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Molecular and Cellular Biology, October 2001, p. 6660-6667, Vol. 21, No. 19
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.19.6660-6667.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Recruitment of the Class II Phosphoinositide 3-Kinase C2 to the Epidermal Growth Factor Receptor: Role of Grb2

Matthew Wheeler and Jan Domin^*

Division of Medicine, Imperial College School of Medicine, London W12 0NN, United Kingdom

Received 19 December 2000/Returned for modification 19 February 2001/Accepted 27 June 2001

Previously we demonstrated that the class II phosphoinositide 3-kinase C2 (PI3K-C2) 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-C2 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-C2 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-C2 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-C2 is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2 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-C2 was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2 was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2 mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.

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^* Corresponding author. Mailing address: Renal Section, Division of Medicine, Imperial College School of Medicine, Du Cane Rd., London W12 0NN, United Kingdom. Phone: 020 8383 2357. Fax: 020 8383 2062. E-mail: j.domin{at}ic.ac.uk.

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Molecular and Cellular Biology, October 2001, p. 6660-6667, Vol. 21, No. 19
0270-7306/01/$04.00+0   DOI: 10.1128/MCB.21.19.6660-6667.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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