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)

To delineate the signaling pathway leading to glucose transport protein (GLUT4) translocation, we examined the effect of microinjection of the nonhydrolyzable GTP analog, guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), into 3T3-L1 adipocytes. Thirty minutes after the injection of 5 mM GTPgammaS, 40% of injected cells displayed surface GLUT4 staining indicative of GLUT4 translocation compared with 55% for insulin-treated cells and 10% in control IgG-injected cells. Treatment of the cells with the phosphatidylinositol 3-kinase inhibitor wortmannin or coinjection of GST-p85 SH2 fusion protein had no effect on GTPgammaS-mediated GLUT4 translocation. On the other hand, coinjection of antiphosphotyrosine antibodies (PY20) blocked GTPgammaS-induced GLUT4 translocation by 65%. Furthermore, microinjection of GTPgammaS led to the appearance of tyrosine-phosphorylated proteins around the periphery of the plasma membrane, as observed by immunostaining with PY20. Treatment of the cells with insulin caused a similar phosphotyrosine-staining pattern. Electroporation of GTPgammaS stimulated 2-deoxy-D-glucose transport to 70% of the extent of insulin stimulation. In addition, immunoblotting with phosphotyrosine antibodies after electroporation of GTPgammaS revealed increased tyrosine phosphorylation of several proteins, including 70- to 80-kDa and 120- to 130-kDa species. These results suggest that GTPgammaS acts upon a signaling pathway either downstream of or parallel to activation of phosphatidylinositol 3-kinase and that this pathway involves tyrosine-phosphorylated protein(s).
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PMID:Ligand-independent GLUT4 translocation induced by guanosine 5'-O-(3-thiotriphosphate) involves tyrosine phosphorylation. 942 34

We propose a novel model for the regulation of the p85/pl10alpha phosphatidylinositol 3'-kinase. In insect cells, the p110alpha catalytic subunit is active as a monomer but its activity is decreased by coexpression with the p85 regulatory subunit. Similarly, the lipid kinase activity of recombinant glutathione S-transferase (GST)-p110alpha is reduced by 65 to 85% upon in vitro reconstitution with p85. Incubation of p110alpha/p85 dimers with phosphotyrosyl peptides restored activity, but only to the level of monomeric p110alpha. These data show that the binding of phosphoproteins to the SH2 domains of p85 activates the p85/p110alpha dimers by inducing a transition from an inhibited to a disinhibited state. In contrast, monomeric p110 had little activity in HEK 293T cells, and its activity was increased 15- to 20-fold by coexpression with p85. However, this apparent requirement for p85 was eliminated by the addition of a bulky tag to the N terminus of p110alpha or by the growth of the HEK 293T cells at 30 degrees C. These nonspecific interventions mimicked the effects of p85 on p110alpha, suggesting that the regulatory subunit acts by stabilizing the overall conformation of the catalytic subunit rather than by inducing a specific activated conformation. This stabilization was directly demonstrated in metabolically labeled HEK 293T cells, in which p85 increased the half-life of p110. Furthermore, p85 protected p110 from thermal inactivation in vitro. Importantly, when we examined the effect of p85 on GST-p110alpha in mammalian cells at 30 degrees C, culture conditions that stabilize the catalytic subunit and that are similar to the conditions used for insect cells, we found that p85 inhibited p110alpha. Thus, we have experimentally distinguished two effects of p85 on p110alpha: conformational stabilization of the catalytic subunit and inhibition of its lipid kinase activity. Our data reconcile the apparent conflict between previous studies of insect versus mammalian cells and show that p110alpha is both stabilized and inhibited by dimerization with p85.
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PMID:Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. 948 53

We have used the yeast two-hybrid system to identify proteins that interact with the intracellular domain of the insulin-like growth factor-I receptor (IGFIR). In a search of a human fetal brain library we identified a cDNA encoding a protein that is the human homologue of mouse p55PIK, a regulatory subunit of phosphatidylinositol 3-kinase (hp55 gamma). The hp55 gamma protein interacts strongly with the activated IGFIR but not with the kinase-negative mutant receptor. hp55 gamma also interacts with the insulin receptor (IR) in the yeast two-hybrid system. The putative hp55 gamma protein is composed of a unique amino terminal region followed by a proline-rich motif and two Src homology 2 (SH2) domains, which are highly homologous to those in mouse p55PIK, rat p55 gamma, human p85 alpha and bovine p85 beta; it contains no SH3 domain. hp55 gamma mRNAs are expressed in most human fetal and adult tissues with particularly high abundance in adult testis. Splice variant(s) of hp55 gamma, one of which has a deletion of 36 amino acids at the amino terminus and another which has an insertion of 59 amino acids at position 256 between the SH2 domains, were also identified. A GST-hp55 gamma fusion protein interacts in vitro with both the activated IGFIR and IR derived from mammalian cells. Our findings suggest that hp55 gamma interacts with the IGFIR and IR and may be involved in PI 3-kinase activation by these receptors.
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PMID:Cloning of human p55 gamma, a regulatory subunit of phosphatidylinositol 3-kinase, by a yeast two-hybrid library screen with the insulin-like growth factor-I receptor. 952 59

