SCF Ubiquitin Ligases in Cell Cycle Control and Chromosome Stability

项目总结摘要 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 通过细胞周期蛋白和细胞周期蛋白激酶抑制物(CKI)，它们在细胞周期进程中振荡，并决定 细胞周期蛋白依赖性激酶的激活动力学，推动细胞周期向前。定义路径， 正常细胞周期中蛋白质丰度动态的网络和机制是必不可少的 了解增殖控制。泛素是真核生物蛋白质降解的主要调节因子， 在细胞周期进程中起着至关重要且高度保守的作用。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者，并塑造了细胞周期后的蛋白质格局 翻译过来就是为了促进扩散。SCF连接酶结合一组~70个可互换的底物受体 称为F-box蛋白，它决定了SCF靶点的选择。同名的Cyclin F是 与经典的CDK激活剂中发现的一样，具有周期蛋白同源结构域。宁可 然后通过磷酸化激活CDK来促进细胞周期，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的mRNA和蛋白水平在细胞周期中显著振荡 比其他F-box编码基因范围更大。细胞周期蛋白F是小鼠存活所必需的，也是 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，它的突变与 神经退行性疾病肌萎缩侧索硬化症。尽管如此，仍然有大量的知识 细胞间隙与细胞周期蛋白F控制的途径和调控方式有关。定义衬底和机构 细胞周期蛋白F的研究对于从根本上理解细胞周期以及人类健康具有重要意义。在.期间 在之前的资金周期中，我们的实验室确定了几种细胞周期蛋白F底物，强调了它在细胞周期中的重要性 控制力。在这里，我们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 与疾病相关，同时也开始剖析细胞周期蛋白F通过的复杂机制 控制住了。基于创新的、公正的、计算的和蛋白质组策略，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，以及其调控的未知机制。在目标1中，我们 研究细胞周期蛋白F通过调节肿瘤抑制因子在G1/S细胞周期中的作用 视网膜母细胞瘤(RB)-口袋蛋白家族，RBL2/p130。我们将确定细胞周期蛋白F 控制RB途径，以及它对基因表达、细胞周期和增殖控制的贡献。在……里面 目标2和3，我们将注意力转向收敛于细胞周期蛋白F以调节其丰度的机制 以及潜在的活动。目标2的重点是通过激酶调节细胞周期蛋白F，以及它们如何决定细胞周期蛋白 F稳定性。在目标3中，我们扩展了对细胞周期蛋白F翻译后调节的分析，确定了它是如何 受去泛素酶调节。总而言之，这些研究将为周期蛋白F的变化提供全面的分析 其自身的调控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.