TI - Tyrosine 474 of ZAP-70 is required for association with the Shc adaptor and for T - cell antigen receptor -dependent gene activation . AB - The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation . Following receptor engagement , ZAP-70 is recruited to the phosphorylated subunits of the T-cell antigen receptor ( TCR ) . This event results in ZAP-70 activation and in association of ZAP-70 with a number of signaling proteins . Among these is the Shc adaptor , which couples the activated TCR to Ras . Shc interaction with ZAP-70 is mediated by the Shc PTB domain . The inhibitory effect of a Shc mutant containing the isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling . Here , we show that a point mutation ( Phe474 ) of the putative Shc binding site on ZAP-70 , spanning tyrosine 474 , prevented ZAP-70 interaction with Shc and the subsequent binding of Shc to phospho-zeta . Neither ZAP-70 catalytic activity nor the pattern of protein phosphorylation induced by TCR triggering was affected by this mutation . However expression of the Phe474 ZAP-70 mutant resulted in impaired TCR -dependent gene activation . ZAP-70 could effectively phosphorylate Shc in vitro . Only the CH domain , which contains the two Grb2 binding sites on Shc , was phosphorylated by ZAP-70 . Both Grb2 binding sites were excellent substrates for ZAP-70 . The data show that Tyr474 on ZAP-70 is required for TCR signaling and suggest that Shc association with ZAP-70 and the resulting phosphorylation of Shc might be an obligatory step in linking the activated TCR to the Ras pathway .