Regulation of Centrosome Assembly by Phosphorylation

磷酸化调节中心体组装

• 批准号: 8290705
• 负责人: Mi Hye Song
• 金额: $ 12.47万
• 依托单位: MICHIGAN TECHNOLOGICAL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-10 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8290705
• 关键词: 26S proteasome; Animals; Biochemical; Biochemistry; Biological; Biological Sciences; Biology; Brain; Caenorhabditis elegans; Cell Cycle; Cell division; Cells; Cellular biology; Centrioles; Centrosome; Chromosome Segregation; Complement; Congenital Disorders; Development; Diagnosis; Diagnostic; Embryo; Ensure; Environment; Event; Genetic; Genomic Instability; Goals; Healthcare; Homologous Gene; Human; Image; Interdisciplinary Study; Knowledge; Link; Malignant Neoplasms; Mass Spectrum Analysis; Michigan; Microcephaly; Microscope; Microtubule-Organizing Center; Mission; Mitotic spindle; Molecular; Molecular Genetics; Mutation; Organism; Outcome; Phosphoproteins; Phosphorylation; Phosphorylation Site; Physiological; Play; Process; Protein phosphatase; Proteins; Proteomics; Public Health; Regulation; Research; Research Personnel; Resolution; Role; Site; Specific qualifier value; System; Testing; Therapeutic; Universities; Work; base; brain size; ciliopathy; experience; fly; genetic analysis; graduate student; human disease; improved; in vivo Model; insight; kinetosome; tumor; undergraduate student

DESCRIPTION (provided by applicant): Centrosomes play a critical role in establishing bipolar spindles. For the fidelity of cell division centrosomes must duplicate precisely once per cell cycle. Errors in this process result in mis- segregation of chromosomes. Aberrant centrosomes are often associated with genomic instability, a feature of many cancers. The proposed research uses the C. elegans embryo as an in vivo model to perform genetics-phosphoproteomic analyses of centrosome assembly. Among 5 essential centrosome factors in C. elegans, SAS-5 plays a key role in the assembly of new centrosomes, and its functional homologs (Ana2 and SIL/STIL) are also required for mitotic spindle organization. A mutation in the putative human homolog (SIL/STIL) of SAS-5 is linked to primary microcephaly (MCPH), an autosomal- recessive congenital disorder with reduced brain size. While we realize the great impact of SAS-5 for cell division and brain development, the molecular and biochemical mechanisms by which SAS-5 regulates centrosome assembly remain elusive. Our long-term goal is to elucidate the molecular and genetic mechanisms of the centrosome assembly. The objective is to understand regulatory mechanism of SAS-5 in centrosome assembly. Proper levels of centrosome proteins (SAS-6, and Plk4/Sak) are critical for the correct number of centrosomes, which is regulated by proteasomal destruction. Recent work proposed that protein phosphatase 2A (PP2A) targets SAS-5 to regulate centrosome assembly. Our central hypothesis is that SAS-5 is regulated by PP2A-dependent phosphorylation, which directs SAS-5 to 26S proteasome to ensure its proper level and localization. Our rationale is that identifying the sites dephosphorylated by PP2A and defining their critical role will reveal how site-specific phosphorylation events contribute to the regulation of SAS-5 activity and the fidelity of the centrosome assembly. We plan to test our central hypothesis by pursuing the following two specific aims: 1) Identify all phosphorylation sites of SAS-5 and specify the sites that are targeted by protein phosphatase 2A (PP2A). 2) Determine the biological impact of site-specific phosphorylation on SAS-5 in centrosome assembly. Toward these aims, we will use genetics, biochemistry, high- resolution imaging, and phosphoproteomics. We expect to identify phosphorylation sites of SAS-5 and their physiological roles responsible for proper activity of SAS-5 in centrosome assembly. The proposed research is significant, because understanding the mechanisms of centrosome assembly will likely provide insight relevant to the diagnosis and treatment of human diseases such as cancers and ciliopathies that are associated with centrioles/basal bodies, in addition to fundamentally advancing the field of centrosome biology. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health because the molecular and biochemical mechanisms of regulated levels of centrosome proteins in C. elegans are expected to enhance our knowledge of centrosome regulation in human diseases with broad and important healthcare ramifications related to cancers and ciliopathies. Thus, the proposed research is relevant to the part of NIH's mission that pertains to developing knowledge that will help developing diagnostic and therapeutic inventions for human diseases.

描述（由申请人提供）：中心体在建立双极纺锤体中发挥着关键作用。为了保证细胞分裂的保真度，中心体必须在每个细胞周期精确复制一次。此过程中的错误会导致染色体错误分离。异常的中心体通常与基因组不稳定有关，这是许多癌症的一个特征。拟议的研究使用秀丽隐杆线虫胚胎作为体内模型来进行中心体组装的遗传学-磷酸蛋白质组学分析。在线虫的 5 个必需中心体因子中，SAS-5 在新中心体的组装中起着关键作用，其功能同源物（Ana2 和 SIL/STIL）也是有丝分裂纺锤体组织所必需的。 SAS-5 的假定人类同源物 (SIL/STIL) 中的突变与原发性小头畸形 (MCPH) 有关，这是一种大脑尺寸缩小的常染色体隐性先天性疾病。虽然我们认识到 SAS-5 对细胞分裂和大脑发育的巨大影响，但 SAS-5 调节中心体组装的分子和生化机制仍然难以捉摸。我们的长期目标是阐明中心体组装的分子和遗传机制。目的是了解 SAS-5 在中心体组装中的调控机制。中心体蛋白（SAS-6 和 Plk4/Sak）的适当水平对于中心体的正确数量至关重要，中心体数量受蛋白酶体破坏的调节。最近的研究提出，蛋白磷酸酶 2A (PP2A) 靶向 SAS-5 来调节中心体组装。我们的中心假设是 SAS-5 受到 PP2A 依赖性磷酸化的调节，该磷酸化将 SAS-5 引导至 26S 蛋白酶体以确保其适当的水平和定位。我们的理由是，识别 PP2A 去磷酸化的位点并定义其关键作用将揭示位点特异性磷酸化事件如何有助于 SAS-5 活性的调节和中心体组装的保真度。我们计划通过追求以下两个具体目标来检验我们的中心假设：1）识别 SAS-5 的所有磷酸化位点并指定蛋白磷酸酶 2A (PP2A) 靶向的位点。 2) 确定中心体组装中位点特异性磷酸化对 SAS-5 的生物学影响。为了实现这些目标，我们将利用遗传学、生物化学、高分辨率成像和磷酸蛋白质组学。我们期望确定 SAS-5 的磷酸化位点及其在中心体组装中负责 SAS-5 适当活性的生理作用。拟议的研究意义重大，因为除了从根本上推进中心体生物学领域之外，了解中心体组装机制还可能为与中心粒/基体相关的人类疾病（例如癌症和纤毛病）的诊断和治疗提供相关见解。 公共健康相关性：拟议的研究与公共健康相关，因为线虫中心体蛋白调节水平的分子和生化机制预计将增强我们对人类疾病中心体调节的了解，这些疾病与癌症和纤毛病相关，具有广泛而重要的医疗保健影响。因此，拟议的研究与 NIH 使命的一部分相关，即开发有助于开发人类疾病诊断和治疗发明的知识。