Further evidence for the involvement of insulin receptor substrates in epidermal growth factor-induced activation of phosphatidylinositol 3-kinase

In accordance with our recent results obtained with cultured rat hepatocyte s [Fujioka, T. & Ui. M. (2001) Eur J. Biochem. 268, 25-34], epidermal growt h factor (EGF) gave rise to transient tyrosine phosphorylation of insulin r eceptor substrates (IRS-1 and IRS-2), thereby activating the bound phosphat idylinositol 3-kinase in human epidermoid carcinoma A431 cells normally abu ndant in EGF receptors (EGFR) and Chinese hamster ovary (CHO) cells transfe cted with full-length EGFR. These actions of EGF, although much smaller in magnitude than those of insulin or IGF-I in the same cells. were accompanie d by tyrosine phosphorylation of EGFR rather than insulin or IGF-I receptor s, never observed in wild-type CHO cells expressing no EGFR, and totally in hibited by an inhibitor of EGFR kinase, AG1478, that was without effect on insulin or IGF-I actions. Recombinant IRS-1 was phosphorylated on tyrosines upon incubation with purified EGFR from A431 cells and P-32-labeled ATR Wh en CHO cells were transfected with C-terminal truncated EGFR lacking three NPXY motifs responsible for direct binding to phosphotyrosine-binding domai ns of IRSs, no effect of EGF could be observed. We suggest that tyrosine ph osphorylation of IRS-1 or IRS-2 could mediate EGFR-induced activation of ph osphatidylinositol 3-kinase in mammalian cells.