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

描述(申请人提供)：哺乳动物SCF(Skp1/Cul1/F-box蛋白)泛素连接酶对许多细胞过程的激活和减弱至关重要。它们以精确、快速和局部的方式指导重要调控元件的蛋白质分解，从而控制复杂的分子机器。在GM57587的最初几年中，我们专注于细胞周期调控中起关键作用的细胞周期蛋白依赖蛋白(CDK)的调节，并定义了导致CDK抑制剂p21和p27通过F-box蛋白Skp2降解的关键步骤。此外，我们还发现，APC/CCdh1是一种类似SCF的连接酶，控制着Skp2的稳定性。我们还证明了另一种F-box蛋白？TrCP通过靶向CDc25A、Claspin、REST和Emi1降解，允许精确调控关键的有丝分裂调节因子CDK1。最后，我们发现，在细胞周期的不同阶段，S和G2中SCF泛素连接酶靶向的三种底物都被APC/C降解(M中的p21，G1中的cdc25A和claspin)。为了拓宽我们对SCF复合体CDK中心作用的理解，我们使用了无偏见的筛选，发现SCF除了控制细胞周期外，还监测和调节多条看似不同的细胞通路，将细胞周期控制与蛋白质合成、核糖体生物发生、细胞存活、DNA损伤检查点和生物钟联系起来。例如，我们发现：翻译抑制物Pdcd4和促凋亡蛋白BimEL在对生长和生存因素的反应中以依赖TrCP的方式降解；Fbxl10和Fbxl11有助于表观遗传调节；Cyclin F/Fbxo1通过靶向CP110进行降解来防止中心体过度复制；以及Fbxl3需要通过促进转录抑制物Cry1和Cry2的蛋白分解来重置生物钟。我们现在提出一个项目，探索干细胞因子控制的细胞周期网络与DNA复制和DNA损伤反应的整合。通过蛋白质组筛选，我们已经确定了参与这些过程的新的假定的SCF和APC/C底物。我们将在DNA复制控制(目标1)、从遗传毒性应激中恢复(目标1和2)以及有丝分裂(目标3)的背景下描述这些潜在底物降解的机制和调节。

DESCRIPTION (provided by applicant): Mammalian SCF (Skp1/Cul1/F-box protein) ubiquitin ligases are critical for the activation and attenuation of many cellular processes. They control complex molecular machines by directing the proteolysis of important regulatory elements in a precise, rapid, and localized manner. During the initial years of GM57587, we focused on the regulation of cyclin-dependent kinases (CDKs), which play pivotal roles in cell cycle control, and defined the key steps leading to the degradation of the CDK inhibitors p21 and p27 via the F-box protein Skp2. Moreover, we found that APC/CCdh1, an SCF-like ligase, controls Skp2 stability. We also demonstrated that ¿TrCP, another F-box protein, allows precise regulation of the critical mitosis regulator Cdk1 by targeting Cdc25A, Claspin, Rest, and Emi1 for degradation. Finally, we found that three substrates that are targeted by SCF ubiquitn ligases in S and G2 are degraded via APC/C during different phases of the cell cycle (p21 in M, and Cdc25A and Claspin in G1). To broaden our understanding beyond CDK-centric roles of SCF complexes, we used unbiased screens and found that SCF, in addition to controlling the cell cycle, monitors and regulates multiple, seemingly disparate, cellular pathways, linking cell cycle control to protein synthesis, ribosomal biogenesis, cell survival, DNA-damage checkpoints, and the circadian clock. For example, we found that: the translation inhibitor Pdcd4 and the pro-apoptotic protein BimEL are degraded in a ¿TrCP-dependent manner in response to growth and survival factors; Fbxl10 and Fbxl11 contribute to epigenetic regulation; Cyclin F/Fbxo1 prevents centrosome overduplication by targeting CP110 for degradation; and Fbxl3 is required to reset the circadian clock by promoting the proteolysis of the transcriptional repressors Cry1 and Cry2. We now propose a project exploring the integration of SCF-controlled cell cycle networks with DNA replication and DNA damage response. Via proteomic screens, we have identified novel putative SCF and APC/C substrates involved in these processes. We will characterize the mechanism and regulation of the degradation of these potential substrates in the context of DNA replication control (Aim 1), recovery from genotoxic stress (Aims 1 and 2), and mitosis (Aim 3).