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One mechanism for transducing signals downstream of lymphocyte receptor activation involves the stable association between signaling proteins. To identify protein ligands of the signal activator phospholipase Cgamma1 (PLCgamma1), we screened T cell cDNA libraries with the PLCgamma1-SH3 domain by the yeast two-hybrid assay. We observed association between the PLCgamma1-SH3 domain and the human Ras guanine nucleotide exchange factor son-of-sevenless-2 (hSos2) through a proline-rich domain interaction. Stable and abundant hSos2 / PLCgamma1 and hSos1 / PLCgamma1 complexes were observed in unstimulated T cells. The interaction between these enzymes was augmented following engagement of the T cell antigen receptor (TCR / CD3). The kinetics of protein complex enhancement correlated with TCR / CD3-induced tyrosine phosphorylation of PLCgamma1; however, those PLCgamma1 molecules in complex with hSos2 were non-phosphorylated after TCR / CD3 stimulation, in contrast to the phosphorylated PLCgamma1 associated with the linker for activation of T cells, LAT. The Grb2 adapter protein was detected in complex with hSos / PLCgamma1, suggesting a regulatory role for Grb2. SH3 domains from both Grb2 and PLCgamma1, but not RasGAP, bound directly to hSos homologues. The SH2 domain from Grb2 formed an association with the hSos / PLCgamma1 complex, which was enhanced following TCR / CD3 ligation. Together, the data suggest a mechanism for the son-of-sevenless and PLCgamma1 signal transducing enzymes in recruitment to protein complexes with potentially differential signaling consequences in T lymphocytes.

The negative regulation of T- or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca2+ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3zeta, ZAP-70, Syk, and phospholipase Cgammal but not the Src family tyrosine kinase p56(lck). By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.