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PMID: 11533253 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

Recruitment of the class II phosphoinositide 3-kinase C2beta to the epidermal growth factor receptor: role of Grb2.

Molecular and cellular biology ·Vol. 21 ·No. 19 ·2001-10-00 ·Pages 6660-7

Wheeler M, Domin J

Abstract

Previously we demonstrated that the class II phosphoinositide 3-kinase C2beta (PI3K-C2beta) 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-C2beta 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-C2beta 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-C2alpha 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-C2beta is indirect, we sought to identify an adaptor molecule that could mediate their association. In addition to the EGF receptor, PI3K-C2beta(2-298) also isolated both Shc and Grb2 from A431 cell lysates. Recombinant Grb2 directly bound PI3K-C2beta 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-C2beta was isolated by coimmunoprecipitation with anti-Grb2 antibody from numerous cell lines. Using immobilized, phosphorylated EGF receptor, recombinant PI3K-C2beta was only purified in the presence of Grb2. We conclude that proline-rich motifs within the N terminus of PI3K-C2beta mediate the association of this enzyme with activated EGF receptor and that this interaction involves the Grb2 adaptor.

MeSH Terms
Adaptor Proteins, Signal Transducing Adaptor Proteins, Vesicular Transport Amino Acid Motifs Binding Sites Binding, Competitive Cell Line ErbB Receptors/metabolism GRB2 Adaptor Protein Humans Macromolecular Substances Peptides/metabolism Phosphatidylinositol 3-Kinases/chemistry,metabolism Proline/metabolism Protein Transport Proteins/chemistry,metabolism,physiology Shc Signaling Adaptor Proteins Src Homology 2 Domain-Containing, Transforming Protein 1 Tumor Cells, Cultured src Homology Domains
Chemicals
Adaptor Proteins, Signal Transducing Adaptor Proteins, Vesicular Transport GRB2 Adaptor Protein GRB2 protein, human Macromolecular Substances Peptides Proteins SHC1 protein, human Shc Signaling Adaptor Proteins Src Homology 2 Domain-Containing, Transforming Protein 1 Proline Phosphatidylinositol 3-Kinases ErbB Receptors
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Wheeler M
Division of Medicine, Imperial College School of Medicine, London W12 0NN, United Kingdom.
Domin J
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.21.19.6660-6667.2001
Published
2001-10-00
Pages
6660-7
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC99811
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.21.19.6660-6667.2001 PMC Full Text View journal details
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