Role of Tyr319 and ZAP-70 kinase activity in TCR-ζ phosphorylation. (A) TCR-ζ was immunoprecipitated from p116 Jurkat cells (ZAP^–/Syk^–) expressing similar levels of ectopic ZAP-70 (ZAP^E/Syk^–) or Syk (ZAP^–/Syk^E) following CD3 cross-linking (+), and the level of associated Lck was determined by immunoblotting with an Lck pAb. The positions of the p56 and p59 Lck isoforms are indicated, and the level of immunoprecipitated TCR-ζ is shown. (B) TCR-ζ was immunoprecipitated following CD3 cross-linking or pervanadate treatment. The levels of TCR-ζ–associated ZAP-70 and Syk phosphorylated on Tyr319 and Tyr352, respectively, were monitored using a pAb recognizing these homologous phosphorylated tyrosine residues. The levels of total TCR-ζ–associated ZAP-70 and Syk were revealed with ZAP-70– and Syk-specific antibodies, respectively. (C) TCR-ζ phosphorylation was monitored in whole cell lysates of unstimulated (–) or CD3-stimulated (+) p116 cells stably expressing WT ZAP-70, Y319F ZAP-70, or a kinase-dead (KD) ZAP-70. The relative levels of ZAP-70 phosphorylated on Tyr319, total ZAP-70, phosphorylated Erk1/Erk2 (P-MAPK), and total Erk2 were assessed by immunoblotting with the appropriate antibodies.