C-SRC AND PHOSPHATIDYLINOSITOL 3-KINASE ARE INVOLVED IN NGF-DEPENDENTTYROSINE PHOSPHORYLATION OF SHC IN PC12 CELLS

The adaptor protein She exists in three isoforms; p46, p52, and p66, a nd is a key regulator of a variety of biological processes. Our previo us studies have shown that the tyrosine kinase c-Src phosphorylates Sh e in a phosphatidylinositol (PtdIns) 4,5-bisphosphate-dependent manner . Here we demonstrate that PtdIns 3,4,5-trisphosphate stimulates phosp horylation of She by c-Src. The phosphorylation is blocked by a glutat hione S-transferase fusion protein containing She phosphotyrosine bind ing (PTB) domain or a phosphotyrosine-containing She PTB domain-bindin g peptide. In rat pheochromocytoma cell line PC12, nerve growth factor (NGF) stimulates tyrosine phosphorylation of both Triton-soluble and -insoluble She which was maximal at 2-5 min after NGF treatment. We fi nd that pretreatment of PC12 cells with the PtdIns 3-kinase inhibitor wortmannin or LY294002 results in almost half inhibition of the NGF-de pendent tyrosine phosphorylation of only Triton-insoluble She. Similar inhibitory effect is observed with tyrosine kinase inhibitors geniste in and PP1. Upon NGF stimulation, c-Src also becomes tyrosine-phosphor ylated and accumulates in the Triton-insoluble fraction. The c-Src eve nts are insensitive to wortmannin but sensitive to genistein. These re sults suggest that coordinate action of PtdIns 3-kinase and/or PtdIns 3,4,5-trisphosphate and c-Src can function as positive regulator in ty rosine phosphorylation of She in vitro and in vivo. (C) 1998 Academic Press.