The Amino-Terminal Src Homology 2 Domain of Phospholipase Cγ1 Is Essential for TCR-Induced Tyrosine Phosphorylation of Phospholipase Cγ1

Abstract

AbstractTCR engagement activates phospholipase Cγ1 (PLCγ1) via a tyrosine phosphorylation-dependent mechanism. PLCγ1 contains a pair of Src homology 2 (SH2) domains whose function is that of promoting protein interactions by binding phosphorylated tyrosine and adjacent amino acids. The role of the PLCγ1 SH2 domains in PLCγ1 phosphorylation was explored by mutational analysis of an epitope-tagged protein transiently expressed in Jurkat T cells. Mutation of the amino-terminal SH2 domain (SH2(N) domain) resulted in defective tyrosine phosphorylation of PLCγ1 in response to TCR/CD3 perturbation. In addition, the PLCγ1 SH2(N) domain mutant failed to associate with Grb2 and a 36- to 38-kDa phosphoprotein (p36–38), which has previously been recognized to interact with PLCγ1, Grb2, and other molecules involved in TCR signal transduction. Conversely, mutation of the carboxyl-terminal SH2 domain (SH2(C) domain) did not affect TCR-induced tyrosine phosphorylation of PLCγ1. Furthermore, binding of p36–38 to PLCγ1 was not abrogated by mutations of the SH2(C) domain. In contrast to TCR/CD3 ligation, treatment of cells with pervanadate induced tyrosine phosphorylation of either PLCγ1 SH2(N) or SH2(C) domain mutants to a level comparable with that of the wild-type protein, indicating that pervanadate treatment induces an alternate mechanism of PLCγ1 phosphorylation. These data indicate that the SH2(N) domain is required for TCR-induced PLCγ1 phosphorylation, presumably by participating in the formation of a complex that promotes the association of PLCγ1 with a tyrosine kinase.