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.
Zhang
Y
Y
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912, USA.
Akhtar
R A
RA
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002478:Cells, Cultured; D003315:Cornea / Q000201:enzymology*; D004815:Epidermal Growth Factor / Q000494:pharmacology*; D004848:Epithelium / Q000201:enzymology*; D017830:Octoxynol; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000819:agonists* / Q000037:antagonists & inhibitors; D019000:Phosphotyrosine / Q000032:analysis; D011092:Polyethylene Glycols / Q000494:pharmacology; D011817:Rabbits; D000077191:Wortmannin
D000730:Androstadienes; D004815:Epidermal Growth Factor; D017830:Octoxynol; D019869:Phosphatidylinositol 3-Kinases; D018129:Phosphatidylinositol Phosphates; D017853:Phosphotransferases (Alcohol Group Acceptor); D019000:Phosphotyrosine; D011092:Polyethylene Glycols; D000077191:Wortmannin; C010615:Nonidet P-40