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Proteomic characterization of proteins associated with the 14-3-3 family members

与 14-3-3 家族成员相关的蛋白质的蛋白质组学表征

基本信息

批准号：
12680748
负责人：
ICHIMURA Tohru
金额：
$ 1.34万
依托单位：
Tokyo Metropolitan University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2002
项目状态：
已结题

项目摘要

The 14-3-3 protein is a family of acidic, dimeric proteins distributed widely among eukaryotic cells. This protein family binds to a variety of proteins in a phosphorylation-dependent manner and participants in the regulation of cell proliferation, differentiation and function. In this study, we have attempted to screen 14-3-3-binding proteins in PC12 cells by means of proteomic techniques. We developed a novel multiple affinity tag, called myc-TEV-FLAG (MEF), and transected the MEF-tagged 14-3-3η cDNA into PC12 cells. The proteins associated with the expressed MEF-14-3-3η protein were pulled from the PC12 extracts and were analyzed by SDS-PAGE and mass spectrometry. This procedure allowed us to identify 121 proteins that include 14 proteins already reported as known targets of 14-3-3 and 107 novel interacting partners. These proteins include a series of components in the growth factor signaling pathways such as the growth factor receptor in the plasma membrane and the effectors of small GTP-binding proteins, suggesting that the 14-3-3 protein may act as a coordinator that creates functional signaling complexes in the coupled with the action of protein kinases. It was also observed that one of the identified proteins, kinesin light chain 2 (KLC2), was directly associated with 14-3-3, and their association was dependent on the phosphorylation of KLC2 at Ser575.

14-3-3蛋白是广泛分布于真核细胞中的酸性二聚体蛋白家族。该蛋白质家族以磷酸化依赖的方式与多种蛋白质结合，并参与细胞增殖、分化和功能的调节。在本研究中，我们试图通过蛋白质组学技术在PC 12细胞中筛选14-3-3结合蛋白。我们开发了一种新的多亲和标签，称为myc-TEV-FLAG（MEF），并将MEF标记的14-3-3η cDNA转染到PC 12细胞中。从PC 12提取物中提取与表达的MEF-14-3-3η蛋白相关的蛋白质，并通过SDS-PAGE和质谱分析。这个程序使我们能够识别121种蛋白质，其中包括14种已经报道为14-3-3和107种新的相互作用伴侣的已知靶点的蛋白质。这些蛋白质包括一系列在生长因子信号通路中的组分，如质膜上的生长因子受体和小GTP结合蛋白的效应物，这表明14-3-3蛋白可能作为一个协调器，在与蛋白激酶的作用偶联中产生功能性信号复合物。还观察到一种鉴定的蛋白质，驱动蛋白轻链2（KLC 2），与14-3-3直接相关，并且它们的相关性依赖于KLC 2在Ser 575处的磷酸化。