Determination of the tyrosine phosphorylation sites in the T cell transmembrane glycoprotein CD5

Studies of CD5-deficient mice indicate that the transmembrane glycoprotein CD5 negatively regulates antigen receptor-mediated signals in thymocytes, l ymph node T cells and B1a cells, CD5 contains four tyrosine residues in its cytoplasmic domain and is phosphorylated on tyrosine residues following an tigen receptor ligation, Recently it has been proposed that CD5 function is dependent on the recruitment of the tyrosine phosphatase SHP-1 to tyrosine -phosphorylated CD5 and subsequent dephosphorylation of signaling molecules . In this study we investigated the requirements for, and sites of, CD5 tyr osine phosphorylation. Using a T cell line deficient in the tyrosine kinase p58(lck) and the same cell line reconstituted with this kinase, we show th at p56(lck) expression is required for efficient CD5 tyrosine phosphorylati on, Using tyrosine-phosphorylated peptides corresponding to CD5 cytoplasmic sequences we also show that the Src homology 2 (SH2) domain of p58(lck) bi nds prominently to pY429SQP with 30-fold less affinity to pY483DLQ and not to pY441PAL, A number of murine CD5 Y --> F and deletion mutants were expre ssed in Jurkat T cells. The Y441F mutant was tyrosine phosphorylated at lev els comparable to wild-type, but the Y429F and Y463F mutants were phosphory lated at lower levels, Two deletion mutants, which contain only one tyrosin e residue (Y378) located at the interface of the transmembrane and cytoplas mic domains, were not tyrosine phosphorylated, suggesting that Y378 is not readily available for phosphorylation. Taken together these results suggest that both Y429 and Y463 can recruit p56(lck), and that these residues are the only prominent sites for CD5 tyrosine phosphorylation.