Tyrosine 319 in the interdomain B of ZAP-70 is a binding site for the Src homology 2 domain of Lck

T-cell antigen receptor-induced signaling requires both ZAP-70 and Lck protein-tyrosine kinases, One essential function of Lck in this process is to phosphorylate ZAP-70 and up-regulate its catalytic activity. We have previously shown that after T-cell antigen receptor stimulation, Lck binds to ZAP-70 via its Src homology 2 (SH2) domain (LckSH2) and, more recently, that Tyr(319) of ZAP-70 is phosphorylated in vivo and plays a positive regulatory role. Here, we investigated the possibility that Tyr(319) mediates the SH2-dependent interaction between Lck and ZAP-70. We show that a phosphopeptide encompassing the motif harboring Tyr(319), YSDP, interacted with LckSH2, although with a lower affinity compared with a phosphopeptide containing the optimal binding motif, YEEI. Moreover, mutation of Tyr(319) to phenylalanine prevented the interaction of ZAP-70 with LckSH2. Based on these results, a gain-of-function mutant of ZAP-70 was generated by changing the sequence (YSDP)-S-319 into (YEEI)-E-319. As a result of its increased ability to bind LckSH2, this mutant induced a dramatic increase in NFAT activity in Jurkat T-cells, was hyperphosphorylated, and displayed a higher catalytic activity compared with wild-type ZAP-70. Collectively, our findings indicate that Tyr(319)-mediated binding of the SH2 domain of Lck is crucial for ZAP-70 activation and consequently for the propagation of the signaling cascade leading to T-cell activation.