Class II phosphoinositide 3-kinases are downstream targets of activated polypeptide growth factor receptors

The class II phosphoinositide 3-kinases (PI3K) PI3K-C2 alpha and PI3K-C2 be ta are two recently identified members of the large PI3K family. Both enzym es are characterized by the presence of a C2 domain at the carboxy terminus and, in vitro, preferentially utilize phosphatidylinositol and phosphatidy linositol 4-monophosphate as lipid substrates. Little is understood about h ow the catalytic activity of either enzyme is regulated in vivo. In this st udy, we demonstrate that PI3K-C2 alpha and PI3K-C2 beta represent two downs tream targets of the activated epidermal growth factor (EGF) receptor in hu man carcinoma-derived A431 cells. Stimulation of quiescent cultures with EG F resulted in the rapid recruitment of both enzymes to a phosphotyrosine si gnaling complex that contained the EGF receptor and Erb-B2. Ligand addition also induced the appearance of a second, more slowly migrating band of PI3 K-C2 alpha and PI3K-C2 beta immunoreactivity on sodium dodecyl sulfate-poly acrylamide gel electrophoresis. Since both PI3K enzymes can utilize Ca2+ as an essential divalent cation in lipid kinase assays and since the catalyti c activity of PI3K-C2 alpha is refractory to the inhibitor wortmannin, thes e properties were used to confirm the recruitment of each PI3K isozyme to t he activated EGF receptor complex. To examine this interaction in greater d etail, PI3K-C2 beta was chosen for further investigation. EGF and platelet- derived growth factor also stimulated the association of PI3K-C2 beta with their respective receptors in other cells, including epithelial cells and f ibroblasts. The use of EGF receptor mutants and phosphopeptides derived fro m the EGF receptor and Erb-B2 demonstrated that the interaction with recomb inant PI3K-C2 beta occurs through E(p)YL/I phosphotyrosine motifs. The N-te rminal region of PI3K-C2 beta was found to selectively interact with the EG F receptor in vitro, suggesting that it mediates the association of this PI 3K with the receptor. However, the mechanism of this interaction remains un clear. We conclude that class II PI3K enzymes may contribute to the generat ion of 3' phosphoinositides following the activation of polypeptide growth factor receptors in vivo and thus mediate certain aspects of their biologic al activity.