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)

CD43, the most abundant membrane protein of T lymphocytes, is able to initiate signal transduction pathways that lead to Ca2+ mobilization and interleukin-2 production, yet the molecular events involved in CD43's signal transduction pathway are poorly understood. In the present report we show that activation of both purified T lymphocytes and Jurkat cells, through CD43 cross-linking with the anti-CD43 L10 monoclonal antibody, induced CD43 association to Fyn kinase. This association is mediated by the Src homology 3 (SH3) domain of Fyn, since a glutathione S-transferase-Fyn SH3 fusion protein was able to precipitate CD43 from lysates of CD43-activated T cells. A synthetic peptide containing the SH3 binding sites of p85, located within the amino acid sequence 300ERQPAPALPPKPPKP314, was able to inhibit binding of CD43 to Fyn as well as to the glutathione S-transferase-Fyn SH3 fusion protein. We also provide evidence that upon CD43 cross-linking, Fyn is tyrosine-phosphorylated in a time-dependent manner. Our results suggest that CD43 cross-linking on the T cell surface induces the interaction between CD43 and Fyn, presumably through the Fyn SH3 domain and a putative SH3 binding site in CD43, leading to Fyn tyrosine phosphorylation and signal propagation.
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PMID:CD43-specific activation of T cells induces association of CD43 to Fyn kinase. 891 Mar 42

Human CD7 is a 40 kDa protein expressed on thymocytes, early T, B, NK and myeloid lineage cells in bone marrow, and on mature T and NK cells. Previous studies suggested human CD7 may be involved in T and NK cell activation and/or adhesion, and that CD7-mediated cell activation may be transduced via the lipid kinase phosphatidylinositol 3-kinase (Pi3-kinase), a heterodimeric cytosolic protein consisting of an 85 kDa adaptor subunit that is coupled to a 110 kDa catalytic subunit. It has recently been shown that a sequence motif present in the cytoplasmic tall of both human and mouse CD7 bound with high affinity to recombinant SH2 domains present in the p85 subunit of Pi3-kinase. In this work, we used co-precipitation with anti-CD7 mAb 3A1 and recombinant p85 SH2-GST fusion proteins and peptide competition analysis to demonstrate that the cytoplasmic tail of CD7 interacts with a functional Pi3-kinase via the pTyr-X-X-Met motif. Furthermore, we show that cross-linking of CD7 markedly increased the amount of Pi3-kinase activity associated with CD7. The interaction of CD7 with the Pi3-kinase signal transduction pathway provides a mechanism for the previously observed functional responses attributed to CD7-mediated T and NK cell activation.
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PMID:Functional association of CD7 with phosphatidylinositol 3-kinase: interaction via a YEDM motif. 891 88

We reported that ecto-NAD+ glycohydrolase activity induced upon differentiation of HL-60 cells with retinoic acid is localized on the extracellular domain of CD38 and that CD38 ligation by a specific monoclonal antibody, HB-7, is followed by rapid tyrosine phosphorylation of cellular proteins including a proto-oncogene product, Cbl. In the present study, we investigated intracellular signaling linked to the HB-7-induced Cbl phosphorylation in dibutyryl cAMP-treated THP-1 cells. The 85-kDa regulatory subunit (p85) of phosphatidylinositol (PI) 3-kinase was immunoprecipitated with anti-Cbl antibody in a manner dependent on the tyrosine phosphorylation of Cbl. PI 3-kinase activity was also observed in the immunoprecipitated fractions containing tyrosine-phosphorylated Cbl. The phosphorylated form of Cbl, which had been separated from the CD38-stimulated cells, was capable of directly binding to a recombinant p85 fused to glutathione S-transferase. Thus, the direct association of tyrosine-phosphorylated Cbl with PI 3-kinase, possibly leading to the kinase activation, appeared to be involved in intracellular signaling caused by the CD38 ligation.
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PMID:Association of phosphatidylinositol 3-kinase with the proto-oncogene product Cbl upon CD38 ligation by a specific monoclonal antibody in THP-1 cells. 894 25

