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

描述（由申请人提供）：哺乳动物SCF（Skp 1/Cul 1/F-box蛋白）泛素连接酶对于许多细胞过程的激活和减弱至关重要。它们通过以精确、快速和局部化的方式指导重要调控元件的蛋白水解来控制复杂的分子机器。在GM 57587的最初几年，我们专注于细胞周期蛋白依赖性激酶（CDK）的调节，这在细胞周期控制中起着关键作用，并定义了导致CDK抑制剂p21和p27通过F-box蛋白Skp 2降解的关键步骤。此外，我们发现APC/CCDh 1，SCF样连接酶，控制Skp 2的稳定性。我们还证明，TrCP是另一种F-box蛋白，通过靶向Cdc 25 A、Claspin、Rest和Cdk 1进行降解，可以精确调节关键的有丝分裂调节因子Cdk 1。最后，我们发现S和G2中SCF泛素连接酶靶向的三种底物在细胞周期的不同阶段通过APC/C降解（M中的p21，G1中的Cdc 25 A和Claspin）。 为了拓宽我们对SCF复合物以CDK为中心的作用的理解，我们使用公正的筛选发现，SCF除了控制细胞周期外，还监测和调节多种看似不同的细胞途径，将细胞周期控制与蛋白质合成、核糖体生物发生联系起来。细胞生存、DNA损伤检查点和昼夜节律钟。例如，我们发现：翻译抑制剂Pdcd 4和促凋亡蛋白BimEL在响应生长和存活因子时以<$TrCP依赖的方式降解; Fbxl 10和Fbxl 11有助于表观遗传调控; Cyclin F/Fbxo 1通过靶向CP 110降解来防止中心体过度复制;并且需要Fbxl 3通过促进转录抑制因子Cry 1和Cry 2的蛋白水解来重置生物钟。 我们现在提出一个项目，探索SCF控制的细胞周期网络与DNA复制和DNA损伤反应的整合。通过蛋白质组学筛选，我们已经确定了新的推定SCF和APC/C基板参与这些过程。我们将在DNA复制控制（目标1）、遗传毒性应激恢复（目标1和2）和有丝分裂（目标3）的背景下表征这些潜在底物降解的机制和调控。 公共卫生相关性：细胞依赖于网络化分子机器集合的正常功能来控制从细胞增殖到细胞死亡再到分化的各种过程。泛素系统可以快速降解这些机器的模块化调节组件，有助于复杂细胞过程的精确操作和同步。鉴于其关键作用，泛素系统在癌细胞中经常被解除管制。因此，预计拟议研究的结果将对基础科学和癌症生物学产生影响。

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). PUBLIC HEALTH RELEVANCE: Cells depend on the proper functioning of an ensemble of networked, molecular machines to control diverse processes, ranging from cell proliferation to cell death to differentiation. The ubiquitin system can rapidly degrade the modular regulatory components of these machines, contributing to the precise operation and synchronization of complex cellular processes. Given its critical role, the ubiquitin system is often deregulated in cancer cells. Thus, it is anticipated that the results of the proposed studies will have an impact on both basic science and cancer biology.