Zhang Y, Akhtar R A
Department of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta 30912, USA.
Exp Eye Res. 1996 Sep;63(3):265-75. doi: 10.1006/exer.1996.0115.
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.
我们研究了表皮生长因子（EGF）对SV - 40转化的兔角膜上皮细胞（RCEC）和原代培养的正常角膜上皮细胞中磷脂酰肌醇3激酶（PI 3激酶）活性的影响。通过薄层层析（TLC）以及脱酰基后在高效液相色谱（HPLC）上分离来鉴定酶产物3 - 磷酸化磷脂酰肌醇。向用³²P标记的SV - 40转化或原代上皮细胞培养物中添加30 ng/ml EGF，与对照孵育相比，磷脂酰肌醇3,4,5 - 三磷酸（PIP3）中的放射性增加了约96%。该生长因子还显著增加了磷脂酰肌醇4 - 磷酸[PI(4)P]、磷脂酰肌醇4,5 - 二磷酸[PI(4,5)P2]和磷脂酸中的放射性，这在两种细胞类型中非常相似。EGF介导的RCEC中PIP3形成的增加具有剂量和时间依赖性。用抗PI 3激酶抗体处理RCEC裂解物，免疫沉淀出的蛋白质可将PI、PI(4)P和PI(4,5)P2磷酸化为相应的3 - 磷酸化磷脂酰肌醇。用EGF孵育RCEC导致与免疫沉淀物相关的PI 3激酶活性呈剂量和时间依赖性增加。非离子去污剂Nonidet P - 40强烈抑制免疫沉淀物中的PI 3激酶活性，但它刺激细胞裂解物中的PI 4激酶活性。真菌代谢产物渥曼青霉素对PI 3激酶活性也有剂量依赖性抑制作用。通过蛋白质免疫印迹法（Western blot）分析PI 3激酶免疫沉淀物，未显示在EGF处理的细胞中PI 3激酶85 kDa亚基的浓度有任何增加。然而，用抗磷酸酪氨酸抗体探测免疫印迹显示该酶85 kDa亚基的磷酸化大幅增加（145%）。从这些数据可以得出结论，RCEC中存在PIP3和其他3 - 磷酸化磷脂酰肌醇，并且EGF可能通过增加该酶85 kDa亚基的酪氨酸磷酸化来激活PI 3激酶，从而刺激这些脂质的产生。
