Eukaryotic cells divide by a complicated process involving multiple, highly coordinated events. Mitosis, during which duplicated chromosomes are segregated to opposite poles of the cell must occur accurately to ensure that daughter contain an intact copy of the genome. Defects in this process can lead to changes in chromosome number, a driving force behind cancer formation. One class of proteins that plays important roles during mitosis is the chromosomal passenger proteins. The passenger proteins, INCENP, Survivin, Aurora B kinase and Borealin form a complex that binds to centromeres and microtubules and coordinates chromosome segregation and division of the cell during cytokinesis. We have recently identified a phosphorylated form of Borealin in mitotic cells and propose to analyze the role of this modification in the function of the chromosomal passenger complex. Our proposal entails three specific aims.
Aim 1. Determine the role of mitotic phosphorylation on known activities of Borealin. Several activities of Borealin may be regulated by mitotic phosphorylation. Using electrophoresis to detect the mitotic phosphorylated form of Borealin we will analyze the impact of phosphorylation on some of the known activities of Borealin including binding to INCENP, oligomerization, and binding to DNA.
Aim 2.. Analyze the impact of Borealin phosphorylation on protein stability. Our preliminary studies indicate that Borealin may be stabilized during mitosis and targeted for degradation by the anaphase promoting complex. Mutations in phosphorylation sites that we have identified increase the amount of Borealin protein. We hypothesize that phosphorylation of Borealin during mitosis protects it from degradation, possibly by interfering with recognition by the APC. This hypothesis will be tested by transfecting cells with phospho-site mutants of Borealin as well as activators and inhibitors of APC-mediated degradation. Metabolic stability of the Borealin protein will be analyzed under these conditions.
Aim 3. Identify the phosphatase that dephosphorylates Borealin during mitotic exit. Exposure of asynchronously growing or S-phase blocked cells to cyclohexamide induces phosphorylation of Borealin. Phosphorylation of Borealin also occurs when cells blocked in S-phase are exposed to the broad spectrum phosphatase inhibitor NaF. This suggests that a labile phosphatase keeps Borealin dephosphorylated during interphase, and that inactivation of the phosphatase during mitosis causes Borealin to become phosphorylated. We propose to identify the interphase Borealin phosphatase by analyzing candidate phosphatases and if necessary use biochemical purification to identify the phosphatase. These experiments should uncover the basis of mitosis specific phosphorylation of Borealin.
More than half a million people in the US die every year due to cancer, a disease characterized by uncontrolled cell division, and inaccurate segregation of chromosomes. The chromosomal passenger protein Borealin plays an essential role in cell division, and understanding how it is regulated will provide insight into how human cancer cells divide and possibly how to kill them. Increased proliferation of fibroblasts and smooth muscle cells has also been implicated in diseases of the cardiovascular system, and knowing how Borealin is regulated may also provide insight into this spectrum of diseases.
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