Role of the Src homology 2 domains and interdomain regions in ZAP-70 phosphorylation and enzymatic activity

The protein tyrosine kinase ZAP-70 which mediates T-cell antigen receptor ( TCR) signalling, contains three distinct functional modules, two tandemly a rranged SH2 domains, a kinase domain and a linker region (interdomain B) th at connects them. ZAP-70 enzymatic activation is strictly dependent on the binding, via its SH2 domains. to the triggered TCR and on tyrosine phosphor ylation. Here we utilized recombinant ZAP-70 and carried out a mutational a nalysis to understand the structural requirements for its activation. We sh ow that deletion of both SH2 domains corresponding to the first 254 residue s moderately increases ZAP-70 enzymatic activity on an exogenous substrate in vitro, results in increased tyrosine phosphorylation and produces subtle conformational changes, as judged by altered SDS/PAGE migration. Mutation of Tyr292, 315 and 319 to Phe in the interdomain B region, which constitute the major phosphorylation sites both in vitro and in vivo, did not affect ZAP-70 enzymatic activity. Moreover, deletion analysis of the interdomain B region established residues 320-619 as a minimal region endowed with full kinase activity. We propose that binding of ZAP-70 to the TCR promotes, thr ough conformational changes, its extensive phosphorylation on tyrosine. How ever, Tyr292, 315 and 319 do not affect ZAP-70 enzymatic activity and may i nfluence ZAP-70 signalling only indirectly by mediating its association wit h intracellular transducers.