We have developed a polyclonal antibody that activates the heterodimeric p85-p110 phosphatidylinositol (PI) 3'-kinase in vitro and in microinjected cells. Affinity purification revealed that the activating antibody recognized the N-terminal SH2 (NSH2) domain of p85, and the antibody increased the catalytic activity of recombinant p85-p110 dimers threefold in vitro. To study the role of endogenous PI 3'-kinase in intact cells, the activating anti-NSH2 antibody was microinjected into GRC + LR73 cells, a CHO cell derivative selected for tight quiescence during serum withdrawal. Microinjection of anti-NSH2 antibodies increased bromodeoxyuridine (BrdU) incorporation fivefold in quiescent cells and enhanced the response to serum. These data reflect a specific activation of PI 3'-kinase, as the effect was blocked by coinjection of the appropriate antigen (glutathione S-transferase-NSH2 domains from p85 alpha), coinjection of inhibitory anti-p110 antibodies, or treatment of cells with wortmannin. We used the activating antibodies to study signals downstream from PI 3'-kinase. Although treatment of cells with 50 nM rapamycin only partially decreased anti-NSH2-stimulated BrdU incorporation, coinjection with an anti-p70 S6 kinase antibody effectively blocked anti-NSH2-stimulated DNA synthesis. We also found that coinjection of inhibitory anti-ras antibodies blocked both serum- and anti-NSH2-stimulated BrdU incorporation by approximately 60%, and treatment of cells with a specific inhibitor of MEK abolished antibody-stimulated BrdU incorporation. We conclude that selective activation of physiological levels of PI 3'-kinase is sufficient to stimulate DNA synthesis in quiescent cells. PI 3'-kinase-mediated DNA synthesis requires both p70 S6 kinase and the P21ras/MEK pathway.
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PMID:Specific activation of p85-p110 phosphatidylinositol 3'-kinase stimulates DNA synthesis by ras- and p70 S6 kinase-dependent pathways. 897 5

Actin-binding proteins such as profilin and gelsolin bind to phosphatidylinositol (PI) 4,5-bisphosphate (PI 4,5-P2) and regulate the concentration of monomeric actin. We report here that profilin and gelsolin stimulate PI 3-kinase-mediated phosphorylation of PI 4,5-P2 (lipid kinase activity) in a concentration-dependent manner. This effect is specific to profilin and gelsolin because other cytoskeletal proteins such as tau or actin do not affect PI 3-kinase activity. In addition to lipid kinase activity, PI 3-kinase also has protein kinase activity: it phosphorylates proteins (p85 subunit of PI 3-kinase). However, the protein kinase activity of PI 3-kinase was not affected in the presence of profilin. Kinetic analysis, as a function of varying concentrations of ATP and PI 4,5-P2, showed that profilin affects the Vmax of PI 3-kinase without affecting k(m). Profilin may also affect PI 3-kinase activity by its direct association to the enzyme because dot-blot analysis using antibody to glutathione S-transferase (GST) suggested that GST-85 kDa, a fusion protein of PI 3-kinase, binds to profilin. However, PI 3-kinase did not affect the actin-sequestering ability of profilin (determined by pyrene-labeled actin), which indicates that actin and p85 do not share a common binding site on profilin. These studies suggest that profilin and gelsolin may control the generation of 3-OH phosphorylated phosphoinositides, which in turn may regulate the actin polymerization.
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PMID:Profilin and gelsolin stimulate phosphatidylinositol 3-kinase activity. 898 88

Insulin receptor substrate 1 (IRS-1), and its structural relative IRS-2, are both phosphorylated on tyrosine following treatment of cells with interleukin-4 (IL-4) and insulin. We have investigated whether both IRS-1 and IRS-2 are expressed in murine lymphohemopoietic cells. T and B lymphocytes and macrophages from primary cultures expressed only IRS-2, which became phosphorylated on tyrosine following stimulation with both IL-4 and insulin. Likewise, the murine myeloid cell line FD-5 expressed only IRS-2, which was tyrosine phosphorylated in response to IL-4 and insulin, as well as interleukin-3 and granulocyte-macrophage colony stimulating factor. Neither IRS-1 nor IRS-2 were expressed at detectable levels in primary bone marrow mast cells although these cells do respond to IL-4. Moreover, a factor-dependent lymphocyte cell line, CT.4S, which grows continuously in IL-4, did not express detectable levels of IRS-1 or IRS-2. IRS-2 from FD-5 cells stimulated with either IL-4 or insulin bound to glutathione S-transferase fusion proteins of the p85 subunit of phosphoinositol 3'-kinase, Grb2, and Syp, paralleling reported associations of IRS-1 with these molecules and indicating phosphorylation of the corresponding residues on IRS-2.
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PMID:Insulin receptor substrate-2 is the major 170-kDa protein phosphorylated on tyrosine in response to cytokines in murine lymphohemopoietic cells. 899 47

