Abstract
ZAP-70 is an 70-kDa protein tyrosine kinase, expressed exclusively in T cells and NK cells, and plays a critical role in mediating T cell activation in response to T cell receptor engagement. The strong correlation between tyrosine phosphorylation of ZAP-70 and its acquisition of increased kinase activity suggests that is 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 individually to phenylalanine. When expressed in COS cells, Y493F-mutated ZAP-70 demonstrated normal basal kinase activity, but, unlike wild type ZAP-70, could not be activated by tyrosine phosphorylation induced by incubation with pervanadate or by co-expression of constitutively activated Lck. This suggests that Tyr-493 phosphorylation is required for 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 reveal that critical tyrosine residues in the kinase domain of ZAP-70 are important in regulation of its catalytic activity.
Highlights
Because of the important role played by ZAP-70 in regulating T lymphocyte activation and development, there is much inter
It has been shown that recruitment to the activated TCR is necessary [4], but insufficient for ZAP-70 activation [9, 10], There is a strong correlation between tyrosine phosphorylation of ZAP-70 and activation of its kinase activity [4, 10, 11], and it is likely that after recruitment to the TCR one or more tyrosine residues of ZAP-70 must become phosphorylated in order for ZAP-70 to become activated
We found that tyrosine 493 is necessary for phosphorylation-induced activation of ZAP-70 kinase activity and appears to be requisite for additional tyrosine phosphorylation events
Summary
Because of the important role played by ZAP-70 in regulating T lymphocyte activation and development, there is much inter-. We have recently identified the tyrosine residues in ZAP-70 that become phosphorylated in vivo in Jurkat T cells upon TCR stimulation. A common approach for studying the effects of phosphorylation of a particular tyrosine upon the activity of a kinase is to mutate the tyrosine residue to phenylalanine. Using this approach, tyrosine 416 of' Src, tyrosine 394 of Lck, and tyrosines 1162 and 1163 of the insulin receptor kinase (IRK) were all identified as being critical sites of autophosphorylation and positive regulation within these kinases [15,16,17,18,19,20,21]. The mutated enzymes were expressed in COS cells either alone or with constitutively activated Y505F-Lck, and the ZAP-70-
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