Regulation of the cell cycle by SCF and APC/C ubiquitin ligases

描述(申请人提供)：泛素-蛋白酶体系统对关键细胞周期调节蛋白的时间协调破坏代表了一种重要的调节机制，以确保特定的蛋白质功能在正确的时间、正确的间隔和单向的方式被关闭。细胞周期机制中许多核心成分的蛋白分解是由两类主要的泛素连接酶控制的：SCF(Skp1-Cul1-F-box蛋白)复合体和后期促进复合体/环体(APC/C)。在人类中有68个SCF连接酶，每个连接酶都有一个不同的F-box蛋白亚基，通过直接将底物招募到连接酶的其余部分，并最终招募到泛素结合酶来提供特异性。尽管有大量的F-box蛋白，但只有三种人SCF泛素连接酶(分别包含F-box蛋白betaTrcp、Fbw7和Skp2)具有成熟的底物，其中许多与细胞周期调控有关(例如，CDc25A、Cyclin E、Emi1、p21、p27、Wee1)。我们提出了一个项目，重点是细胞周期网络的新一层控制及其与泛素系统的整合。利用一种新的筛选方法，我们已经鉴定了六种新的假定的SCF底物，并将表征其中之一E2F3的降解机制、调节和生物学功能。E2F3是一种与细胞周期控制密切相关的蛋白质(目标1)。我们将进一步确定和表征那些具有生物学意义的底物，这些底物是F-box蛋白Fbw5破坏的目标，以调节细胞周期进展(目标2)。最后，我们将研究BetaTrcp在控制一种新底物降解中的作用，该底物是通过生化筛选确定的：Claspin，一种属于DMA复制监视机制(AIM 3)的蛋白质。鉴于细胞周期机制的关键作用，细胞周期调节蛋白的改变降解显然是癌细胞典型的不受限制的增殖的决定因素。随着我们继续揭开泛素系统调节蛋白的预定降解如何控制细胞增殖的机制，我们致力于将我们的基础研究成果与对恶性转化的理解相结合。预计我们的研究结果将对基础科学和癌症生物学产生影响。

DESCRIPTION (provided by applicant): Temporally coordinated destruction of key cell cycle regulatory proteins by the ubiquitin-proteasome system represents an important regulatory mechanism to ensure that specific protein functions are turned off at the right time, in the right compartment and in a unidirectional fashion. Proteolysis of many core components of the cell cycle machinery is controlled by two major classes of ubiquitin ligases: the SCF (Skp1-Cul1-F-box protein) complexes and the Anaphase Promoting Complex/Cyclosome (APC/C). In humans there are sixty- eight SCF ligases, each characterized by a different F-box protein subunit that provides specificity by directly recruiting the substrate to the rest of the ligase and, ultimately, to the ubiquitin conjugating enzyme. Despite the large number of F-box proteins, only three human SCF ubiquitin ligases (containing the F-box proteins betaTrcp, Fbw7 and Skp2, respectively) have well-established substrates, many of which are involved in cell cycle control (e.g., Cdc25A, cyclin E, Emi1, p21, p27, Wee1). We propose a project focused on a new tier of control of the cell cycle networks and its integration with the ubiquitin system. Using a novel screen, we have identified six novel putative SCF substrates, and we will characterize the mechanism, regulation and biological function of the degradation of one of them, namely E2F3, a protein intimately involved in the control of the cell cycle (Aim 1). We will furthermore identify and characterize those biologically significant substrates that are targeted for destruction by the F-box protein Fbw5 to regulate cell cycle progression (Aim 2). Finally, we will study the role of betaTrcp in controlling the degradation of a novel substrate identified using a biochemical screen: Claspin, a protein that is part of the DMA replication surveillance machinery (Aim 3). Given the crucial function of the cell cycle machinery, altered degradation of cell cycle regulatory proteins is clearly a contributing determinant of the unrestrained proliferation typical of cancer cells. As we continue to unravel the mechanisms of how the scheduled degradation of regulatory proteins by the ubiquitin system controls cellular proliferation, we are committed to the integration of our basic research results with an understanding of malignant transformation. It is anticipated that the results of our studies will have an impact on both basic science and cancer biology.