ACTIVATING AND INHIBITORY MUTATIONS IN ADJACENT TYROSINES IN THE KINASE DOMAIN OF ZAP-70

ZAP-70 is a 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell act ivation in response to T cell receptor engagement, The strong correlat ion between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that it is positively regulated by tyrosine phosphorylation, Previously, we identified tyrosines 492 and 493 of ZAP-70 as being sites of in vivo phosphorylation in response to T cell receptor engagement, To determine the role of phosphorylation in regulating ZAP-70 activity, we mutated each of these tyrosines indi vidually to phenylalanine, When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild typ e ZAP-70, could not be activated by tyrosine phosphorylation induced b y incubation with pervanadate or by co-expression of constitutively ac tivated Lck, This suggests that Tyr-493 phosphorylation is required fo r the tyrosine phosphorylation-induced activation of ZAP-70, The Y492F mutation resulted in 4-fold higher basal kinase activity, which could be stimulated further by tyrosine phosphorylation, These results reve al that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.