SCF Ubiquitin Ligases in Cell Cycle Control and Chromosome Stability

项目摘要 关键蛋白质丰度和活性的动态振荡驱动细胞周期进程。这是典型的 细胞周期蛋白和细胞周期蛋白激酶抑制剂（CKIs），它们在细胞周期进程中振荡，并决定细胞周期。 细胞周期蛋白依赖性激酶的激活动力学，其推动细胞周期向前。定义路径， 在正常细胞周期中蛋白质丰度的动态网络和机制是至关重要的， 了解增殖控制。泛素是真核生物中蛋白质降解的主要调节因子， 在细胞周期进程中起重要和高度保守的作用。E3的Skp1-Cul1-F-box（SCF）家族 泛素连接酶是细胞周期进程的主要调节因子， 以促进扩散。SCF连接酶与一组约70种可互换的底物受体结合 称为F-box蛋白，其决定SCF靶选择。Epoxy Cyclin F是细胞周期蛋白的创始成员， F-box家族，并具有与典型CDK激活剂中发现的类似的细胞周期蛋白同源结构域。而 除了通过磷酸化激活CDK以促进细胞周期外，细胞周期蛋白F与SCF结合，促进细胞周期 通过泛素化。细胞周期蛋白F mRNA和蛋白水平在细胞周期中显著振荡， 比其他F-box编码基因更大的程度。细胞周期蛋白F是小鼠生存所必需的， 在许多人类细胞系中的生长/存活。此外，它在癌症中过度表达，其突变与癌症相关。 神经变性疾病肌萎缩性侧索硬化症然而，仍然有大量的知识 与细胞周期蛋白F控制什么途径以及如何调节有关的空白。定义基质和机制 对细胞周期蛋白F的研究对于细胞周期的基本认识以及人类健康都具有重要意义。期间 在上一个资助周期中，我们的实验室鉴定了几种细胞周期蛋白F底物，突出了其在细胞周期中的重要性 控制在这里，我们建立在这项工作的基础上，定义了一种在正常细胞周期控制中具有重要作用的新底物 并与疾病相关，同时也开始剖析细胞周期蛋白F的复杂机制， 控制。基于创新的、无偏见的、计算的和蛋白质组学的策略，我们的数据表明， 细胞周期蛋白F在细胞周期中的关键作用，以及其调节的未描述机制。在目标1中，我们 研究细胞周期蛋白F在G1/S进程中的作用，通过调节肿瘤抑制因子， 视网膜母细胞瘤（RB）-口袋蛋白家族，RBL 2/p130。我们将确定细胞周期蛋白F 控制RB通路以及这如何有助于基因表达、细胞周期和增殖控制。在 目标2和3，我们把我们的注意力集中在细胞周期蛋白F的机制，以调节其丰度 潜在的活动。目的2是研究激酶对细胞周期蛋白F的调控及其如何决定细胞周期蛋白的表达。 F稳定性。在目标3中，我们扩展了对细胞周期蛋白F的翻译后调节的分析，确定它是如何被调节的。 由去泛素化酶调节。总之，这些研究将提供一个全面的分析，如何细胞周期蛋白F是 自身调节及其对人类细胞周期的贡献。

PROJECT SUMMARY ABSTRACT Dynamic oscillations in the abundance and activity of key proteins drives cell cycle progression. This is typified by cyclins and cyclin kinase inhibitors (CKIs), which oscillate during cell cycle progression and determine the activation kinetics of Cyclin Dependent Kinases, which propel the cell cycle forward. Defining the pathways, networks and mechanisms underlying dynamics in protein abundance during normal cell cycles is essential to understanding proliferative control. Ubiquitin is the major regulator of protein degradation in eukaryotes and plays an essential and highly conserved role in cell cycle progression. The Skp1-Cul1-F-box (SCF) family of E3 ubiquitin ligases are major regulators of cell cycle progression and sculpt the protein landscape post- translationally to facilitate proliferation. SCF ligases engage a set of ~70 interchangeable substrate receptors termed F-box proteins, which dictate SCF target selection. The eponymous Cyclin F is the founding member of the F-box family and has a cyclin homology domain like that found in the canonical CDK activators. Rather than activate a CDK to promote cell cycle via phosphorylation, cyclin F binds to the SCF, promoting cell cycle through ubiquitination. Cyclin F mRNA and protein levels oscillate significantly during the cell cycle and to a greater extent than other F-box encoding genes. Cyclin F is required for viability in mice and essential for growth/survival in many human cell lines. Further, it is overexpressed in cancer, and its mutation is linked to the neurodegenerative disease amyotrophic lateral sclerosis. Nevertheless, there remain significant knowledge gaps related to what pathways cyclin F controls and how it is regulated. Defining substrates and mechanisms of cyclin F is significant to a fundamental understanding of cell cycle, as well as human health. During the previous funding cycle, our lab identified several cyclin F substrates, highlighting its importance in cell cycle control. Here, we build on that work, defining a new substrate with important roles in normal cell cycle control and with relevance to disease, while also beginning to dissect the complex mechanisms by which cyclin F is controlled. Borne out of innovative, unbiased, computational, and proteomic strategies, our data suggest new, critical roles for cyclin F in cell cycle, and undescribed mechanisms underlying its regulation. In aim 1, we examine the role of cyclin F in G1/S progression through regulation of a tumor suppressor in the retinoblastoma (RB)-pocket protein family, RBL2/p130. We will determine the mechanisms by which cyclin F controls the RB-pathway and how this contributes to gene expression, cell cycle, and proliferative control. In aims 2 and 3, we turn our attention towards mechanisms that converge on cyclin F to regulate its abundance and potentially activity. Aim 2 is focused on the regulation of cyclin F by kinases and how they determine cyclin F stability. In aim 3 we extend this analysis of post-translational regulation of cyclin F, determining how it is regulated by deubiquitinases. Together, these studies will provide a comprehensive analysis for how cyclin F is itself regulated and its contributions to cell cycle in humans.