Role of MPSI Kinase and Yeast Spindle Pole Cycle

描述（申请人提供）：中心体是有丝分裂纺锤杆上的细胞器，负责组织染色体分离所必需的微管。新生中心体的组装是一个严格调控的过程，每个细胞周期只发生一次。这一过程中的缺陷可导致中心体异常，这在肿瘤细胞中经常观察到，并被认为有助于遗传不稳定，这是细胞转化的一个标志。蛋白磷酸化是参与中心体组装和功能的关键信号，尽管其分子细节尚不清楚。使用酿酒酵母中心体（纺锤杆体，SPB）作为模型系统，我们将确定磷酸化事件对功能细胞器的受控组装很重要。关键事件将在脊椎动物细胞中进行保存测试。本提案的第一个具体目标集中在磷酸化对中心体复制的重要调节因子，蛋白激酶Mps1的影响。Mps1在细胞周期中受到严格调控；它受到APC的泛素介导的蛋白水解，并通过细胞周期蛋白依赖性激酶磷酸化来稳定。我们将测试Cdk磷酸化和自磷酸化是否直接阻止体外泛素化，并分析Mps1在这些位点突变的体内后果。第二个具体目标是获得SPB磷酸化状态的全面图谱。磷酸化位点将通过质谱法在纯化的SPBs上鉴定。通过从同步细胞中分离spb，我们将监测在细胞周期中特定点发生的磷酸化变化。此外，我们将用候选激酶磷酸化分离的SPB，以了解它们对SPB整体磷酸化状态的贡献。最后的具体目标集中在保守的γ -微管蛋白复合物。虽然脊椎动物中心体和酵母spb在结构上是不同的，但参与微管成核的成分是保守的。一旦体内和体外γ -微管蛋白复合物磷酸化位点被绘制出来，我们将确定这些位点突变对细胞的影响，以及复合物在体外成核微管的能力。通过了解磷酸化在中心体中的作用，我们将获得中心体组装如何与细胞周期协调以及如何维持有丝分裂的保真度的重要见解。

DESCRIPTION (provided by applicant): Centrosomes are the organelles at mitotic spindle poles that organize microtubules essential for chromosome segregation. Assembly of nascent centrosomes is a tightly regulated process that occurs exactly once each cell cycle. Defects in this process can lead to centrosomal abnormalities, which are frequently observed in tumor cells and are thought to contribute to genetic instability, a hallmark of cellular transformation. Protein phosphorylation is a critical signal involved in centrosome assembly and function, though the molecular details are not well understood. Using the S. cerevisiae centrosome (the Spindle Pole Body, SPB) as a model system, we will identify phosphorylation events that are important to the controlled assembly of a functional organelle. Key events will be tested for conservation in vertebrate cells. The first specific aim of this proposal focuses on the effects phosphorylation has on an important regulator of centrosome duplication, the protein kinase Mps1. Mps1 is tightly regulated through the cell cycle; it is subject to ubiquitin mediated proteolysis by the APC, and stabilized by cyclin dependant kinase phosphorylation. We will test whether phosphorylation by Cdk and autophosphorylation directly prevents ubiquitination in vitro, and assay the in vivo consequences of Mps1 mutated at these sites. The second specific aim is designed to obtain a comprehensive map of the phosphorylation state of the SPB. Phosphorylation sites will be identified on purified SPBs by mass spectrometry. By isolating SPBs from synchronized cells, we will monitor changes in phosphorylation that occur at particular points in the cell cycle. In addition, we will phosphorylate isolated SPBs with candidate kinases to gain an understanding of their contribution to the overall phosphorylation state of the SPB. The final specific aim focuses on the conserved gamma-tubulin complex. Although vertebrate centrosomes and yeast SPBs are structurally distinct, components involved in the nucleation of microtubules are conserved. Once in vivo and in vitro gamma-tubulin complex phosphorylation sites are mapped, we will determine the effects of mutations in these sites on cells, and on the ability of the complex to nucleate microtubules in vitro. By understanding the role phosphorylation plays at centrosomes, we will gain important insights into how centrosome assembly is coordinated with the cell cycle, and how the fidelity of mitosis is maintained.