Effect of epidermal growth factor on phosphatidylinositol 3-kinase activity in rabbit corneal epithelial cells

We investigated the effect of epidermal growth factor (EGF) on phosphatidylinositol 3-kinase (PI 3-kinase) activity in SV-40 transformed rabbit corneal epithelial cells (RCEC) and in normal corneal epithelial cells grown in primary culture. The enzyme products, 3-phosphorylated phosphoinositides, were identified by TLC and by deacylation followed by separation on HPLC. Addition of 30 ng ml-1 EGF to 32P-labeled SV-40 transformed or primary epithelial cell culture, increased the radioactivity in phosphatidylinositol 3,4,5-trisphosphate (PIP3) by about 96% over that of control incubations. The growth factor also significantly increased the radioactivity in phosphatidylinositol 4-phosphate [PI(4)P], phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and phosphatidic acid which was quite similar in the two cell types. The EGF-mediated increase in PIP3 formation in RCEC was dose- and time-dependent. Treatment of RCEC lysate with anti-PI 3-kinase antibody immunoprecipitated proteins that phosphorylated PI, PI(4)P and PI(4,5)P2 to corresponding 3-phosphorylated phosphoinositides. Incubation of RCEC with EGF resulted in a dose- and time-dependent increase in PI 3-kinase activity associated with the immunoprecipitate. Nonionic detergent, Nonidet P-40, strongly inhibited the PI 3-kinase activity in the immunoprecipitate, but it stimulated the PI 4-kinase activity in the cell lysate. Wortmannin, a fungal metabolite, also exerted a dose-dependent inhibitory effect on the activity of PI 3-kinase. Analysis of the PI 3-kinase immunoprecipitate by Western blot did not show any increase in concentration of the 85-kDa subunit of PI 3-kinase in EGF-treated cells. However, probing the immunoblot with anti-phosphotyrosine antibody showed a large increase (145%) in phosphorylation of the 85-kDa subunit of the enzyme. It can be concluded from these data that PIP3 and other 3-phosphoinositides exist in RCEC, and that EGF stimulates the production of these lipids by activation of PI 3-kinase, probably by increased tyrosine phosphorylation of the 85-kDa subunit of the enzyme.