MECHANISM OF ACTIVATION FOR ZAP-70 CATALYTIC ACTIVITY

There is a growing body of evidence, including data from human genetic and T-cell receptor function studies, which implicate a xi-associated protein of M(r) 70,000 (Zap-70) as a critical protein tyrosine kinase in T-cell activation and development, During T-cell activation, Zap-7 0 becomes associated via its src homology type 2 (SH2) domains with ty rosine-phosphorylated immune-receptor tyrosine activating motif (ITAM) sequences in the cytoplasmic xi chain of the T-cell receptor, An intr iguing conundrum is how Zap-70 is catalytically activated for downstre am phosphorylation events, To address this question, we have used puri fied Zap-70, tyrosine phosphorylated glutathione S-transferase (GST)-Z eta, and GST-Zeta-1 cytoplasmic domains, and various forms of ITAM-con taining peptides to see what effect binding of xi had upon Zap-70 tyro sine kinase activity, The catalytic activity of Zap-70 with respect to autophosphorylation increased approximate to 5-fold in the presence o f 125 nM phosphorylated GST-Zeta or GST-Zeta-1 cytoplasmic domain, A 2 0-fold activity increase was observed for phosphorylation of an exogen ous substrate, Both activity increases showed a GST-Zeta concentration dependence, The increase in activity was not produced with nonphospho rylated GST-Zeta, phosphorylated xi, or phosphorylated ITAM-containing peptides, The increase in Zap-70 activity was SH2 mediated and was in hibited by phenylphosphate, Zap-70 SH2, and an antibody specific for Z ap-70 SH2 domains, Since GST-Zeta and GST-Zeta-1 exist as dimers, the data suggest Zap-70 is activated upon binding a dimeric form of phosph orylated xi and not by peptide fragments containing a single phosphory lated ITAM, Taken together, these data indicate that the catalytic act ivity of Zap-70 is most likely activated by a trans-phosphorylation me chanism.