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首页> 外文期刊>Molecular and Cellular Biology >Recruitment of the Class II Phosphoinositide 3-Kinase C2β to the Epidermal Growth Factor Receptor: Role of Grb2
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Recruitment of the Class II Phosphoinositide 3-Kinase C2β to the Epidermal Growth Factor Receptor: Role of Grb2

机译：表皮生长因子受体II类磷酸肌醇3-激酶C2β的招募：Grb2的作用。

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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.
机译：以前，我们证明II类磷酸肌醇3激酶C2β（PI3K-C2β）被迅速募集到磷酸酪氨酸信号复合物，该复合物包含表皮生长因子（EGF）的活化受体。尽管显示这种关联取决于存在于EGF受体上的特定磷酸酪氨酸残基，但其潜在机制仍不清楚。在这项研究中，PI3K-C2β与EGF受体之间的相互作用被PI3K N端区域内三个富含脯氨酸基序的合成肽竞争性削弱。此外，在每个富含脯氨酸的区域之前被截断的一系列N末端PI3K-C2β片段以降低的效率与受体结合。富含脯氨酸的区域无法介导受体缔合。最后，缺乏相似的富含脯氨酸基序的PI3K-C2α的等效N末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，因此我们寻求鉴定一种介导其缔合的衔接子分子。除EGF受体外，PI3K-C2β（2-298）还从A431细胞裂解物中分离了Shc和Grb2。重组Grb2在体外直接结合PI3K-C2β，使用表达为谷胱甘肽 S -转移酶（GST）融合蛋白的SH3结构域可重现该效应。与Grb2的相互作用极大地提高了该PI3K的催化活性。当通过与多种细胞系中的抗Grb2抗体共免疫沉淀分离出PI3K-C2β时，就证实了这种关联的相关性。使用固定的磷酸化EGF受体，仅在Grb2存在下纯化重组PI3K-C2β。我们得出结论，PI3K-C2βN末端内富含脯氨酸的基序介导了该酶与活化EGF受体的缔合，并且这种相互作用涉及Grb2衔接子。

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