MPS1 kinase in the Spindle Pole Cycle

描述(申请人提供)：中心体是细胞中主要的微管组织中心(MTOC)。重要的是，中心体作为有丝分裂纺锤体的极，使负责将染色体正确分离到后代细胞的微管成核。中心体复制或功能的失败会导致基因组的不稳定，并有助于细胞转化。因此，对复制的详细机制理解对于评估这一过程中的错误如何导致中心体缺陷并可能导致疾病状态至关重要。利用遗传上易驯化的芽生酵母，酿酒酵母中的中心体，即纺锤体极体(SPB)被很好地定义，我们建议研究产生新的中心体的组装机制，磷酸化如何调节中心体上的事件，以及特定的调节磷酸化在真核生物中是否保守。我们和其他人之前已经证明，蛋白质磷酸化在中心体的组装和功能中发挥着重要作用。最近，我们分离了完整的酵母SPB，并对它们进行了质谱分析，得到了一个磷酸蛋白质组，该蛋白质组包括18个核心SPB组分中的17个定位的磷酸化位点。我们将利用这一新的资源来发现哪些磷酸化事件对SPB组装和微管成核是重要的。在第一个目标中，我们将测试一个启动酵母SPB复制的模型，该模型基于这样的假设，即保守的SPB组分Sfi1的磷酸化既调节其与现有SPB的逐步添加，也调节其招募形成新生SPB的SPB组分的能力。此外，我们将测试Sfi1及其结合伙伴CDc31(中心素)是否足以启动SPB组装。第二个目标集中在有丝分裂停滞细胞中SPBS的扩张，因为这种扩张类似于中心体成熟。在这两种情况下，SPBS和中心体之间保守的成分被添加到结构中，中心体微管成核能力增加。我们将确定哪些SPB组分、磷酸化事件和调节因子有助于CDC20耗竭细胞中SPBS的扩展。这项工作将揭示SPB的核心组装机制及其调控。第三个也是最终目标集中在磷酸化在3-微管蛋白复合体的组装和功能中的作用，3-微管蛋白复合体由三个负责微管成核的保守蛋白(Tub4、Spc97和Spc98)组成。另一种重要的蛋白质Spc110将3-微管蛋白复合体连接到SPB上，我们将确定Spc110磷酸化是否对3-微管蛋白复合体的组装和/或微管成核至关重要。最后，我们将询问人类3-微管蛋白复合体中的保守残基是否也被磷酸化，并以类似于酵母3-微管蛋白复合体的方式发挥作用。总而言之，我们完整的酵母SPB磷酸蛋白质组使我们能够超越特定磷酸化位点的图谱，进入更复杂的问题，即磷酸化调节如何在中心体组装、结构和功能中发挥作用。

DESCRIPTION (provided by applicant): Centrosomes are the major microtubule organizing centers (MTOC) in cells. Importantly, as poles of the mitotic spindle, centrosomes nucleate the microtubules responsible for proper chromosome segregation to the progeny cells. Failure in centrosome duplication or function leads to genomic instability and contributes to cellular transformation. Therefore, a detailed mechanistic understanding of duplication is critical in assessing how errors in this process result in centrosomal defects and potentially lead to a disease state. Using the genetically tractable budding yeast, Saccharomyces cerevisiae, in which the centrosome, known as the spindle pole body (SPB), is well defined, we propose to investigate the assembly mechanisms that produce new centrosomes, how phosphorylation regulates events at the centrosome, and whether specific regulatory phosphorylations are conserved among eukaryotes. We, and others, have previously shown that protein phosphorylation plays a significant role in the assembly and function of centrosomes. Recently, we isolated intact yeast SPBs and subjected them to mass spectrometric analysis, yielding a phosphoproteome that includes 297 mapped phosphorylation sites across 17 of the 18 core SPB components. We will use this novel resource to discover which phosphorylation events are important for SPB assembly and microtubule nucleation. In the first aim, we will test a model for the initiation of yeast SPB duplication based on the hypothesis that phosphorylation of the conserved SPB component Sfi1 regulates both its step-wise addition to the existing SPB and its ability to recruit SPB components that form the nascent SPB. In addition, we will test whether Sfi1 and its binding partner Cdc31 (centrin) alone are sufficient for initiation of SPB assembly. The second aim focuses on the expansion of SPBs in mitotically-arrested cells, as this expansion is similar to centrosome maturation. In both cases, components that are conserved between SPBs and centrosomes are added to the structures, and centrosome microtubule nucleation capacities increase. We will determine which SPB components, phosphorylation events, and regulators contribute to the expansion of SPBs in cdc20- depleted cells. This work will reveal core SPB assembly mechanisms and their regulation. The third and final aim focuses on the role of phosphorylation in the assembly and function of the 3-tubulin complex, consisting of three conserved proteins (Tub4, Spc97, Spc98) responsible for microtubule nucleation. Another important protein, Spc110, attaches the 3-tubulin complex to the SPB, and we will determine whether Spc110 phosphorylation is critical for 3-tubulin complex assembly and/or microtubule nucleation. Finally, we will ask whether conserved residues within the human 3-tubulin complex are also phosphorylated and function in a manner similar to those in the yeast 3-tubulin complex. In summary, our completed yeast SPB phosphoproteome allows us to move beyond the mapping of specific phosphorylation sites to the more complex questions of how phospho-regulation acts in centrosome assembly, structure and function.