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.