The adaptor protein Shc exists in three isoforms; p46, p52, and p66, and is a key regulator of a variety of biological processes. Our previous studies have shown that the tyrosine kinase c-Src phosphorylates Shc in a phosphatidylinositol (PtdIns) 4,5-bisphosphate-dependent manner. Here we demonstrate that PtdIns 3,4,5-trisphosphate stimulates phosphorylation of Shc by c-Src. The phosphorylation is blocked by a glutathione S-transferase fusion protein containing Shc phosphotyrosine binding (PTB) domain or a phosphotyrosine-containing Shc PTB domain-binding peptide. In rat pheochromocytoma cell line PC12, nerve growth factor (NGF) stimulates tyrosine phosphorylation of both Triton-soluble and -insoluble Shc which was maximal at 2-5 min after NGF treatment. We find that pretreatment of PC12 cells with the PtdIns 3-kinase inhibitor wortmannin or LY294002 results in almost half inhibition of the NGF-dependent tyrosine phosphorylation of only Triton-insoluble Shc. Similar inhibitory effect is observed with tyrosine kinase inhibitors genistein and PP1. Upon NGF stimulation, c-Src also becomes tyrosine-phosphorylated and accumulates in the Triton-insoluble fraction. The c-Src events are insensitive to wortmannin but sensitive to genistein. These results suggest that coordinate action of PtdIns 3-kinase and/or PtdIns 3,4,5-trisphosphate and c-Src can function as positive regulator in tyrosine phosphorylation of Shc in vitro and in vivo.