Reciprocal regulation of SH3 and SH2 domain binding via tyrosine phosphorylation of a common site in CD3ε

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3 epsilon. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3 epsilon to Eps8L1 and Nck SH3 domains. CD3 epsilon] bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3 epsilon. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lek kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3 epsilon in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3 epsilon to interact with either SH3 or SH2 domain-containing proteins.