Abstract
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.
Highlights
The protein tyrosine kinase ZAP-70 plays a central role in T-cell activation
Since its identification as a tyrosine phosphoprotein associated with the chain of the activated T-cell antigen receptor (TCR),1 the protein tyrosine kinase (PTK) ZAP-70 has emerged as a central molecule in the generation and propagation of critical early signaling events initiated by TCR triggering and culminating in T-cell proliferation and differentiation [1,2,3,4,5]
To assess the relevance to TCR signaling of residue Tyr474 on ZAP-70, a ZAP-70 mutant was generated by sitespecific mutagenesis, carrying a tyrosine to phenylalanine substitution at position 474 (Phe474ZAP-70)
Summary
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. Activation of ZAP-70 requires plasma membrane localization, which is achieved through a complex interaction of the tandem SH2 domains of ZAP-70 with specific tyrosine-phosphorylated motifs, termed immunoreceptor tyrosine-based activation motifs (ITAMs) [6], on the CD3-bound chain [7, 8]. Upon TCR triggering, Shc binds to the phosphorylated TCR chain through its carboxyl-terminal SH2 domain and becomes in turn phosphorylated, an event that results in recruitment of Grb2/Sos [22, 23]
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