Distinctive regulation of v-Src-associated phosphatidylinositol 3-kinase during PC12 cell differentiation.

In chicken embryo fibroblasts, the binding of v-Src to PtdIns 3-kinase requires Src homology domains, SH3, SH2 and the SH1 or kinase domain, which induces the cytoskeletal disruption associated with fibroblast transformation. In the rat phaeochromocytoma PC12 cell line, v-Src has a different effect on the cytoskeleton, inducing neurite extension rather than cytoskeletal disruption. Here we show that v-Src-induced neurite outgrowth is suppressed by the selective PtdIns 3-kinase inhibitor LY294002, suggesting that this effect of v-Src in PC12 cells also requires the activity of the lipid kinase. However, in contrast with chicken embryo fibroblasts, the association of PtdIns 3-kinase with v-Src in PC12 cells is delayed until several hours after activating the v-Src tyrosine kinase. Furthermore the v-Src-associated p85 regulatory subunit of PtdIns 3-kinase is not phosphorylated on tyrosine in PC12 cells and associates only weakly with isolated v-Src homology domains (SH3/SH2) in a Src kinase-independent manner. However, p85 and v-Src both associate with an unidentified protein (of molecular mass approx. 68 kDa; termed p68), which becomes tyrosine phosphorylated concomitantly with the association of both p85 and PtdIns 3-kinase with v-Src in PC12 cells. Thus we conclude that the mode of regulation of v-Src-associated PtdIns 3-kinase is cell-context-dependent and that p68 might act as an adaptor protein to mediate the association of p85 and v-Src in PC12 cells. The different regulation of PtdIns 3-kinase in PC12 and in chicken embryo fibroblasts in response to v-Src activity might reflect the different cytoskeletal rearrangements induced by this oncoprotein in the two cell types.