A CONSERVED AMINO-TERMINAL SHC DOMAIN BINDS TO PHOSPHOTYROSINE MOTIFSIN ACTIVATED RECEPTORS AND PHOSPHOPEPTIDES

Background: Signal transduction by growth factor receptor protein-tyro sine kinases is generally initiated by autophosphorylation on tyrosine residues following ligand binding. Phosphotyrosines within activated receptors form binding sites for the Src homology 2 (SH2) domains of c ytoplasmic signalling proteins. One such protein, Shc, is tyrosine pho sphorylated in response to a large number of growth factors and cytoki nes. Phosphorylation of Shc on tyrosine residue Y317 allows binding to the SH2 domain of Grb2, and hence stimulation of the Ras pathway. Shc is therefore implicated as an adaptor protein able to couple normal a nd oncogenic protein-tyrosine kinases to Ras activation. Shc itself co ntains an SH2 domain at its carboxyl terminus, but the function of the amino-terminal half of the protein is unknown. Results: We have found that the Shc amino-terminal region binds to a number of tyrosine-phos phorylated proteins in v-src-transformed cells. This domain also bound directly to the activated epidermal growth factor (EGF) receptor. A p hosphotyrosine (pY)-containing peptide modeled after the Shc-binding s ite in polyoma middle T antigen (LLSNPTpYSVMRSK) was able to compete e fficiently with the activated EGF receptor for binding to the Shc amin o terminus. This competition was dependent on phosphorylation of the t yrosine residue within the peptide, and was abrogated by deletion of t he leucine residue at position -5. The Shc amino-terminal domain also bound to the autophosphorylated nerve growth factor receptor (Trk), bu t bound significantly less well to a mutant receptor in which tyrosine Y490 in the receptor's Shc-binding site had been substituted by pheny lalanine. Conclusion: These data implicate the amino-terminal region o f Shc in binding to activated receptors and other tyrosine-phosphoryla ted proteins. Binding appears to be specific for phosphorylated tyrosi ne residues within the sequence NPXpY, which is conserved in many Shc- binding sites. The Shc amino-terminal region bears only very limited s equence identity to known SH2 domains, suggesting that it represents a new class of phosphotyrosine-binding modules. Consistent with this vi ew, the amino-terminal Shc domain is highly conserved in a Drosophila Shc homologue. Binding of Shc to activated receptors through its amino terminus could leave the carboxy-terminal SH2 domain free for other i nteractions. In this way, Shc may Function as an adaptor protein to br ing two tyrosine-phosphorylated proteins together.