Recruitment of the class II phosphoinositide 3-kinase C2 beta to the epidermal growth factor receptor: Role of Grb2

Previously we demonstrated that the class II phosphoinositide 3-kinase C2 b eta (PI3K-C2 beta) is rapidly recruited to a phosphotyrosine signaling comp lex containing the activated receptor for epidermal growth factor (EGF). Al though this association was shown to be dependent upon specific phosphotyro sine residues present on the EGF receptor, the underlying mechanism remaine d unclear. In this study the interaction between PI3K-C2 beta and the EGF r eceptor is competitively attenuated by synthetic peptides derived from each of three proline-rich motifs present within the N-terminal region of the P I3K. Further, a series of N-terminal PI3K-C2 beta fragments, truncated prio r to each proline-rich region, bound the receptor with decreased efficiency . A single proline-rich region was unable to mediate receptor association. Finally, an equivalent N-terminal fragment of PI3K-C2 alpha that lacks simi lar proline-rich motifs was unable to affinity purify the activated EGF rec eptor from cell lysates. Since these findings revealed that the interaction between the EGF receptor and PI3K-C2 beta is indirect, we sought to identi fy an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2 beta (2-298) also isolated both Shc and Grb2 fro m A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2 beta in vitro, and this effect was reproduced using either SH3 domain expressed as a glut athione S-transferase (GST) fusion. Interaction with Grb2 dramatically incr eased the catalytic activity of this PI3K. The relevance of this associatio n was confirmed when PI3K-C2 beta was isolated by coimmunoprecipitation wit h anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphory lated EGF receptor, recombinant PI3K-C2 beta was only purified in the prese nce of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2 beta mediate the association of this enzyme with activated EGF rece ptor and that this interaction involves the Grb2 adaptor.