PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE STIMULATES PHOSPHORYLATION OF THE ADAPTER PROTEIN SHC BY C-SRC

The adaptor protein She was prepared as glutathione S-transferase fusi on proteins (GST-Shc) and used as in vitro substrate for c-Src. Since phosphotyrosine-binding domain of She has been shown to bind phosphati dyl-inositol 4,5-bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Natu re 378, 584-592], effect of PtdIns(4,5)P2 on the phosphorylation of GS T-Shc by c-Src was examined, PtdIns(4,5)P2 stimulated the phosphorylat ion of GST-Shc without any effect on the c-Src activity as judged by b oth its autophosphorylation and phosphorylation of exogenous substrate , Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic ac id, phosphatidylinositol, and phosphatidylinositol 4-phosphate but not phosphatidylcholine stimulated the c-Src activity itself. K-m for GST -Shc in the presence of 1 mu M PtdIns(4,5)P2 was calculated to be 90 n M. The PtdIns(4,5)P2-dependent phosphorylation of GST-Shc was inhibite d by a GST-fusion protein containing the phosphotyrosine-binding domai n of She. These results suggest that PtdIns(4,5)P2 can act as a regula tor of phosphorylation of She by c-Src through its binding to She. (C) 1997 Federation of European Biochemical Societies.