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Mechanism of Activation of Signaling Enzymes in Inositol Lipid Metabolism Following Ligation of Cell Surface Receptors in T Lymphocytes

T 淋巴细胞细胞表面受体连接后肌醇脂质代谢中信号酶的激活机制

基本信息

批准号：
02807029
负责人：
SASAKI Terukatsu
金额：
$ 1.22万
依托单位：
Sapporo Medical College
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

1. An increase in the cytoplasmic free Ca^<2+> concentration([Ca^<2+>]i)occurs on ligation with antibody of the T cell receptor (TCR) / CD3 complex of a human T cell leukemia line, Jurkat. It was found that the[Ca^<2+>]i response is inhibited by staurosporine(Stsp). Stsp markedly inhibited tyrosine phosphorylation of proteins found on ligation of the TCR/CD3 complex. Stsp had no effect on the[Ca^<2+>]i response in fibroblasts following the stimulation of cell surface receptors coupled to G-proteins, while the[Ca^<2+>]i response to the activation of receptor tyrosine kinase was inhibited by Stsp. These results indicate that a protein tyrosine kinase is involved in the[Ca^<2+>]i response to ligation of the TCR/CD3 complex.2. The ligation of the TCR/CD3 complex induces tyrosine phosphorylation of the zeta chain of the complex. Tyrosine-phosphorylated zeta chain may function as a binding site of proteins with src homology region 2(SH2)involved in the signal transduction in T lymphocytes. In order to screen such proteins, rat zeta chain and human p59^<fyn> are co-expressed in baculovirus system. The expression of zeta chain was confirmed by immunoblotting with anti zeta peptide(residues 110-122)antibody. Simultaneous expression of zeta chain and p59^<fyn> in SF9 cells resulted in the tyrosinephosphorylation of zeta chain. Screening for the proteins containing SH2 region are in progress by the use of lambdagt11 library of cDNA from Jurkat cells and the phosphorylated zeta chain, labeled with[ ^<32>P]phosphate at tyrosine residues, as a probe.3. Binding specificity of SH2 regions present in phospholipase Cgamma(PLCgamma)and nonreceptor protein tyrosine kinases, p56^<1ck> and p59^<fyn>, is studied. The SH2 regions of these proteins were expressed in E. coli as fusion proteins with the trpE gene product and affinity column of the SH2 regions were prepared. The SH2 region of PLC had different binding specificity from those of the SH2 regions of p56^<1ck> and p59^<fyn>.

1.细胞质游离Ca ^2+浓度（[Ca ^2 +] i）的增加发生在与人T细胞白血病细胞系Jurkat的T细胞受体（TCR）/CD3复合物的抗体连接时。结果发现，[Ca ^<2 +>] i反应可被Staurosporine（Stsp）抑制. Stsp显著抑制TCR/CD3复合物连接时发现的蛋白质酪氨酸磷酸化。Stsp对G蛋白偶联的细胞表面受体刺激后的成纤维细胞[Ca ^2 +] i反应没有影响，而Stsp抑制受体酪氨酸激酶激活的[Ca ^2 +] i反应。这些结果表明蛋白酪氨酸激酶参与了对TCR/CD3复合物连接的[Ca ^（2+）] i应答。TCR/CD3复合物的连接诱导复合物的ζ链的酪氨酸磷酸化。酪氨酸磷酸化zeta链可能是src同源区2（SH 2）蛋白的结合位点，参与T淋巴细胞信号转导。为了筛选这类蛋白质，在杆状病毒系统中共表达大鼠zeta链和人p59 α。<fyn>通过用抗zeta肽（残基110 - 122）抗体的免疫印迹证实zeta链的表达。在SF9细胞中同时表达zeta链和p59~+导致zeta链的酪氨酸磷酸化。<fyn>利用Jurkat细胞的cDNA文库，以磷酸化的zeta链为探针，在酪氨酸残基上标记[^P]磷酸，筛选含有SH2区的蛋白.<32>研究了磷脂酶C γ（PLC γ）和非受体蛋白酪氨酸激酶p56 ^和p59 ^中SH2区的结合特异性。<1ck><fyn>这些蛋白的SH2区在E. coli作为融合蛋白，制备了trpE基因产物和SH2区的亲和柱。PLC的SH2区与p56和p59的SH2区具有不同的结合特异性。<1ck><fyn>