Regulation of Borealin Function by Mitotic Phosphorylation

描述(申请人提供)：真核细胞通过一个复杂的过程分裂，涉及多个高度协调的事件。有丝分裂，在此期间，复制的染色体被分离到细胞的相反两极，必须准确地发生，以确保女儿包含完整的基因组副本。这个过程中的缺陷可能会导致染色体数量的变化，而染色体数量是癌症形成的驱动力。在有丝分裂过程中起重要作用的一类蛋白质是染色体乘客蛋白。乘客蛋白INCENP、Survivin、Aurora B Kinase和Borealin形成一个复合体，与着丝粒和微管结合，协调细胞胞质分裂过程中的染色体分离和分裂。我们最近在有丝分裂细胞中发现了一种Borealin的磷酸化形式，并建议分析这种修饰在染色体乘客复合体功能中的作用。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Borealin已知活性的作用。Borealin的几种活性可能受有丝分裂磷酸化的调节。使用电泳法检测Borealin的有丝分裂磷酸化形式，我们将分析磷酸化对Borealin的一些已知活性的影响，包括与INCENP结合、寡聚和与DNA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可能在有丝分裂过程中稳定下来，并被后期促进复合体靶向降解。我们已经确定的磷酸化位点的突变增加了Borealin蛋白的数量。我们假设，在有丝分裂过程中，Borealin的磷酸化可能通过干扰APC的识别来保护它免受降解。这一假设将通过将Borealin的磷酸位点突变体以及APC介导的降解的激活剂和抑制剂导入细胞来验证。在此条件下，将对Borealin蛋白的代谢稳定性进行分析。目的3.鉴定在有丝分裂退出过程中使Borealin去磷酸化的磷酸酶。将异步化生长或S时相阻断的细胞暴露于环己胺可诱导去甲肾上腺素的磷酸化。当被阻断在S期的细胞暴露于广谱磷酸酶抑制剂NaF时，Borealin的磷酸化也会发生。这表明，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，如果必要的话，还可以使用生化提纯来鉴定该磷酸酶。这些实验应该会揭示Borealin有丝分裂特异性磷酸化的基础。 与公共卫生相关：美国每年有超过50万人死于癌症，这种疾病的特点是细胞分裂不受控制，染色体分离不准确。染色体乘客蛋白Borealin在细胞分裂中起着至关重要的作用，了解它是如何调控的，将有助于深入了解人类癌细胞是如何分裂的，以及可能如何杀死它们。成纤维细胞和平滑肌细胞的增殖增加也与心血管系统的疾病有关，了解Borealin是如何调节的也可能提供对这一疾病谱的洞察。

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.