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

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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.