Structural and functional dissection of the cytoplasmic domain of the transmembrane adaptor protein SIT (SHP2-interacting transmembrane adaptor protein)

SIT (SHP2-interacting transmembrane adaptor protein) is a recently identifi ed transmembrane adaptor protein, which is expressed in lymphocytes. Its st ructural properties, in particular the presence of five potential tyrosine phosphorylation sites, suggest involvement of SIT in TCR-mediated recruitme nt of SH2 domain-containing intracellular signaling molecules to the plasma membrane. Indeed, it has recently been demonstrated that SIT inducibly int eracts with the SH2-containing protein tyrosine phosphatase 2 (SHP2) via an immunoreceptor tyrosine-based inhibition motif (ITIM). Moreover, SIT is ca pable to inhibit TCR-mediated signals proximal of activation of protein kin ase C. However, inhibition of T cell activation by SIT occurs independently of SHP2 binding. The present study was performed to further characterize t he molecular interaction between SIT and intracellular effector molecules a nd to identify the protein(s) mediating its inhibitory function. We demonst rate that SIT not only interacts with SHP2 but also with the adaptor protei n Grb2 via two consensus YxN motifs. However, mutation of both Grb2-binding sites also does not influence the inhibitory function of SIT. In contrast, mutation of the tyrosine-based signaling motif Y-168 ASV completely abroga tes the ability of SIT to inhibit T cell activation. Co-precipitation exper iments revealed that the tyrosine kinase p50(csk) could represent the negat ive regulatory effector molecule that binds to this motif.