Functional analysis of complex type ubiquitin ligase

调节蛋白的遍在化和随后的蛋白酶体降解控制多种细胞过程，包括细胞周期进程、基因转录和信号转导。泛素与靶蛋白的缀合通过三种酶E1、E2和E3加工。E3负责识别和募集多聚泛素化的靶蛋白。基于Cullin的E3复合物是E3的良好表征，被认为调节许多细胞事件。在本研究中，我们试图阐明基于Cu 12的E3之一ECV 1的功能<Fem1B>。现在我们表明，ECV 1<Fem1B>负责调节Nek 2的细胞水平，Nek 2作为M期激酶发挥作用，通过靶向其进行泛素化和蛋白水解。在Fem 1B敲低细胞中，Nek 2的消除受损，导致蛋白质的异常积累。免疫共沉淀分析还显示，Fem 1B与Nek 2在体内相互作用。WT Nek 2的过表达促进了Nek 2的降解。最后，纯化的重组ECV-1<Fem1B>复合物在体外介导Nek 2泛素化。因此，这些观察结果表明，ECV 2<Fem1B>复合物通过确定Nek 2的丰度在细胞周期进程中起重要作用。

Ubiquitylation and subsequent proteasomal degradation of regulatory proteins control a variety of cellular processes, including cell cycle progression, gene transcription, and signal transduction. The ubiquitin conjugation to target proteins is processed by three enzymes, El, E2, and E3. The E3s are responsible for recognizing and recruiting target proteins for polyubiquitylation. Cullin-based E3 complexes, that are well characterized E3s, are thought to regulate many cellular events. In this research, we try to clarify the function of one of Cu12-based E3s, ECV^<Fem1B>. Now we show that ECV^<Fem1B> is responsible for regulating the cellular level of Nek2, which functions as M-phase kinase, by targeting it for ubiquitylation and proteolysis. The elimination of Nek2 was impaired in Fem1B knockdown cells, resulting in abnormal accumulation of the protein. Coimmunoprecipitation analysis also revealed that Fem1B interacts with Nek2 in vivo. Overexpression of WT Nek2 promoted degradation of Nek2. Finally, the purified recombinant ECV^<Fem1B> complex mediated Nek2 ubiquitylation in vitro. These observations thus demonstrate that the ECV^<Fem1B> complex plays an important role in cell-cycle progression by determining the abundance of Nek2.