MUTATION OF TYROSINES-492 493 IN THE KINASE DOMAIN OF ZAP-70 AFFECTS MULTIPLE T-CELL RECEPTOR SIGNALING PATHWAYS/

The protein-tyrosine kinase ZAP-70 is implicated, together with the Sr c kinase p56(lck), in controlling the early steps of the T-cell antige n receptor (TCR) signaling cascade. To help elucidate further the mech anism by which ZAP-70 regulates these initial events, we used a domina nt-negative mutant approach. We overexpressed in the Jurkat T-cell lin e ZAP-70 mutated on Tyr-492 and Tyr-493 in the putative regulatory loo p of its kinase domain. This mutant inhibited TCR induced activation o f nuclear factor of activated T cells by interfering with both intrace llular calcium increase and Ras-regulated activation of extracellular signal-regulated kinases. Moreover, TCR-induced phosphorylation of pp3 6-38, thought to play a role upstream of these pathways, was found to be reduced. In contrast, overexpression of wildtype ZAP-70 induced con stitutive activation of nuclear factor of activated T cells. The ZAP-7 0 mutant studied here could be phosphorylated on tyrosine when associa ted to the TCR zeta chain and was able to bind p56(lck). This result d emonstrates that Tyr-492 and Tyr-493 are not responsible for the Src h omology domain a-mediated association of p56(lck) with ZAP-70. Our dat a are most consistent with a model in which recruitment to the TCR all ows ZAP-70 autophosphorylation and binding to p56(lck), which in turn phosphorylates Tyr-492 and/or Tyr-493 with consequent up-regulation of the ZAP-70 kinase activity. ZAP-70 will then be able to effectively c ontrol phosphorylation of its substrates and lead to gene activation.