Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation

Epidermal growth factor (EGF) induces tyrosine phosphorylation of the She adapter protein, which plays an important role in EGF-stimulated mitogenesis. She stimulates Ras/mitogen-activated protein kinase (MAPK) through forming a complex with Grb2 at the phosphorylated tyrosine (Y) residue 317. In this study, we identified novel phosphorylation sites of She, at Y239 and Y240, To define the She pathway further, we used NIH 3T3 cells expressing the previously characterized mutant EGF receptor (EGF-R) which lacks all known autophosphorylation sites but retains EGF-stimulated mitogenesis with selective phosphorylation of She. We constructed wild-type (WT) or mutant She cDNAs in which Y317 or/and Y239 and Y240 are replaced with phenylalanine (F) and introduced them into NIH 3T3 cells expressing WT or mutant EGF-R. In the WT EGF-R-expressing cells, the Y239/240/317F She, but not Y317F or Y239/240F She, decreased EGF-stimulated cell growth. In the mutant EGF-R-expressing cells, Y317F She or Y239/240F She decreased EGF-stimulated cell growth significantly, though Y317F was a little more potent than Y239/240F, Although cells expressing the Y317F She hardly activated MAPK in response to EGF, cells expressing the Y239/240F She fully activated MAPK. In contrast, Y239/240F She, but not Y317F She, reduced the EGF-induced c-myc message. These results suggest that She activates two distinct signaling pathways, Y317 to Ras/MAPK and Y239 and Y240 to another pathway including Myc, and that both are involved in EGF-induced mitogenic signaling.