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)

The Crk adapter proteins are assumed to play a role in T lymphocyte activation because of their induced association with tyrosine-phosphorylated proteins, such as ZAP-70 and Cbl, and with the phosphatidylinositol 3kinase regulatory subunit, p85, following engagement of the T cell antigen receptor. Although the exact mechanism of interaction between these molecules has not been fully defined, it has been generally accepted that Crk, ZAP-70, and p85 interact with tyrosine-phosphorylated Cbl, which serves as a major scaffold protein in activated T lymphocytes. Our present results demonstrate a cell activation-dependent reciprocal co-immunoprecipitation of CrkII and p85 from lysates of Jurkat T cells and a direct binding of CrkII to p85 in an overlay assay. The use of bead-immobilized GST fusion proteins indicated a complex mechanism of interaction between CrkII and p85 involving two distinct and mutually independent regions in each molecule. A relatively high affinity binding of the CrkII-SH3(N) domain to p85 and the p85-proline-B cell receptor-proline (PBP) region to CrkII was observed in lysates of either resting or activated T cells. Direct physical interaction between the CrkII-SH3(N) and the p85-PBP domain was demonstrated using recombinant fusion proteins and was further substantiated by binding competition studies. In addition, immobilized fusion proteins possessing the CrkII-SH2 and p85-SH3 domains were found to pull down p85 and CrkII, respectively, but only from lysates of activated T cells. Nevertheless, the GST-CrkII-SH2 fusion protein was unable to mediate direct association with p85 from lysates of either resting or activated T cells. Our results support a model in which T cell activation dependent conformational changes in CrkII and/or p85 promote an initial direct or indirect low affinity interaction between the two molecules, which is then stabilized by a secondary high affinity interaction mediated by direct binding of the CrkII-SH3(N) to the p85-PBP domain.
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PMID:T cell activation induces direct binding of the Crk adapter protein to the regulatory subunit of phosphatidylinositol 3-kinase (p85) via a complex mechanism involving the Cbl protein. 1141 12

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

Tie1 is an orphan receptor tyrosine kinase that is expressed almost exclusively in endothelial cells and that is required for normal embryonic vascular development. Genetic studies suggest that Tie1 promotes endothelial cell survival, but other studies have suggested that the Tie1 kinase has little to no activity, and Tie1-mediated signaling pathways are unknown. To begin to study Tie1 signaling, a recombinant glutathione S-transferase (GST)-Tie1 kinase fusion protein was produced in insect cells and found to be autophosphorylated in vitro. GST-Tie1 but not a kinase-inactive mutant associated with a recombinant p85 SH2 domain protein in vitro, suggesting that Tie1 might signal through phosphatidylinositol (PI) 3-kinase. To study Tie1 signaling in a cellular context, a c-fms-Tie1 chimeric receptor (fTie1) was expressed in NIH 3T3 cells. Ligand stimulation of fTie1 resulted in Tie1 autophosphorylation and downstream activation of PI 3-kinase and Akt. Stimulation of fTie1-expressing cells potently inhibited UV irradiation-induced apoptosis in a PI 3-kinase-dependent manner. Moreover, both Akt phosphorylation and inhibition of apoptosis were abrogated by mutation of tyrosine 1113 to phenylalanine, suggesting that this residue is an important PI 3-kinase binding site. These findings are the first biochemical demonstration of a signal transduction pathway and corresponding cellular function for Tie1, and the antiapoptotic effect of Tie1 is consistent with the results of previous genetic studies.
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PMID:The endothelial receptor tyrosine kinase Tie1 activates phosphatidylinositol 3-kinase and Akt to inhibit apoptosis. 1186 50

