MPS1 kinase in the Spindle Pole Cycle

描述（由申请方提供）：中心体是细胞中主要的微管组织中心（MTOC）。重要的是，作为有丝分裂纺锤体的两极，中心体使微管成核，负责将染色体正确分离到后代细胞。中心体复制或功能的失败导致基因组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该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。使用遗传上易于处理的芽殖酵母，酿酒酵母，其中的中心体，被称为纺锤体极体（SPB），是明确的，我们建议调查的装配机制，产生新的中心体，磷酸化如何调节事件的中心体，以及是否特定的监管磷酸化是真核生物之间的保守。我们和其他人先前已经表明，蛋白质磷酸化在中心体的组装和功能中起着重要作用。最近，我们分离出完整的酵母SPB，并将它们进行质谱分析，产生一个磷酸化蛋白质组，其中包括297个映射的磷酸化位点的17个核心SPB组件。我们将使用这种新的资源，以发现磷酸化事件是重要的SPB组装和微管成核。在第一个目标中，我们将测试一个模型的基础上启动酵母SPB复制的保守SPB组件Sfi 1磷酸化调节其逐步除了现有的SPB和它的能力，以招募SPB组件，形成新生的SPB的假设。此外，我们将测试是否Sfi 1和它的结合伙伴Cdc 31（中心蛋白）单独足以启动SPB大会。第二个目标集中在有丝分裂停滞细胞中SPB的扩增，因为这种扩增类似于中心体成熟。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、磷酸化事件和调节剂有助于cdc 20缺失细胞中SPB的扩增。这项工作将揭示核心SPB组装机制及其调控。第三个也是最后一个目标集中在3-微管蛋白复合物的组装和功能中磷酸化的作用，该复合物由负责微管成核的三种保守蛋白（Tub 4，Spc 97，Spc 98）组成。另一个重要的蛋白质，Spc 110，重视3-微管蛋白复合物的SPB，我们将确定是否Spc 110磷酸化是至关重要的3-微管蛋白复合物组装和/或微管成核。最后，我们将问是否保守残基内的人3-微管蛋白复合物也磷酸化和功能的方式类似于那些在酵母3-微管蛋白复合物。总之，我们完成的酵母SPB磷酸化蛋白质组使我们能够超越特定磷酸化位点的映射到更复杂的问题，即磷酸化调节如何在中心体组装，结构和功能中起作用。

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.