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

Phosphatidylinositol (PI) 3-kinase plays an important role in transducing the signals of various growth factor receptors. However, the regulatory mechanism of PI3-kinase activity by these growth factor receptors is not completely understood. Therefore, we attempted to clarify the regulatory mechanism of PI3-kinase using insulin and 3T3 L1 fibroblasts. Our results showed that insulin stimulated PI3-kinase activity seven-fold and concomitantly phosphorylated a p85 subunit at the tyrosine residue. However, this tyrosine phosphorylation was not significant in the activation of PI3-kinase as the PI3-kinase pulled down by the overexpressed GST-p85 fusion protein showed as high an activity as the immunoprecipitated one. The p110 subunit was phosphorylated at both serine and tyrosine residues without insulin treatment. Since the phosphorylation state was not changed by insulin. The results suggested that phosphorylation of the p110 subunit does not control PI3-kinase activity. Finally, it was shown that the insulin receptor substrate-1 (IRS-1) binding to PI3-kinase was not sufficient for full activation because the amount of IRS-1 pulled down by the GST-p85 fusion protein reached almost maximum, after incubation with insulin-treated cell lysates for 20 min, whereas PI3-kinase activity reached its maximum only after incubation for 5 h. All results suggest that the phosphorylation of p85 subunit at tyrosine residues and phosphorylation of p110 subunit at tyrosine or serine residues are not functionally significant in the regulation of PI3-kinase activity. They also suggest that P13-kinase is needed to bind to other protein(s) as well as the insulin receptor substrate-1 for full activation.
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PMID:The regulatory mechanism of phosphatidylinositol 3-kinase by insulin in 3T3 L1 fibroblasts: phosphorylation-independent activation of phosphatidylinositol 3-kinase. 989 59

To examine the molecular mechanism of insulin receptor trafficking, we investigated the intracellular signaling molecules that regulate this process in Rat1 fibroblasts overexpressing insulin receptors. Cellular localization of insulin receptors was assessed by confocal laser microscopy with indirect immunofluorescence staining. Insulin receptors were visualized diffusely in the basal state. Insulin treatment induced the change of insulin receptor localization to perinuclear compartment. This insulin-induced insulin receptor trafficking was not affected by treatment of the cells with PI3-kinase inhibitor (wortmannin), whereas treatment with MEK [mitogen-activated protein (MAP) kinase-Erk kinase] inhibitor (PD98059) partly inhibited the process in a dose-dependent manner. Interestingly, treatment with both wortmannin and PD98059 almost completely inhibited insulin receptor trafficking. The functional importance of PI3-kinase and MAP kinase in the trafficking process was directly assessed by using single cell microinjection analysis. Microinjection of p85-SH2 and/or catalytically inactive MAP kinase ([K71A]Erk1) GST fusion protein gave the same results as treatment with wortmannin and PD98059. Furthermore, to determine the crucial step for the requirement of PI3-kinase and MAP kinase pathways, the effect of wortmannin and PD98059 on insulin receptor endocytosis was studied. Insulin internalization from the plasma membrane and subsequent insulin degradation were not affected by treatment with wortmannin and PD98059. In contrast, insulin receptor down-regulation from the cell surface and insulin receptor degradation, after prolonged incubation with insulin, were markedly impaired by the treatment. These results suggest that PI3-kinase and MAP kinase pathways synergistically regulate insulin receptor trafficking at a step subsequent to the receptor internalization.
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PMID:Synergistic role of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase cascade in the regulation of insulin receptor trafficking. 1043 44

