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

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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>.