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节的过程，细胞通过该过程分裂并将其遗传物质均匀分布到子细胞中。染色体分离错误可导致非整倍性和基因组不稳定性，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。敲除NudC导致Aurora B从动粒的错误定位。NudC缺陷细胞还表现出减弱的纺锤体检查点和过早的后期开始，提出了有趣的可能性，即Aurora B对NudC的磷酸化可能参与调节这些过程。我将检验极光B磷酸化的NudC调节纺锤体检查点活性的假设。为此，Aim 1将首先确定Aurora B对NudC的磷酸化。我将识别NudC中的Aurora B磷酸化位点，突变这些位点并通过体外IP激酶测定确认其真实性。我将通过GST-下拉和co-IP分析绘制NudC与Aurora B相互作用的结构域。此外，我将产生抗磷酸化NudC抗体，以检查有丝分裂中Aurora B磷酸化NudC的表达和定位。Aurora B RNAi和Aurora B激酶抑制剂将用于体内确认NudC的Aurora B磷酸化。目的2研究Aurora B磷酸化的NudC在纺锤体组装检查点中的作用。我将使用活细胞成像来确定NudC缺陷细胞中后期发作的时间。将使用NudC敲低然后用NudC突变体拯救的方法来确定Aurora B-磷酸化的NudC是否可以拯救过早的后期发作。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作是否是由于着丝粒依赖性途径（检查点蛋白募集到着丝粒），或不依赖于着丝粒的途径（胞质有丝分裂检查点复合物[MCC]），或两者兼而有之。这些研究将阐明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的完整性。 与公共卫生的相关性：癌症是美国的主要死因之一，每年约有50万人死亡。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/或有助于肿瘤发生过程的因素。在为癌症患者设计治疗计划时，这些分子的鉴定为更定制的药物方案提供了机会。

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.