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

Cytokine-dependent activation of distinct signaling pathways is a common scheme thought to be required for the subsequent programmation into cell proliferation and survival. The PI 3-kinase/Akt, Ras/MAP kinase, Ras/NFIL3 and JAK/STAT pathways have been shown to participate in cytokine mediated suppression of apoptosis in various cell types. However the relative importance of these signaling pathways seems to depend on the cellular context. In several cases, individual inhibition of each pathway is not sufficient to completely abrogate cytokine mediated cell survival suggesting that cooperation between these pathways is required. Here we showed that individual inhibition of STAT5, PI 3-kinase or MEK activities did not or weakly affected the IL-3 dependent survival of the bone marrow derived Ba/F3 cell line. However, the simultaneous inhibition of STAT5 and PI 3-kinase activities but not that of STAT5 and MEK reduced the IL-3 dependent survival of Ba/F3. Analysis of the expression of the Bcl-2 members indicated that phosphorylation of Bad and Bcl-x expression which are respectively regulated by the PI 3-kinase/Akt pathway and STAT5 probably explain this cooperation. Furthermore, we showed by co-immunoprecipitation studies and pull down experiments with fusion proteins encoding the GST-SH2 domains of p85 that STAT5 in its phosphorylated form interacts with the p85 subunit of the PI 3-kinase. These results indicate that the activations of STAT5 and the PI 3-kinase by IL-3 in Ba/F3 cells are tightly connected and cooperate to mediate IL-3-dependent suppression of apoptosis by modulating Bad phosphorylation and Bcl-x expression.
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PMID:Cooperation between STAT5 and phosphatidylinositol 3-kinase in the IL-3-dependent survival of a bone marrow derived cell line. 1071 4

Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), a primary output signal of phosphoinositide (PI) 3-kinase, plays a crucial role in diverse cellular processes. Evidence indicates that PIP(3) exerts downstream signaling, in part, by recruiting effector proteins to plasma membranes. Consequently, identification of signaling enzymes with PIP(3)-binding motifs represents a viable approach to understand the mechanism by which specificity of the PI 3-kinase-mediated signaling network is maintained. To address this issue, we have developed biotinylated derivatives of PIP(3) as affinity probes for the purification and characterization of PIP(3)-binding proteins. Considering the relaxed requirement for the acyl moiety in PIP(3) recognition, these biotinylated PIP(3) analogues display two structural features. First, they contain short acyl side chains (C(4) and C(8)), allowing them to be soluble in aqueous milieu. This desirable feature avoids the formation of lipid aggregates, which minimizes nonspecific hydrophobic interactions with proteins. Second, the appended biotin is located at the terminus of the sn-1 acyl side chain, thereby maintaining the integrity of the phosphoinositol head group essential for selective recognition. The utility of these affinity ligands is validated by the purification of recombinant PIP(3)-binding proteins, expressed as GST fusion proteins, to homogeneity from bacterial lysates. These include the C-terminal SH2 domain of the p85 subunit of PI 3-kinase and the N-terminal PH domain of PLCgamma1. The efficiency of biotinylated PIP(3) analogues in the purification of these recombinant proteins was approximately 20% of that of glutathione beads
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PMID:Biotinylated phosphatidylinositol 3,4,5-trisphosphate as affinity ligand. 1079 Mar 14

The binding of von Willebrand factor (vWF) to glycoprotein (GP) Ib-IX-V stimulates transmembrane signaling events that lead to platelet adhesion and aggregation. Recent studies have revealed that the signaling protein 14-3-3 zeta binds directly to the cytoplasmic domain of GP Ib alpha. In this study, the dynamic association of 14-3-3 zeta with GP Ib-IX, the phosphoinositide 3-kinase (PI 3-kinase), or both, was investigated in resting, thrombin, or vWF and botrocetin-stimulated platelets by analysis of discrete subcellular fractions. Results of this study demonstrate maximal coimmunoprecipitation of 14-3-3 zeta with GP Ib-IX in the nonstimulated cytosolic fraction and in the actin cytoskeletal fraction of thrombin- or vWF-stimulated human platelets. Immunoprecipitated 14-3-3 zeta or GP Ib from cytosolic fractions contained PI 3-kinase enzyme activity and an 85-kd polypeptide recognized by antibodies to the p85 subunit of PI 3-kinase. After platelet activation, the level of association between these species decreased in the cytosolic fraction. However, increased complex formation between 14-3-3 zeta and GP Ib-IX and between PI 3-kinase and GP Ib-IX was detected in actin cytoskeletal fractions derived from thrombin- or vWF-stimulated platelets. Recombinant glutathione S-transferase-14-3-3 zeta fusion protein (14-3-3 zeta-GST) inhibited affinity-captured PI 3-kinase enzyme activity up to 70% at 2 mcmol/L 14-3-3 zeta-GST. However, increasing concentrations up to 5 mcmol/L 14-3-3 zeta-GST resulted in the 3-fold enhancement of PI 3-kinase enzyme activity. We propose that the association between PI 3-kinase and 14-3-3 zeta with GP Ib-IX serves to promote the rapid translocation of these signaling proteins to the activated cytoskeleton, thereby regulating the formation of 3-position phosphoinositide-signaling molecules in this subcellular compartment. (Blood. 2000;96:577-584)
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PMID:Phosphoinositide 3-kinase forms a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3zeta. 1088 21


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