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Phosphorylation of Tyr319 in ZAP‐70 is required for T‐cell antigen receptor‐dependent phospholipase C‐γ1 and Ras activation

Accumulating evidence indicates that the interdomain B regions of ZAP‐70 and Syk play pivotal roles in the coupling of T‐cell antigen receptor (TCR) stimulation to the activation of downstream signaling pathways. The interdomain B region of ZAP‐70 contains at least three candidate sites of tyrosine phosphorylation. In this report, we identify Tyr319 as a functionally important phosphorylation site in the ZAP‐70 interdomain B region. TCR crosslinkage triggered a rapid increase in the phosphorylation of Tyr319 in Jurkat T cells. Although mutation of Tyr319 to Phe had no effect on the tyrosine kinase activity of ZAP‐70, the resulting ZAP(Y319→F) mutant failed to reconstitute TCR‐dependent Ca2+ mobilization, Ras activation, CD69 expression and NFAT‐dependent transcription in ZAP‐70‐deficient Jurkat cells. These defects were correlated with reduced tyrosine phosphorylation of phospholipase C (PLC)‐γ1 and the LAT adapter protein in the ZAP(Y319→F)‐expressing cells. On the other hand, ZAP(Y319→F)‐expressing cells displayed normal increases in SLP‐76 phosphorylation and ERK activation during TCR stimulation. Phosphorylation of Tyr319 promoted the association of ZAP‐70 with the SH2 domains of two key signaling molecules, Lck and PLC‐γ1. These studies suggest that Tyr319 phosphorylation is required for the assembly of a ZAP‐70‐containing signaling complex that leads to the activation of the PLC‐γ1‐ and Ras‐dependent signaling cascades in antigen‐stimulated T cells.