TYROSINE-474 OF ZAP-70 IS REQUIRED FOR ASSOCIATION WITH THE SHC ADAPTER AND FOR T-CELL ANTIGEN RECEPTOR-DEPENDENT GENE ACTIVATION

The protein tyrosine kinase ZAP-70 plays a central role in T-cell acti vation. Following receptor engagement, ZAP-70 is recruited to the phos phorylated subunits of the T-cell antigen receptor (TCR). This event r esults 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 t he Shc PTB domain. The inhibitory effect of a Shc mutant containing th e isolated PTB domain suggests that Shc interaction with ZAP-70 might be required for TCR signaling. Here, we show that a point mutation (Ph e(474)) 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 patt ern of protein phosphorylation induced by TCR triggering was affected by this mutation. However expression of the phe(474) ZAP-70 mutant res ulted in impaired TCR-dependent gene activation. ZAP-70 could effectiv ely phosphorylate Shc in vitro. Only the CH domain, which contains the two Grb2 binding sites on Shc, was phosphorylated by ZAP-70. Both Grb 2 binding sites were excellent substrates for ZAP-70, The data show th at Tyr(474) On ZAP-70 is required for TCR signaling and suggest that S hc association with ZAP-70 and the resulting phosphorylation of Shc mi ght be an obligatory step in linking the activated TCR to the Ras path way.