We have examined the ability of epidermal growth factor (EGF)-stimulated ERK activation to regulate Grb2-associated binder-1 (Gab1)/phosphatidylinositol 3-kinase (PI3K) interactions. Inhibiting ERK activation with the MEK inhibitor U0126 increased the EGF-stimulated association of Gab1 with either full-length glutathione S-transferase-p85 or the p85 C-terminal Src homology 2 (SH2) domain, a result reproduced by co-immunoprecipitation of the native proteins from intact cells. This increased association of Gab1 and the PI3K correlates with an increase in PI3K activity and greater phosphorylation of Akt. This result is in direct contrast to what we have previously reported following HGF stimulation where MEK inhibition decreased the HGF-stimulated association of Gab1 and p85. In support of this divergent effect of ERK on Gab1/PI3K association following HGF and EGF stimulation, U0126 decreased the HGF-stimulated association of p85 and the Gab1 c-Met binding domain but did not alter the EGF-stimulated association of p85 and the c-Met binding domain. An examination of the mechanism of this effect revealed that the treatment of cells with EGF + U0126 increased the tyrosine phosphorylation of Gab1 as well as its association with another SH2-containing protein, SHP2. Furthermore, overexpression of a catalytically inactive form of SHP2 or pretreatment with pervanadate markedly increased EGF-stimulated Gab1 tyrosine phosphorylation. These experiments demonstrate that EGF and HGF-mediated ERK activation result in divergent effects on Gab1/PI3K signaling. HGF-stimulated ERK activation increases the Gab1/PI3K association, whereas EGF-stimulated ERK activation results in a decrease in the tyrosine phosphorylation of Gab1 and a decreased association with the PI3K. SHP2 is shown to associate with and dephosphorylate Gab1, suggesting that EGF-stimulated ERK might act through the regulation of SHP2.
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PMID:ERK negatively regulates the epidermal growth factor-mediated interaction of Gab1 and the phosphatidylinositol 3-kinase. 1189 55

The 68 kDa Src substrate associated during mitosis (Sam68) is an RNA binding protein with Src homology (SH) 2 and 3 domain binding sites. We have recently found that Sam68 is a substrate of the insulin receptor (IR) that translocates from the nucleus to the cytoplasm and that Tyr-phosphorylated Sam68 associates with the SH2 domains of p85 PI3K and GAP, in vivo and in vitro. In the present work, we have further demonstrated the cytoplasmic localization of Sam68, which is increased in cells overexpressing IR. Besides, we sought to further study the association of Sam68 with the Ras-GAP pathway by assessing the interactions with SH3 domains of Grb2. We employed GST-fusion proteins containing the SH3 domains of Grb2 (N or C), and recombinant Sam68 for in vitro studies. In vivo studies of protein-protein interaction were assessed by co-immunoprecipitation experiments with specific antibodies against Sam68, GAP, Grb2, SOS, and phosphotyrosine; and by affinity precipitation with the fusion proteins (SH3-Grb2). Insulin stimulation of HTC-IR cells promotes phosphorylation of Sam68 and its association with the SH2 domains of GAP. Sam68 is constitutively associated with the SH3 domains of Grb2 and it does not change upon insulin stimulation, but Sam68 is Tyr-phosphorylated and promotes the association of GAP with the Grb2-SOS complex. In vitro studies with fusion proteins showed that Sam68 association with Grb2 is preferentially mediated by the C-terminal SH3 domains of Grb2. In conclusion, Sam68 is a substrate of the IR and may have a role as a docking protein in IR signaling, recruiting GAP to the Grb2-SOS complex, and in this way it may modulate Ras activity.
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PMID:Sam68 associates with the SH3 domains of Grb2 recruiting GAP to the Grb2-SOS complex in insulin receptor signaling. 1211 20

We previously reported (Rani, M. R., Asthagiri, A. R., Singh, A., Sizemore, N., Sathe, S. S., Li, X., DiDonato, J. D., Stark, G. R., and Ransohoff, R. M. (2001) J. Biol. Chem. 276, 44365-44368) that IFN-beta induction of beta-R1 in fibrosarcoma cells required transcription factors ISGF-3 and NF-kappa B. IFN-beta treatment did not augment the abundance of NF-kappa B, but led to phosphorylation of the NF-kappa B subunit p65 and induced a nuclear activity capable of phosphorylating a p65-GST fusion construct in the carboxy-terminal transactivation domain (TAD), residues 354-551. We now present evidence for the involvement of phosphoinositide 3-kinase (PI3K) in this pathway. Pretreatment of HT1080-derived fibrosarcoma cells with pharmacological inhibitors of PI3K (wortmannin or LY294002) selectively inhibited IFN-beta-induced beta-R1 mRNA accumulation in a dose-dependent manner. In stably transfected cell lines, bovine p85, the regulatory subunit of PI3K, functioned as a dominant-negative inhibitor of interferon (IFN) signaling via PI3K and selectively suppressed IFN-beta-mediated induction of beta-R1. Overexpression of PTEN (phosphatase and tensin homologue mutated on chromosome ten), an antagonist of PI3K, blocked induction of a beta-R1 promoter-reporter construct. Studies with PTEN mutants suggested that the lipid kinase activity of PI3K was essential for IFN-beta-induced transcription of beta-R1. Consistent with this finding, a dominant-negative mutant of the serine-threonine kinase Akt, a downstream effector of PI3K, selectively blocked IFN-beta-mediated induction of the beta-R1 promoter reporter. Furthermore, IFN-beta-mediated phosphorylation of GST-p65 was blocked by pretreatment with LY294002. These data suggest that IFN-beta acts through PI3K to enhance the transactivation competence of NF-kappa B complexes through phosphorylation of p65 within the TAD. The results provide novel insight into the role of PI3K in the transcriptional response to IFN-beta.
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PMID:Requirement of phosphoinositide 3-kinase and Akt for interferon-beta-mediated induction of the beta-R1 (SCYB11) gene. 1216 89