The human proto-oncogene product c-Cbl and a similar protein in Caenorhabditis elegans (Sli-1) contain a proline-rich COOH-terminal region that binds Src homology 3 (SH3) domains of proteins such as the adapter Grb2. Cb1-Grb2 complexes can be recruited to tyrosine-phosphorylated epidermal growth factor (EGF) receptors through the SH2 domain of Grb2. Here we identify by molecular cloning a Drosophila cDNA encoding a protein (Drosophila Cbl [D-Cbl]) that shows high sequence similarity to the N-terminal region of human c-Cbl but lacks proline-rich sequences and fails to bind Grb2. Nonetheless, in COS-1 cells, expression of hemagglutinin epitope-tagged D-Cbl results in its coimmunoprecipitation with EGF receptors in response to EGF. EGF also caused tyrosine phosphorylation of D-Cbl in such cells, but no association of phosphatidylinositol 3-kinase was detected in assays using anti-p85 antibody. A point mutation in D-Cbl (G305E) that suppresses the negative regulation of LET-23 by the Cbl homolog Sli-1 in C. elegans prevented tyrosine phosphorylation of D-Cbl as well as binding to the liganded EGF receptor in COS-1 cells. Colocalization of EGF receptors with both endogenous c-Cbl or expressed D-Cbl in endosomes of EGF-treated COS-1 cells is also demonstrated by immunofluorescence microscopy. In lysates of adult transgenic Drosophila melanogaster, GST-DCbl binds to the tyrosine-phosphorylated 150-kDa torso-DER chimeric receptor. Expression of D-Cbl directed by the sevenless enhancer in intact Drosophila compromises severely the development of the R7 photoreceptor neuron. These data suggest that despite the lack of Grb2 binding sites, D-Cbl functions as a negative regulator of receptor tyrosine kinase signaling in the Drosophila eye by a mechanism that involves its association with EGF receptors or other tyrosine kinases.
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PMID:Interactions of Drosophila Cbl with epidermal growth factor receptors and role of Cbl in R7 photoreceptor cell development. 912 72

Expression cloning is an effective approach for isolating genes encoding proteins that associate with a target species. Several molecules have been isolated by expression cloning, including CRE-BP1 associating with Jun (Macgregor et al., 1990); Grb1, identical to p85 PI3-kinase, with the EGF receptor (Skolnik et al., 1991); and Max with Myc (Blackwood and Eisenman, 1991). Expression cloning involves induction of proteins from a lambda gt11 cDNA expression library and screening the proteins on nitrocellulose membranes using a peptide probe (Macgregor et al., 1990). With this method, we previously isolated an Lck tyrosine kinase-associated protein, LckBP1, which is identical to HS1 (Kitamura et al., 1989, 1995; Takemoto et al., 1995). In those experiments, we used a glutathione S-transferase (GST)-Lck SH3 domain fusion protein as a probe, followed by detection of the complex with anti-GST polyclonal antibody. Whereas the ease of obtaining the fusion construct and high-titer anti-GST polyclonal antibody represented clear advantages, the system suffered from high background and low sensitivity. Here we show that pretreatment of nitrocellulose filters with NaDodSO4 reduces background and, in turn, increases sensitivity.
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PMID:A simple improvement in expression cloning. 921 73

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.
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PMID:Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa. 926 59

We have observed previously the co-immunoprecipitation of the p85 subunit of phosphatidylinositol-3 kinase (PI3K) and SHP2 in murine lymphohemopoietic cells after stimulation with interleukin-3. We have investigated this interaction in more detail and now report the identification of a potentially novel 100-kDa protein (termed p100), which is inducibly phosphorylated on tyrosine after interleukin-3 treatment and which co-immunoprecipitates with both p85 PI3K and SHP2. The Src homology region 2 domains of both p85 and SHP2 appear to mediate their interactions with p100. Sequential precipitation analyses suggest that these interactions are direct and do not involve Grb2, and that the same p100 protein, or a portion of it, interacts with both p85 and SHP2, implying that p100 may serve to link these two proteins. Far Western blotting with both full-length p85 and isolated p85 Src homology region 2 domains supports this view. Interestingly, p100 also appears to be a substrate for the SHP2 phosphatase activity. In addition, p100 is precipitated by Grb2-glutathione S-transferase fusion proteins, an interaction largely mediated by the Grb2 SH3 domains. p100 appears to be distinct from JAK2, Vav, STAT5, and c-Cbl. Although largely cytosolic, p100 can be detected associated with SHP2 and PI3K in crude membrane fractions after interleukin-3 stimulation. We propose that p100 plays a role as an adaptor molecule, linking PI3K and SHP2 in IL-3 signaling.
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PMID:Interleukin-3 induces association of the protein-tyrosine phosphatase SHP2 and phosphatidylinositol 3-kinase with a 100-kDa tyrosine-phosphorylated protein in hemopoietic cells. 936 Oct 8


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