The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein-protein interactions

Background: Signal transduction initiated by a wide variety of extracellular signals involves the activation of protein-tyrosine kinases. Phosphorylated tyrosine residues in activated receptors or docking proteins then function as binding sites for the Src homology 2 (SH2) or phosphotyrosine-binding (PTB) domains of cytoplasmic signalling proteins. She is an adaptor protein that contains both PTB and SH2 domains and becomes phosphorylated on tyrosine in response to many different extracellular stimuli. These results have suggested that She is a prominent effector of protein-tyrosine kinase signalling. Thus far, only a single She phosphorylation site, the tyrosine at position 317 (Y317) has been identified. Phosphorylation of Y317 has been implicated in Grb2 binding and activation of the Ras pathway. Results: Here, we report the identification of two major and novel She tyrosine phosphorylation sites, Y239 and Y240. These residues are present in the central proline-rich (CHI) region and are conserved in all isoforms of She. Y239/240 are co-ordinately phosphorylated by the Src protein-tyrosine kinase in vitro, and in response to epidermal growth factor stimulation or in v-src-transformed cells in vivo. Mutagenesis studies indicate that Y239/240 make an important contribution to the association of She with Grb2. Phosphopeptide-binding studies suggest that these two tyrosine residues may be involved in interactions with a number of cellular proteins. Conclusions: She is the most prominent general substrate for protein-tyrosine kinases in vivo. The identification of two novel She phosphorylation sites indicates that She has the potential to interact with multiple downstream effectors. She Y239/240 are highly conserved in evolution, suggesting that the phosphorylation of these residues is of fundamental importance. We propose that distinct She phosphorylation isomers form different signalling complexes and thereby activate separate downstream signalling cascades.