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

Aurora B 的新型底物 NudC 在纺锤体检查点活动中的功能

• 批准号: 7880809
• 负责人: Kimberly Nicole Weiderhold
• 金额: $ 3.41万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7880809
• 关键词: Address; Anaphase; Aneuploidy; Antibodies; Back; Biological Assay; Cancer Patient; Cause of Death; Cell Proliferation; Cells; Chemicals; Chromosome Segregation; Chromosomes; Co-Immunoprecipitations; Complex; Consensus; Cytokinesis; Dynein ATPase; Ensure; Exhibits; Genetic Materials; Genome Stability; Genomic Instability; Genomics; Goals; Image; Immunofluorescence Microscopy; Immunoprecipitation; In Vitro; Kinetochores; Laboratories; Lead; Life; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Microtubules; Mitosis; Mitotic; Mitotic Activity; Mitotic Checkpoint; Molecular; Molecular Motors; Motor; Mutate; Pathway interactions; Peptides; Pharmaceutical Preparations; Phosphopeptides; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Process; Proteins; Public Health; RNA Interference; Role; Site; Site-Directed Mutagenesis; Testing; Time; Treatment Protocols; United States; aurora B kinase; cellular imaging; daughter cell; design; domain mapping; dynactin; human PLK1 protein; in vitro Assay; in vivo; inhibitor/antagonist; interest; kinase inhibitor; mimetics; mutant; novel; premature; treatment planning; tumorigenesis

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.

描述(申请人提供)：有丝分裂是一个高度调控的过程，通过这个过程，细胞分裂并将其遗传物质均匀地分配给子细胞。染色体分离的错误可能导致非整倍体和基因组不稳定，这是癌症的一个标志。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，是实现正确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器是如何控制染色体分离以确保基因组稳定的。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B(Aurora B)的一种新底物。NudC基因的敲除导致Aurora B从着丝粒错误定位。缺失nudC的细胞也表现出纺锤体检查点的减弱和早期的后期启动，这增加了Aurora B对nudC的磷酸化可能参与调控这些过程的有趣的可能性。我将测试Aurora B-磷酸化的nudC调节纺锤体检查点活动的假设。为此，目标1将首先确定Aurora B对nudC的磷酸化。我将在nudC中鉴定Aurora B磷酸化位点(S)，突变这些位点，并通过体外IP-K试验确认它们的真实性。我将通过GST-Pull-Down和co-IP分析绘制NudC与Aurora B相互作用的区域。此外，我将产生抗磷酸化裸体C抗体，以检测极光B磷酸化裸体C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确认Aurora B对nudC的磷酸化。目的2研究极光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。采用nudC基因敲除，然后用nudC突变体拯救的方法将被用来确定Aurora B-磷酸化的nudC是否可以挽救早期的后期发病。我将进一步研究NudC缺陷细胞中纺锤体检查点的减弱和早期后期发病是由于动粒依赖途径(检查点蛋白募集到动粒)，还是由于动粒非依赖途径(胞浆有丝分裂检查点复合体[MCC])，或者两者兼而有之。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的完整性。 与公共卫生相关：癌症是美国的主要死亡原因之一，每年夺走大约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有助于阐明促进和/或促进肿瘤形成过程的因素。在为癌症患者设计治疗计划时，对这些分子的识别为更多定制药物方案提供了机会。