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

14-3-3蛋白是一个酸性二聚体蛋白家族，广泛分布于真核细胞中。该蛋白家族以磷酸化依赖的方式与多种蛋白结合，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筛选PC12细胞中的14-3-3结合蛋白。我们开发了一种新的多重亲和标签myc-TEV-FLAG (MEF)，并将MEF标记的14-3-3η cDNA横切到PC12细胞中。从PC12提取物中提取MEF-14-3-3η蛋白相关蛋白，采用SDS-PAGE和质谱分析。这一过程使我们鉴定出121种蛋白质，其中包括14种已经报道为14-3-3已知靶标的蛋白质和107种新的相互作用伙伴。这些蛋白包括一系列生长因子信号通路中的组分，如质膜中的生长因子受体和小gtp结合蛋白的效应器，表明14-3-3蛋白可能作为协调者，与蛋白激酶的作用偶联产生功能性信号复合物。我们还观察到其中一个已鉴定的蛋白，激酶蛋白轻链2 (KLC2)，与14-3-3直接相关，并且它们的关联依赖于KLC2在Ser575位点的磷酸化。

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.