Wheeler M, Domin J
Division of Medicine, Imperial College School of Medicine, London W12 0NN, United Kingdom.
Mol Cell Biol. 2001 Oct;21(19):6660-7. doi: 10.1128/MCB.21.19.6660-6667.2001.
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.
先前我们证明，II类磷酸肌醇3激酶C2β（PI3K - C2β）可迅速募集至一个含表皮生长因子（EGF）活化受体的磷酸酪氨酸信号复合物。尽管这种关联显示依赖于EGF受体上存在的特定磷酸酪氨酸残基，但其潜在机制仍不清楚。在本研究中，PI3K - C2β与EGF受体之间的相互作用被源自PI3K N端区域内三个富含脯氨酸基序中每一个的合成肽竞争性减弱。此外，一系列在每个富含脯氨酸区域之前被截短的PI3K - C2β N端片段与受体结合的效率降低。单个富含脯氨酸区域无法介导与受体的关联。最后，缺乏类似富含脯氨酸基序的PI3K - C2α的等效N端片段无法从细胞裂解物中亲和纯化活化的EGF受体。由于这些发现揭示了EGF受体与PI3K - C2β之间的相互作用是间接的，我们试图鉴定一种能够介导它们关联的衔接分子。除了EGF受体外，PI3K - C2β(2 - 298)还从A431细胞裂解物中分离出了Shc和Grb2。重组Grb2在体外直接与PI3K - C2β结合，并且使用作为谷胱甘肽S - 转移酶（GST）融合蛋白表达的任何一个SH3结构域都能重现这种效应。与Grb2的相互作用显著增加了该PI3K的催化活性。当通过与抗Grb2抗体共免疫沉淀从众多细胞系中分离出PI3K - C2β时，这种关联的相关性得到了证实。使用固定化的、磷酸化的EGF受体，重组PI3K - C2β仅在存在Grb2的情况下被纯化。我们得出结论，PI3K - C2β N端的富含脯氨酸基序介导了该酶与活化的EGF受体的关联，并且这种相互作用涉及Grb2衔接分子。
