Recruitment of the Class II Phosphoinositide 3-Kinase C2β to the Epidermal Growth Factor Receptor: Role of Grb2

Abstract

Previously we demonstrated that the class II phosphoinositide 3-kinase C2β (PI3K-C2β) is rapidly recruited to a phosphotyrosine signaling complex containing the activated receptor for epidermal growth factor (EGF). Although this association was shown to be dependent upon specific phosphotyrosine residues present on the EGF receptor, the underlying mechanism remained unclear. In this study the interaction between PI3K-C2β and the EGF receptor is competitively attenuated by synthetic peptides derived from each of three proline-rich motifs present within the N-terminal region of the PI3K. Further, a series of N-terminal PI3K-C2β fragments, truncated prior 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-C2α that lacks similar proline-rich motifs was unable to affinity purify the activated EGF receptor from cell lysates. Since these findings revealed that the interaction between the EGF receptor and PI3K-C2β is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2β(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2β in vitro, and this effect was reproduced using either SH3 domain expressed as a glutathione S-transferase (GST) fusion. Interaction with Grb2 dramatically increased the catalytic activity of this PI3K. The relevance of this association was confirmed when PI3K-C2β was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2β was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2β mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.

ACKNOWLEDGMENTS

We thank Tony Pawson for his generous provision of human Grb2 pcDNA3.0 cDNA. We are grateful to Charles Coombes, Imperial College School of Medicine, for provision of the T47D, SKBr3, and HMT3522 breast cancer cell lines and to L. Racusen, The John Hopkins University School of Medicine, Baltimore, Md., for the human renal tubular epithelial cell line HKC-8.

This work was supported by grants from The Wellcome Trust and BBSRC.