Regulation of Borealin Function by Mitotic Phosphorylation

描述（由申请人提供）：真核细胞分裂是一个复杂的过程，涉及多个高度协调的事件。有丝分裂，在此过程中，复制的染色体被分离到细胞的相反两极，必须准确地进行，以确保子代包含完整的基因组拷贝。这一过程中的缺陷会导致染色体数目的改变，而染色体数目是癌症形成背后的驱动力。在有丝分裂过程中起重要作用的一类蛋白质是染色体客运蛋白。载客蛋白，INCENP, Survivin， Aurora B激酶和Borealin形成一个复合物，与着丝粒和微管结合，并在细胞质分裂期间协调染色体分离和细胞分裂。我们最近在有丝分裂细胞中发现了Borealin的磷酸化形式，并建议分析这种修饰在染色体乘客复合物功能中的作用。我们的建议包含三个具体目标。目的1。确定有丝分裂磷酸化对已知Borealin活性的作用。Borealin的一些活性可能受到有丝分裂磷酸化的调节。利用电泳检测有丝分裂磷酸化形式的Borealin，我们将分析磷酸化对Borealin的一些已知活性的影响，包括与INCENP的结合，寡聚化和与DNA的结合。目标2 . .分析Borealin磷酸化对蛋白质稳定性的影响。我们的初步研究表明，Borealin可能在有丝分裂期间稳定，并被后期促进复合物降解。我们已经确定的磷酸化位点的突变增加了北方realin蛋白的数量。我们假设有丝分裂过程中Borealin的磷酸化可能通过干扰APC的识别来保护它免受降解。这一假设将通过用Borealin的磷酸化位点突变体以及apc介导的降解的激活剂和抑制剂转染细胞来验证。在这些条件下，将分析Borealin蛋白的代谢稳定性。目标3。鉴定在有丝分裂结束时脱磷酸化Borealin的磷酸酶。不同步生长或s期阻滞细胞暴露于环己胺可诱导Borealin磷酸化。当被阻断在s期的细胞暴露于广谱磷酸酶抑制剂NaF中时，Borealin也会发生磷酸化。这表明一种不稳定的磷酸酶在间期保持Borealin去磷酸化，而有丝分裂期间磷酸酶的失活导致Borealin被磷酸化。我们建议通过分析候选磷酸酶来鉴定间期Borealin磷酸酶，必要时使用生化纯化来鉴定磷酸酶。这些实验将揭示有丝分裂特异性磷酸化Borealin的基础。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.