MECHANISM OF T LYMPHOCYTE ACTIVATION--REGULATION OF PLCR1 ACTIVATION

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Perturbation of the T cell receptor (TCR) by antigen/MHC or by anti-receptor antibodies (Ab) initiates a cascade of events which include protein tyrosine kinase activation and the phosphorylation and activation of phospholipase Cg-1 (PLCg1). PLCg-1 contains a number of src-homology (SH) domains. These domains function as protein docking sites and are likely to play a role in coupling PLCg-1 to the TCR/CD3 complex and in regulating its activity. Our goal is to establish a structure-function relationship for PLCg1 activation in T lymphocytes. Specific aims of this project are: 1). The identification of T-cell proteins that interact with PLCg1 SH domains. 2). The identification of the functional role of PLCg1 SH domains in T-lymphocyte activation. The project's strategy is as follows: 1). Expression of PLCg1 domains as GST- fusion proteins, as a screening tool for the identification of candidate binding proteins. 2). Demonstration of the interaction between native proteins. 3). Characterization of the function of individual domains by mutational anlaysis of an epitope-tagged PLCg1. PLCg1 GST-SH2(N) domain bound a phosphoprotein of about 38 kDa, while the SH2(C) domain bound phosphoproteins of about 38, 75, and 120 kDa and, to a lesser extent, 60, 85 and 100 kDa phosphoproteins. The GST-SH3 domain bound a prominent tyrosine phosphorylated protein of 120 kDa. Phospho- proteins of about 38 and about 120 kDa were coprecipitated with native PLCg1 from CD3-activated cells and a phosphoprotein of about 75 kDa was observed in cells treated with pervanadate, a strong inducer of protein tyrosine phosphorylation used for mimicry of T-cell activation. The 120 kDa SH3 domain-binding phospho- protein was identified as cbl, a protein of unknown function, but rapidly phosphorylated in response to mitogens or Ab-mediated ligation of the receptor. A constitutive interaction between native PLCg1 and cbl was shown by co-precipitation in Jurkat cells and displacement by a peptide encoding an SH3-binding motif of cbl. The function of cbl in T cell activation is addressed as a separate project (Z01 BO 04002-02 LIB). Single amino acid mutations of PLCg1 SH domains were performed on the basis of loss-of-function mutations of conserved amino acids in the SH domains of Grb2. A prominent defect in CD3-induced PLCg1 tyrosine phosphorylation was associated with the mutation of the SH2(N), but not the SH2(C) or SH3 domains. The SH2(N) domain binds exclusively to a about 38 kDa phospho- protein, suggesting that this protein, likely to be the recently cloned protein, Lnk, may be critical for phosphorylation of PLCg1 in response to CD3 ligation. Mutation of PLCg1 SH2(N) domain did not affect tyrosine phosphorylation in response to treatment with pervanadate, suggesting that the mutated PLCg1 could be phosphorylated by alternate mechanism(s).