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起着关键作用。因为细胞分裂的忠诚度必须精确复制每个细胞周期。在此过程中的错误导致染色体的错误分离。异常的中心体通常与基因组不稳定性有关，这是许多癌症的特征。拟议的研究使用秀丽隐杆线虫的胚胎作为体内模型，对中心体组装进行遗传学 - 磷酸蛋白质组学分析。在秀丽隐杆线虫中的5个基本中心体因子中，SAS-5在新的中心体组装中起关键作用，其功能同源物（ANA2和SIL/STIL）也需要有丝分裂纺锤体组织。 SAS-5的假定人同源物（SIL/ETIL）突变与原发性小头畸形（MCPH）有关，这是一种脑大小降低的常染色体 - 常见先天性疾病。虽然我们意识到SAS-5对细胞分裂和脑发育的巨大影响，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。目的是了解中心体组装中SAS-5的调节机制。适当水平的中心体蛋白（SAS-6和PLK4/SAK）对于正确数量的中心体数量至关重要，这受蛋白酶体破坏的调节。最近的工作提出，蛋白质磷酸酶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PP2A依赖性磷酸化的调节，该磷酸化将SAS-5至26S蛋白酶体定向以确保其适当的水平和定位。我们的理由是，确定由PP2A脱磷酸化的位点并定义其关键作用将揭示特定位点特异性磷酸化事件如何有助于调节SAS-5活性和中心体组装的忠诚度。我们计划通过追求以下两个具体目的来检验中心假设：1）确定SAS-5的所有磷酸化位点，并指定蛋白质磷酸酶2a（PP2A）靶向的位点。 2）确定位点特异性磷酸化对中心体组装中SAS-5的生物学影响。针对这些目标，我们将使用遗传学，生物化学，高分辨率成像和磷蛋白质组学。我们希望确定SAS-5的磷酸化位点及其生理作用，使SAS-5在中心体组装中适当活性。拟议的研究很重要，因为了解中心体组装的机制可能还会提供与诊断和治疗人类疾病（如癌症和纤毛病）相关的洞察力，这些疾病与中心三醇/基础体外有关，除了从根本上促进中心体生物学领域。 公共卫生相关性：拟议的研究与公共卫生有关，因为预计秀丽隐杆线虫中受调节水平的中心体蛋白质的分子和生化机制有望增强我们对与癌症和纤毛病有关的广泛医疗保健后果的人类疾病中的中心体调节的了解。因此，拟议的研究与NIH使命的一部分有关，该部分与发展知识有关，这些知识将有助于开发人类疾病的诊断和治疗性发明。