DISTINCT TYROSINE PHOSPHORYLATION SITES IN ZAP-70 MEDIATE ACTIVATION AND NEGATIVE REGULATION OF ANTIGEN RECEPTOR FUNCTION

Biochemical and genetic evidence has implicated two families of protei n tyrosine kinases (PTKs), the Src- and Syk-PTKs, in T- and B-cell ant igen receptor signaling. ZAP-70 is a member of the Syk-PTKs that assoc iates with the T-cell antigen receptor and undergoes tyrosine phosphor ylation following receptor activation, Three tyrosine residues, Tyr-29 2, -492, and -493, have been identified as sites of phosphorylation fo llowing T-cell antigen receptor engagement. Utilizing ZAP-70- and Syk- deficient lymphocytes (Syk(-) DT40 cells), we provide biochemical and functional evidence that heterologous trans-phosphorylation of Tyr-493 by a Src-PTK is required for antigen receptor-mediated activation of both the calcium and ras pathways. In contrast, cells expressing mutat ions at Tyr-292 or -492 demonstrate hyperactive T- and B-cell antigen receptor phenotypes. Thus, phosphorylation of ZAP-70 mediates both act ivation and inactivation of antigen receptor signaling.