimulation of ENaC-mediated Na+ transport as well as for the production of the phosphorylated form of SGK. In A6 kidney cells, mineralocorticoid induction of the phosphorylated form of SGK preceded the increase in Na+ transport, and specific inhibition of PI3K inhibited both phosphorylation of SGK and mineralocorticoid-induced Na+ transport. Insulin both augmented SGK phosphorylation and synergized with mineralocorticoids in stimulating Na+ transport. In a Xenopus laevis oocyte coexpression assay, SGK-stimulated ENaC activity was also markedly reduced by PI3K inhibition. Finally, in vitro-translated SGK specifically interacted with the ENaC subunits expressed in Escherichia coli as glutathione S-transferase fusion proteins. These data suggest that SGK is a PI3K-dependent integrator of insulin and mineralocorticoid actions that interacts with ENaC subunits to control Na+ entry into kidney collecting duct cells.
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PMID:SGK integrates insulin and mineralocorticoid regulation of epithelial sodium transport. 1120 6

Etk/Bmx, a member of the Tec family of nonreceptor protein-tyrosine kinases, is characterized by an N-terminal pleckstrin homology domain and has been shown to be a downstream effector of phosphatidylinositol 3-kinase. P21-activated kinase 1 (Pak1), another well characterized effector of phosphatidylinositol 3-kinase, has been implicated in the progression of breast cancer cells. In this study, we characterized the role of Etk in mammary development and tumorigenesis and explored the functional interactions between Etk and Pak1. We report that Etk expression is developmentally regulated in the mammary gland. Using transient transfection, coimmunoprecipitation and glutathione S-transferase-pull down assays, we showed that Etk directly associates with Pak1 via its N-terminal pleckstrin homology domain and also phosphorylates Pak1 on tyrosine residues. The expression of wild-type Etk in a non-invasive human breast cancer MCF-7 cells significantly increased proliferation and anchorage-independent growth of epithelial cancer cells. Conversely, expression of kinase-inactive mutant Etk-KQ suppressed the proliferation, anchorage-independent growth, and tumorigenicity of human breast cancer MDA-MB435 cells. These results indicate that Pak1 is a target of Etk and that Etk controls the proliferation as well as the anchorage-independent and tumorigenic growth of mammary epithelial cancer cells.
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PMID:Etk/Bmx tyrosine kinase activates Pak1 and regulates tumorigenicity of breast cancer cells. 1138 70

Based on our previous observations that active ERK associates with and phosphorylates Gab1 in response to HGF, and the prediction that the ERK phosphorylation site is adjacent to one of the phosphatidylinositol 3-kinase (PI3K) SH2 binding motifs, we examined the possibility that ERK phosphorylation can regulate the Gab1/PI3K association. The HGF-mediated association of Gab1 with either full-length GST-p85 or its isolated N- or C-terminal SH2 domains was inhibited by approximately 50% in the setting of ERK inhibition, a result confirmed by co-immunoprecipitation of the native proteins. A 14-amino acid peptide encoding (472)YVPMTP(477) (one of the major p85 binding sites in Gab1 and the predicted ERK phosphorylation site) was synthesized with either phosphotyrosine alone (pY), or phosphotyrosine + phosphothreonine (pYT). In both pull-down assays and competition assays, pYT demonstrated a higher affinity for p85 than did pY alone. Finally, examination of the phosphorylation state of Akt after HGF stimulation revealed that ERK inhibition resulted in a decrease in Akt activation at both 5 and 10 min. These results suggest that activated ERK can phosphorylate Gab1 in response to HGF stimulation and thereby potentiate the Gab1/PI3K association and subsequent PI3K activation.
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PMID:ERK regulates the hepatocyte growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. 1144 78


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