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

1.与人T细胞白血病细胞株Jurkat的T细胞受体(TCR)/CD3复合体抗体结合后，胞浆内游离钙浓度([Ca^&lt；2+&gt；]i)增加。结果表明，星形孢菌素(STSP)可抑制[Ca~(2+)&gt；]i反应。STSP显著抑制TCR/CD3复合体连接蛋白的酪氨酸磷酸化。STSP对G蛋白偶联的细胞表面受体刺激的成纤维细胞的[Ca^&lt；2+&gt；]i反应无影响，但对受体酪氨酸激酶激活的[Ca^&lt；2+&gt；]i反应有抑制作用。这些结果表明蛋白酪氨酸激酶参与了[Ca^&lt；2+&gt；]i对TCR/CD3复合体的连接反应。TCR/CD3复合体的连接诱导复合体Zeta链的酪氨酸磷酸化。酪氨酸磷酸化的Zeta链可能是参与T淋巴细胞信号转导的Src同源区2(SH2)蛋白的结合部位。为了筛选这类蛋白，在杆状病毒系统中共表达了大鼠Zeta链和人P59；用抗Zeta多肽(残基110-122)抗体免疫印迹证实了Zeta链的表达。在SF9细胞中同时表达Zeta链和P59^&lt；fyn&gt；导致Zeta链的酪氨酸磷酸化。利用Jurkat细胞cDNAlambdagt11文库和酪氨酸残基标记的磷酸化Zeta链作为探针，对含有SH2区的蛋白质进行筛选。研究了磷脂酶CGamma(PLCGamma)中SH2区与非受体蛋白酪氨酸激酶p56^lt；1ck&gt；和p59^lt；fyn&gt；的结合特异性。将这些蛋白的SH2区在大肠杆菌中表达为与trpE基因产物的融合蛋白，并制备了SH2区的亲和柱。PLC的SH2区与p56^&lt；1ck&gt；和p59^&lt；fyn&gt；的SH2区具有不同的结合特异性。

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>.