EVIDENCE FOR A REQUIREMENT FOR BOTH PHOSPHOLIPID AND PHOSPHOTYROSINE BINDING VIA THE SHC PHOSPHOTYROSINE-BINDING DOMAIN IN-VIVO

The adapter protein She is a critical component of mitogenic signaling pathways initiated by a number of receptors. She can directly bind to several tyrosine-phosphorylated receptors through its phosphotyrosine -binding (PTB) domain, and a role for the PTB domain in phosphotyrosin e-mediated signaling has been well documented, The structure of the Sh e PTB domain demonstrated a striking homology to the structures of ple ckstrin homology domains, which suggested acidic phospholipids as a se cond ligand for the She PTB domain. Here we demonstrate that She bindi ng Bia ifs PTB domain to acidic phospholipids is as critical as bindin g to phosphotyrosine for leading to She phosphorylation, Through struc ture-based, targeted mutagenesis of the She PTB domain, we first ident ified the residues within the PTB domain critical for phospholipid bin ding in vitro. In vivo, the PTB domain was essential for localization of She to the membrane, as mutant She proteins that failed to interact with phospholipids in vitro also failed to localize to the membrane. We also observed that PTB domain-dependent targeting to the membrane p receded the PTB domain's interaction with the tyrosine-phosphorylated receptor and that both events were essential for tyrosine phosphorylat ion of She following receptor activation, Thus, She, through its inter action with two different ligands, is able to accomplish both membrane localization and binding to the activated receptor tia a single PTB d omain.