Activation of ZAP-70 tyrosine kinase due to a structural rearrangement induced by tyrosine phosphorylation and/or ITAM binding

The protein tyrosine kinase ZAP-70 is implicated in the early steps of the T-cell antigen receptor (TCR) signaling. Binding of ZAP-70 to the phosphory lated immunoreceptor tyrosine-based activation motifs (ITAMs) of the TCR ze ta chain through its two src-homology 2 (SH2) domains results in its activa tion coupled to phosphorylation on multiple tyrosine residues, mediated by Src kinases including Lck as well as by autophosphorylation. The mechanism of ZAP-70 activation following receptor binding is still not completely und erstood. Here we investigated the effect of intramolecular interactions and autophosphorylation by following the kinetics of recombinant ZAP-70 activa tion in a spectrophotometric substrate phosphorylation assay. Under these c onditions, we observed a lag phase of several minutes before full ZAP-70 ac tivation, which was not observed using a truncated form lacking the first 2 54 residues, suggesting that it might be due to an intramolecular interacti on involving the interdomain A and SH2 region. Accordingly, the lag phase c ould be reproduced by testing the truncated form in the presence of recombi nant SH2 domains and was abolished by the addition of diphosphorylated ITAM peptide. Preincubation with ATP or phosphorylation by Lck also abolished t he lag phase and resulted in a more active enzyme. The same results were ob tained using a ZAP-70 mutant lacking the interdomain B tyrosines. These fin dings are consistent with a mechanism in which ZAP-70 phosphorylation/autop hosphorylation on tyrosine(s) other than 292, 315, and 319, as well as enga gement of the SH2 domains by the phosphorylated TCR, can induce a conformat ional change leading to accelerated enzyme kinetics and higher catalytic ef ficiency.