Functional analysis of complex type ubiquitin ligase

泛素化和随后的蛋白酶体降解调节蛋白控制着各种细胞过程，包括细胞周期进程、基因转录和信号转导。泛素与靶蛋白的结合由三种酶El， E2和E3处理。E3s负责识别和招募多泛素化的靶蛋白。以cullin为基础的E3复合体，具有很好的E3特征，被认为可以调节许多细胞事件。在本研究中，我们试图阐明cu12基E3s之一ECV^<Fem1B>的功能。现在，我们发现ECV^<Fem1B>负责调节Nek2的细胞水平，Nek2作为m期激酶，通过靶向其泛素化和蛋白水解。在Fem1B敲低的细胞中，Nek2的消除受损，导致该蛋白的异常积累。共免疫沉淀分析也显示Fem1B在体内与Nek2相互作用。过表达WT Nek2促进了Nek2的降解。最后，纯化的重组ECV^<Fem1B>复合物在体外介导Nek2泛素化。因此，这些观察结果表明，ECV^<Fem1B>复合体通过决定Nek2的丰度，在细胞周期进程中起着重要作用。

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.