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 implied that activation of Src family kinases is involved in GPIb-mediated platelet activation, although the related signal transduction pathway remains poorly defined. This study presents evidence for an important role of Src and GPIb association. In platelet lysates containing Complete, a broad-spectrum protease inhibitor mixture, Src and Lyn dynamically associated with GPIb on VWF-botrocetin stimulation. Cytochalasin D, which inhibits translocation of Src kinases to the cytoskeleton, further increased Src and GPIb association. Similar results were obtained with botrocetin and monomeric A1 domain, instead of intact VWF, with induction of both Src activation and association between GPIb and Src. These findings suggest that ligand binding of GPIb, without receptor clustering, is sufficient to activate Src. Immunoprecipitation studies demonstrated that Src, phosphoinositide 3- kinase (PI 3-kinase), and GPIb form a complex in GPIb-stimulated platelets. When the p85 subunit of PI 3-kinase was immunodepleted, association of Src with GPIb was abrogated. However, wortmannin, a specific PI 3-kinase inhibitor, failed to block complex formation between Src and GPIb. The Src-SH3 domain as a glutathione S-transferase (GST)-fusion protein coprecipitated the p85 subunit of PI 3-kinase and GPIb. These findings taken together suggest that the p85 subunit of PI 3-kinase mediates GPIb-related activation signals and activates Src independently of the enzymatic activity of PI 3- kinase.
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PMID:Interaction between von Willebrand factor and glycoprotein Ib activates Src kinase in human platelets: role of phosphoinositide 3-kinase. 1239 36

Receptor activator of NF-kappaB ligand (RANKL) is essential for differentiation and function of osteoclasts. The negative signaling pathways downstream of RANKL are not well characterized. By retroviral transduction of RAW264.7 cells with a dominant negative Src homology 2 domain-containing phosphatase-1 (SHP-1)(C453S), we studied the role of tyrosine phosphatase SHP-1 in RANKL-induced osteoclastogenesis. Over-expression of SHP-1(C453S) significantly enhanced the number of tartrate-resistant acid phosphatase-positive multinuclear osteoclast-like cells in response to RANKL in a dose-dependent manner. RANKL induced the recruitment of SHP-1 to a complex containing TNFR-associated factor (TRAF)6. GST pull down experiments indicated that the association of SHP-1 with TRAF6 is mediated by SHP-1 lacking the two Src homology 2 domains. RANKL-stimulated IkappaB-alpha phosphorylation, IkappaB-alpha degradation and DNA binding ability of NF-kappaB were increased after over-expression of SHP-1(C453S). However, RANKL-induced phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated protein kinase, was unchanged. In addition, SHP-1 regulated RANKL-stimulated tyrosine phosphorylation of p85 subunit of phosphatidylinositol 3 kinase and the phosphorylation of Akt. Increased numbers of osteoclasts contribute to severe osteopenia in Me(v)/Me(v) mice due to mutation of SHP-1. Like RAW264.7 cells expressing SHP-1(C453S), the bone marrow macrophages of Me(v)/Me(v) mice generated much more osteoclast-like cells than that of littermate controls in response to RANKL. Furthermore compared with controls, RANKL induces enhanced association of TRAF6 and RANK in both RAW264.7 cells expressing SHP-1(C453S) and bone marrow macrophages from Me(v)/Me(v) mice. Therefore, SHP-1 plays a role in signals downstream of RANKL by recruitment to the complex containing TRAF6 and these observations may help to understand the mechanism of osteoporosis in Me(v)/Me(v) mice.
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PMID:Receptor activator of NF-kappa B ligand stimulates recruitment of SHP-1 to the complex containing TNFR-associated factor 6 that regulates osteoclastogenesis. 1450 Jun 59

