Tyrosine 319, a newly identified phosphorylation site of ZAP-70, plays a critical role in T cell antigen receptor signaling

Following T cell antigen receptor (TCR) engagement, the protein tyrosine ki nase (PTK) ZAP-70 is rapidly phosphorylated on several tyrosine residues, p resumably by two mechanisms: an autophosphorylation and a trans-phosphoryla tion by the Src-family PTK Lck, These events have been implicated in both p ositive and negative regulation of ZAP-70 activity and in coupling this PTK to downstream signaling pathways in T cells. We show here that Tyr(315) an d Tyr(319) in the interdomain B of ZAP-70 are autophosphorylated in vitro a nd become phosphorylated in vivo upon TCR triggering. Moreover, by mutation al analysis, we demonstrate that phosphorylation of Tyr(319) is required fo r the positive regulation of ZAP-70 function. Indeed, overexpression in Jur kat cells and in a murine T cell hybridoma of a ZAP-70 mutant in which Tyr( 319) was replaced by phenylalanine (ZAP-70-Y319F) dramatically impaired ant i-TCR-induced activation of the nuclear factor of activated T cells and int erleukin-2 production, respectively. Surprisingly, an analogous mutation of Tyr(315) had little or no effect. The inhibitory effect of ZAP-70-Y319F co rrelated with a substantial loss of its activation-induced tyrosine phospho rylation and up-regulation of catalytic activity, as well as with a decreas ed in vivo capacity to phosphorylate known ZAP-70 substrates, such as SLP-7 6 and LAT, Collectively, our data reveal the pivotal role of Tyr(319) phosphorylation in the positive regulation of ZAP-70 and in TCR-mediated signaling.