Phosphorylation of Tyr319 in ZAP-70 is required for T-cell antigen receptor-dependent phospholipase C-gamma 1 and Ras activation

Accumulating evidence indicates that the interdomain B regions of ZAP-70 an d Syk play pivotal roles in the coupling of T-cell antigen receptor (TCR) s timulation to the activation of downstream signaling pathways. The interdom ain B region of ZAP-70 contains at least three candidate sites of tyrosine phosphorylation, In this report, we identify Tyr319 as a functionally impor tant phosphorylation site in the ZAP-70 interdomain B region. TCR crosslink age 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 ki nase activity of ZAP-70, the resulting ZAP(Y319-->F) mutant failed to recon stitute TCR-dependent Ca2+ mobilization, Ras activation, CD69 expression an d NFAT-dependent transcription in ZAP-70-deficient Jurkat cells. These defe cts were correlated with reduced tyrosine phosphorylation of phospholipase C (PLC)-gamma 1 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 stimulati on. Phosphorylation of Tyr319 promoted the association of ZAP-70 with the S H2 domains of two key signaling molecules, Lck and PLC-gamma 1. These studi es suggest that Tyr319 phosphorylation is required for the assembly of a ZA P-70-containing signaling complex that leads to the activation of the PLC-g amma 1- and Pas-dependent signaling cascades in antigen-stimulated T cells.