Function of NudC, a Novel Subtrate of Aurora B, in Spindle Checkpoint Activity

描述（由申请人提供）：有丝分裂是一个高度调控的过程，细胞通过该过程分裂并将其遗传物质均匀地分配给子细胞。染色体分离错误可能导致非整倍体和基因组不稳定，这是癌症的标志。有丝分裂调节因子（包括有丝分裂激酶和纺锤体组装检查点蛋白）的协调活动是染色体忠实分离所必需的。我的长期目标是了解有丝分裂调节因子如何控制染色体分离以确保基因组稳定性。 NudC 是一种高度保守的蛋白质，调节有丝分裂和胞质分裂。我最近发现 NudC 是极光激酶 B (Aurora B) 的一种新型底物。 NudC 的敲低导致 Aurora B 从着丝粒错误定位。 NudC 缺陷细胞还表现出纺锤体检查点减弱和后期过早开始，这提出了一个有趣的可能性：Aurora B 对 NudC 的磷酸化可能参与调节这些过程。我将检验 Aurora B 磷酸化 NudC 调节纺锤体检查点活性的假设。为此，目标 1 将首先确定 Aurora B 的 NudC 磷酸化。我将识别 NudC 中的 Aurora B 磷酸化位点，对这些位点进行突变，并通过体外 IP 激酶测定来确认其真实性。我将通过 GST 下拉和 co-IP 分析绘制 NudC 与 Aurora B 相互作用的域。此外，我将生成抗磷酸 NudC 抗体来检查 Aurora B 磷酸化 NudC 在有丝分裂中的表达和定位。 Aurora B RNAi 和 Aurora B 激酶抑制剂将用于确认 NudC 体内的 Aurora B 磷酸化。目标 2 将研究 Aurora B-磷酸化 NudC 在纺锤体组装检查点中的功能。我将使用活细胞成像来确定 NudC 缺陷细胞的后期开始时间。 NudC 敲低，然后用 NudC 突变体方法进行挽救，将用于确定 Aurora B 磷酸化 NudC 是否可以挽救过早的后期发作。我将进一步研究 NudC 缺陷细胞中纺锤体检查点减弱和后期早期发生是否是由于着丝粒依赖性途径（检查点蛋白招募到着丝粒）或动粒独立途径（胞质有丝分裂检查点复合物 [MCC]），或两者兼而有之。这些研究应该阐明 NudC 如何通过与 Aurora B 的相互作用来调节有丝分裂进程并确保基因组完整性。 与公共卫生的相关性：癌症是美国的主要原因之一，每年夺去大约 50 万人的生命。癌症的主要特征之一是不受控制的细胞增殖。了解细胞分裂的分子机制将阐明促进和/或促成肿瘤发生过程的因素。在为癌症患者设计治疗计划时，这些分子的识别为制定更有针对性的药物治疗方案提供了机会。

DESCRIPTION (provided by applicant): Mitosis is a highly regulated process by which cells divide and distribute their genetic material evenly to daughter cells. Errors in chromosome segregation can lead to aneuploidy and genomic instability, a hallmark of cancer. The coordinated activities of mitotic regulators, including mitotic kinases and spindle assembly checkpoint proteins, are required for faithful chromosome segregation. My long-term goal is to understand how mitotic regulators control chromosome segregation to ensure genomic stability. NudC, a highly conserved protein, regulates mitosis and cytokinesis. I recently discovered that NudC is a novel substrate of Aurora kinase B (Aurora B). Knockdown of NudC resulted in mislocalization of Aurora B from the kinetochore. NudC-deficient cells also exhibit a weakened spindle checkpoint and premature anaphase onset, raising the interesting possibility that phosphorylation of NudC by Aurora B might be involved in regulating these processes. I will test the hypothesis that Aurora B-phosphorvlated NudC regulates spindle checkpoint activity. To do so, Aim 1 will first determine NudC phosphorylation by Aurora B. I will identify Aurora B phospho-site(s) in NudC, mutate these sites and confirm their authenticity by IP-kinase assays in vitro. I will map domains of NudC interactions with Aurora B by GST-pulldown and co-IP assays. Further, I will generate anti-phospho NudC antibodies to examine the expression and localization of Aurora Bphosphorylated NudC in mitosis. Aurora B RNAi and an Aurora B kinase inhibitor will be used to confirmAurora B phosphorylation of NudC in vivo. Aim 2 will investigate the function of Aurora B-phosphorylated NudC in spindle assembly checkpoint. I will use live-cell imaging to determine the timing of anaphase onset in NudC-deficient cells. A NudC knockdown followed by rescue with NudC mutants approach will be used to determine if Aurora B-phosphorylated NudC can rescue premature anaphase onset. I will further investigate whether the weakened spindle checkpoint and early anaphase onset in NudC-deficient cells is due to a kinetochore-dependent pathway (checkpoint protein recruitment to the kinetochore), or a kinetochoreindependent pathway (cytosolic mitotic checkpoint complex [MCC]), or both. These studies should elucidate how NudC, through its interactions with Aurora B, regulates mitotic progression and ensures genomic integrity. Relevance to Public Health: Cancer is one of the major causes of death in the United States, claiming roughly 500,000 lives every year. One of the major hallmarks of cancer is uncontrolled cell proliferation. Understanding the molecular mechanisms by which cells divide will elucidate factors that promote and/or contribute to the tumorigenesis process. Identification of these molecules offers the opportunity for more tailored drug regimens when designing a treatment plan for cancer patients.