MECHANISM OF T LYMPHOCYTE ACTIVATION--REGULATION OF PLC GAMMA1 ACTIVATION

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Perturbation of the T cell receptor (TCR) by antigen/MHC or by anti-receptor antibodies initiates a cascade of events which includes protein tyrosine kinase activation and the phosphorylation and activation of phospholipase Cg1 (PLCg1). In turn, PLCg1-mediated phosphoinositide (PI) hydrolysis controls calcium mobilization and protein kinase C activation, two obligatory events in lymphokine secretions and cell proliferation. PLCg1 structure includes two src-homology (SH) 2 domains that function as docking sites for tyrosine-phosphorylated proteins. The function of these domains in PLCg1 activation in T cells is unknown. By acting as docking sites for other molecules, they are likely to play a role in coupling PLCg1 to surface receptors or other regulatory molecules that control PLCg1 activation and compartmentalization. The overall goal of this project is to establish a structure-function relationship for PLCg1 activation in T lymphocytes. Specific aims are the identification of T-cell proteins that interact with PLCg1 subdomains and the role of these domains in PLCg1 activation in T cells. The project's strategy includes: 1. The expression of PLCg1 domains as fusion proteins to screen for candidate binding proteins; 2. A mutational analysis of an epitope-tagged PLCg1 expressed in T lymphocytes; 3. The reconstitution of receptor signaling in PLCg1-deficient cells. PLCg1 GST-SH2(N) domain bound exclusively a 38 kDa phosphoprotein, while the SH2(C) domain bound several phosphoproteins. A SH2(N) domain-defective PLCg1 mutant expressed in human Jurkat T cells did not bind p38 and was marginally phosphorylated in response to TCR engagement. Mutations of the SH2(C) had no impact of TCR-induced PLCg1 phosphorylation. Pharmacological treatment with pervanadate bypasses the receptors and resulted in PLCg1 phosphorylation irrespective of the SH2 domain mutations. Therefore, PLCg1 could be phosphorylated by alternate, SH2(N) domain-independent mechanism(s). Wild type (WT) and mutant PLCg1 proteins were transfected into a DT40-derived PLCg1/2-defective chicken B cell line. Consistent with the results observed in Jurkat cells, only the WT and SH2(C) domain mutant, but not the SH2(N) domain mutant, were tyrosine phosphorylated in response to antigen receptor engagement. While the WT protein reconstituted antigen receptor-induced PI hydrolysis, neither SH2 domain mutant reconstituted this activity. These data indicate that PLCg1 SH2 domains serve discrete functions. The SH2(N) domain is required and sufficient for antigen receptor-induced PLCg1 tyrosine phosphorylation, while both domains are required for the enzymatic activation of PLCg1 by the antigen receptors.