TYROSINE RESIDUE-239 AND RESIDUE-240 OF SHC ARE PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE-DEPENDENT PHOSPHORYLATION SITES BY C-SRC

In the previous study (Sate K.-I. et al. (1997) FEES Lett. 410, 136-14 0), we showed that the phosphorylation of Shc protein by c-Src is depe ndent on the binding of phosphatidylinositol 4,5-bisphosphate (PtdIns( 4,5)P2) to the PTB domain of Shc. In this study, we demonstrate that, in contrast to c-Src, v-Src and epidermal growth factor (EGF) receptor can phosphorylate Shc in a PtdIns(4,5)P2-independent manner and at di fferent phosphorylation sites. To determine the phosphorylation sites in Shc, we used mutant Shc proteins in which tyrosine residues (Y) 317 and/or 239 and 240 were replaced by phenylalanine residues (F), We fo und that Y317F Shc but not Y239/240P or Y239/240/317F Shc was phosphor ylated by c-Src. The reaction was PtdIns(4,5)P2-dependent and inhibite d by the addition of PTB domain of Shc. On the other hand, v-Src and E GF receptor were able to phosphorylate both Y317F and Y239/240F but no t Y239/240/317F Shc in a PtdIns (4,5)P2-independent manner. These resu lts highlight the difference between c-Src and v-Src or EGF receptor a nd suggest that c-Src can phosphorylate predominantly on Tyr239/240 of Shc only when Shc PTB domain is bound to PtdIns(4,5)P2. (C) 1997 Acad emic Press.