Perturbation of the T cell receptor (TCR)/CD3 complex by antigen (in the context of the appropriate MHC) or by anti-receptor antibodies (Ab) initiates a cascade of events which includes protein tyrosine kinase activation, tyr osine phosphorylation of an inositol phospholipid (InsPL)-specific phospholipase C (PLC) isozyme, PLC gamma-1 , activation of InsPL hydrolysis, and the generation of second messengers that control protein kinase C activation and calcium mobilization. The objective of this study is to elucidate the mechanism coupling PLC gamma-1 to the TCR/CD3 complex and regulating its activity and effector function. A number of functional subdomains constitutes PLC gamma-1. Some of these domains are homologous to amino acid modules initially defined in the src gene product. These src-homology domains (SH) function as docking sites in protein/protein interactions: SH2 recognizes tyrosine phosphorylated peptides, while SH3 interacts with proline-rich sequences of certain proteins. PLC gamma-1 contains two SH2 and one SH3, whose function is currently unknown. We have expressed both SH2 and the SH3 of PLC gamma-1 (individually or in combination) as fusion proteins (FP) with the glutathione S-transferase (GST) from S. iaponicum. By screening with an anti-phosphotyrosine (PY) Ab, at least two phosphoproteins were detected by coprecipitation with the carboxy-terminal SH2 from lysates of activated human Jurkat T leukemia cells or a mouse T lymphocyte cell line. One distinct phosphoprotein was recognized by the GST-SH3. No detectable phosphoprotein was precipitated with the NH2-terminal SH2 or from non-activated cells. Additive but not synergistic effects on phosphoprotein recruitment was observed with the GST-FP encompassing multiple SH domains. The phosphoprotein recognized by the GST-SH3 was identified by immunoreactivity as c-cbl, a transcription factor. Although cbl phosphorylation was activation-dependent, its interaction with GST- SH3 was not. An additional non-phosphorylated protein coprecipitated with GST-SH3 and competed with cbl for SH3 binding. Identification of this protein and the GST-SH2-precipitated proteins is in progress. Additional PLC gamma-1 subdomains are also tested for their role in protein recruitment and PLC regulation.
