Perturbation of the T- or B-cell antigen receptor (TCR or BCR) by antigen or by anti-receptor antibodies induces protein tyrosine kinase activation, the phosphorylation and activation of phospholipase Cgamma-1 (PLCg1), and the activation of small G-proteins, such as Ras. The activation of PLCg1, in particular, mediates phosphoinositide (PI) hydrolysis which in turn controls Ca2+ mobilization and protein kinase C activation, obligatory events in the activation of T- or B-lymphocytes. The laboratory is concerned with establishing the mechanisms by which immune receptors regulate PLCg1. PLCg1 has two src-homology (SH) 2 domains that bind tyrosine-phosphorylated proteins and one SH3 domain that interacts with proline-rich polipeptides. These domains play a role in coupling proteins to other regulatory molecules. Mutational analysis demonstrated that the amino terminal SH2 (SH2N) domain was required and sufficient for TCR- or BCR-induced tyrosine phosphorylation of PLCg1, and for the association of PLCg1 with the T cell adapter, LAT, or the B cell adapter, BLNK. PLCg1 membrane translocation required the SH2N domain and was decreased by mutation of the SH3 domain. BCR-induced PI hydrolysis was abrogated by mutation of either the SH2N or the carboxyl-terminal SH2 (SH2C) domain and was decreased by mutation of the SH3 domain. The cellular proto-oncogene, Cbl, is an adapter that associates with numerous signaling proteins, including PLCg1, involved in signal transduction by distinct receptors. Cbl is a major target of tyrosine phosphorylation after TCR engagement. We have found that over-expression of Cbl reduced TCR-induced AP1 (a Ras-dependent transcription factor) and NF-AT (a Ca2+-dependent transcription factor) reporter activities, suggesting that Cbl could act at a point shared by both the Ras and PLCg1/Ca2+ pathways. Cbl over-expression decreased TCR-induced PI-hydrolysis, but had no effect on TCR-induced tyrosine phosphorylation of PLCg1. An SH3 binding site and the aa. 655-906 region were required for Cbl-mediated regulation of PLCg1. Over-expression of an oncogenic Cbl mutant, 70Z/3, did not affect TCR-induced NF-AT activation and enhanced PI-hydrolysis. Wild type Cbl and the 70Z/3 Cbl were similarly able to bind PLCg1, indicating that the interaction with PLCg1 does not directly inhibit its activity. These data support a sequential model of immune receptor-induced PLCg1 activation where the earliest event is the engagement of the SH2N domain by a phosphoprotein that recruits PLCg1 in an activation complex within the membrane with ensuing phosphorylation, while the SH3 domain contributes additional membrane anchoring. Up-regulation of PLCg1 activity requires translocation and phosphorylation and is abrogated or reduced by disruption of the SH2N or SH3 domains, respectively. The SH2C domain is exclusively required for receptor-induced PLCg1 activation. Cbl regulates receptor-induced PLCg1 activity by affecting, in an SH3 domain-dependent manner, a point downstream of or independent of PLCg1 phosphorylation.
