Regulation of Borealin Function by Mitotic Phosphorylation

通过有丝分裂磷酸化调节 Borealin 功能

• 批准号: 7456205
• 负责人: William R. Taylor
• 金额: $ 21.6万
• 依托单位: UNIVERSITY OF TOLEDO
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7456205
• 关键词: Binding; Biochemical; CDH1 gene; Cancer Etiology; Cardiovascular system; Cell Cycle; Cell Survival; Cell division; Cells; Centromere; Chromosome Segregation; Chromosomes; Class; Co-Immunoprecipitations; Complex; Condition; Cytokinesis; DNA; Daughter; Defect; Disease; Electrophoresis; Ensure; Eukaryotic Cell; Event; Fibroblasts; Genome; Genome Stability; Human; Immunofluorescence Immunologic; Interphase; Lead; Malignant Neoplasms; Measures; Mediating; Metabolic; Microtubules; Mitosis; Mitotic; Modification; Mutation; Numbers; Phase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Play; Process; Protein Overexpression; Proteins; Public Health; Regulation; Role; Site; Small Interfering RNA; Smooth Muscle Myocytes; Testing; Western Blotting; anaphase-promoting complex; aurora B kinase; base; borealin; cancer cell; daughter cell; driving force; in vitro Assay; inhibitor/antagonist; inner centromere protein; insight; killings; mutant; phosphatase inhibitor; research study; survivin

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.

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，在此期间，复制的染色体被分离到细胞的两极，必须准确地发生，以确保子代包含完整的基因组拷贝。这个过程中的缺陷会导致染色体数量的变化，这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。过客蛋白、INCENP、存活蛋白、极光B激酶和北方蛋白形成复合物，其结合至着丝粒和微管并在胞质分裂期间协调细胞的染色体分离和分裂。我们最近已经确定了一个磷酸化形式的Borealin在有丝分裂细胞，并建议分析这种修饰的染色体乘客复合物的功能中的作用。我们的建议包含三个具体目标。目标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，寡聚化和与DNA结合。瞄准二...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可能是稳定的有丝分裂过程中，并有针对性地降解的后期促进复合物。我们已经鉴定的磷酸化位点的突变增加了Borealin蛋白的量。我们推测，在有丝分裂过程中磷酸化的Borealin保护它免受降解，可能是通过干扰APC的识别。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测试该假设。将在这些条件下分析Borealin蛋白的代谢稳定性。目标3.鉴定在有丝分裂退出过程中使Borealin去磷酸化的磷酸酶。将异步生长或S期阻断的细胞暴露于环己酰胺诱导Borealin的磷酸化。当S期阻滞的细胞暴露于广谱磷酸酶抑制剂NaF时，也会发生Borealin的磷酸化。这表明不稳定的磷酸酶在间期期间保持Borealin去磷酸化，并且在有丝分裂期间磷酸酶的失活导致Borealin变得磷酸化。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Borealin有丝分裂特异性磷酸化的基础。 公共卫生相关性：在美国，每年有50多万人死于癌症，这种疾病的特征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，了解它是如何调节的将有助于了解人类癌细胞如何分裂以及如何杀死它们。成纤维细胞和平滑肌细胞的增殖增加也与心血管系统疾病有关，了解Borealin是如何调节的也可以提供对这种疾病谱的了解。