Regulation of Centrosome Assembly by Phosphorylation

描述(申请人提供)：中心体在建立双极纺锤体中起着关键作用。为了细胞分裂的保真度，中心体必须在每个细胞周期精确复制一次。这一过程中的错误导致染色体的错误分离。中心体的异常通常与基因组不稳定有关，这是许多癌症的一个特征。这项拟议的研究使用线虫胚胎作为体内模型来进行中心体组装的遗传学-磷蛋白组学分析。在线虫的5个重要中心体因子中，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。推测SAS-5的人类同源基因(sIL/STIL)中的一个突变与原发性小头畸形(MCPH)有关，MCPH是一种常染色体隐性遗传性先天性疾病，大脑体积缩小。虽然我们认识到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不清楚。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。适当水平的中心体蛋白(SAS-6和Plk4/SAK)对于中心体的正确数量至关重要，中心体的数量由蛋白酶体破坏来调节。最近的工作提出，蛋白磷酸酶2A(PP2A)以SAS-5为靶点，调节中心体组装。我们的中心假设是，SAS-5受PP2A依赖的磷酸化调控，该磷酸化引导SAS-5到26S蛋白酶体，以确保其适当的水平和定位。我们的基本原理是，确定PP2A去磷酸化的位点并确定它们的关键作用将揭示位点特异性磷酸化事件如何有助于调节SAS-5的活性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验证我们的中心假设：1)确定SAS-5的所有磷酸化位点，并指定蛋白磷酸酶2A(PP2A)的靶点。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的磷酸化位点及其在中心体组装中负责SAS-5适当活性的生理作用。这项拟议的研究意义重大，因为了解中心体组装的机制可能会为诊断和治疗与中心粒/基底相关的人类疾病(如癌症和纤毛疾病)提供洞察力，并从根本上推动中心体生物学领域的发展。 公共卫生相关性：拟议的研究与公共健康相关，因为线虫中心体蛋白调控水平的分子和生化机制有望增强我们对人类疾病中心体调控的了解，并与癌症和纤毛疾病相关的广泛和重要的保健分支。因此，拟议的研究与NIH任务的一部分有关，该部分涉及开发有助于开发人类疾病诊断和治疗发明的知识。

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.