A recent study (Nusrat, A., Chen, J. A., Foley, C. S., Liang, T. W., Tom, J., Cromwell, M., Quan, C., and Mrsny, R. J. (2000) J. Biol. Chem. 275, 29816-29822) suggested that phosphatidylinositol 3-kinase (PI 3-kinase) may interact with occludin; however, there exists no evidence of direct interaction of PI 3-kinase with the tight junctions. Activation of PI 3-kinase by oxidative stress and its role in disruption of tight junctions was examined in Caco-2 cell monolayer. The oxidative stress-induced decrease in electrical resistance, increase in inulin permeability, and redistribution of occludin and ZO-1 were reduced by a PI 3-kinase inhibitor, LY294002. Oxidative stress-induced tyrosine phosphorylation and dissociation from the actin cytoskeleton of occludin and ZO-1 were reduced by LY294002. The regulatory subunit of PI 3-kinase, p85, and the PI 3-kinase activity were co-immunoprecipitated with occludin, which were rapidly increased by oxidative stress. Oxidative stress resulted in increased translocation of p85 from the intracellular compartment into the intercellular junctions. Pair-wise glutathione S-transferase pull-down assay showed that glutathione S-transferase-occludin (C-terminal tail) binds to recombinant p85. This study shows that oxidative stress increases the association of PI 3-kinase with the occludin, and that PI 3-kinase activity is involved in oxidative stress-induced disruption of tight junction.
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PMID:Role of phosphatidylinositol 3-kinase in oxidative stress-induced disruption of tight junctions. 1450 Jul 30

In a previous study (Shin, E. Y., Shin, K. S., Lee, C. S., Woo, K. N., Quan, S. H., Soung, N. K., Kim, Y. G., Cha, C. I., Kim, S. R., Park, D., Bokoch, G. M., and Kim, E. G. (2002) J. Biol. Chem. 277, 44417-44430) we reported that phosphorylation of p85 betaPIX, a guanine nucleotide exchange factor (GEF) for Rac1/Cdc42, is a signal for translocation of the PIX complex to neuronal growth cones and is associated with basic fibroblast growth factor (bFGF)-induced neurite outgrowth. However, the issue of whether p85 betaPIX phosphorylation affects GEF activity on Rac1/Cdc42 is yet to be explored. Here we show that Rac1 activation occurs in a p85 betaPIX phosphorylation-dependent manner. A GST-PBD binding assay reveals that Rac1 is activated in a dose- and time-dependent manner in PC12 cells in response to bFGF. Inhibition of ERK or PAK2, the kinases upstream of p85 betaPIX in the bFGF signaling, prevents Rac1 activation, suggesting that phosphorylation of p85 betaPIX functions upstream of Rac1 activation. To directly address this issue, transfection studies with wild-type and mutant p85 betaPIX (S525A/T526A, a non-phosphorylatable form) were performed. Expression of mutant PIX markedly inhibits both bFGF- and nerve growth factor (NGF)-induced activation of Rac1, indicating that phosphorylation of p85 betaPIX is responsible for activation of this G protein. Both wild-type and mutant p85 betaPIX displaying negative GEF activity (L238R/L239S) are similarly recruited to growth cones, suggesting that Rac1 activation is not essential for translocation of the PIX complex (PAK2-p85 betaPIX-Rac1). However, expression of mutant p85 betaPIX (L238R/L239S) results in retraction of the pre-existing neurites. Our results provide evidence that bFGF- and NGF-induced phosphorylation of p85 betaPIX mediates Rac1 activation, which in turn regulates cytoskeletal reorganization at growth cones, but not translocation of the PIX complex.
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PMID:Basic fibroblast growth factor stimulates activation of Rac1 through a p85 betaPIX phosphorylation-dependent pathway. 1455 70


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