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。有丝分裂调节因子的协调活动，包括有丝分裂激酶和纺锤体组装检查点蛋白，是忠实的染色体分离所必需的。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的稳定性。NudC是一种高度保守的蛋白，调节有丝分裂和细胞分裂。我最近发现NudC是极光激酶B （Aurora B）的一种新型底物。敲低NudC导致着丝点上的极光B定位错误。NudC缺陷细胞也表现出纺锤体检查点减弱和后期过早开始，这提出了一种有趣的可能性，即Aurora B对NudC的磷酸化可能参与调节这些过程。我将测试Aurora b磷酸化NudC调节纺锤体检查点活动的假设。为此，Aim 1将首先确定Aurora B对NudC的磷酸化作用，然后鉴定NudC中的Aurora B磷酸化位点，使这些位点突变，并通过体外ip激酶试验确认其真实性。我将通过gst -pull - down和co-IP分析绘制NudC与Aurora B相互作用的域。此外，我将生成抗磷酸化NudC抗体，以检测极光b磷酸化NudC在有丝分裂中的表达和定位。Aurora B RNAi和Aurora B激酶抑制剂将用于在体内证实maurora B磷酸化NudC。目的2将研究Aurora b磷酸化的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缺陷细胞的后期发病时间。我们将采用敲除NudC然后用NudC突变体进行挽救的方法来确定Aurora b磷酸化的NudC是否可以挽救过早的晚期发作。我将进一步研究在nudc缺陷细胞中纺锤体检查点减弱和早期后期发作是由于着丝点依赖途径（检查点蛋白募集到着丝点），还是由于着丝点独立途径（细胞质有丝分裂检查点复合体[MCC]），或两者兼有。这些研究应该阐明NudC如何通过与Aurora B的相互作用调节有丝分裂进程并确保基因组完整性。

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.