Regulation of the Mps1 Protein Kinase and Centrosome Duplication

描述（由申请人提供）：哺乳动物Mps1蛋白激酶是中心体复制的关键调节因子，是我们拟议项目的重点。中心体对有丝分裂纺锤体的组装至关重要，过多的中心体会导致纺锤体异常，不能忠实地分离染色体。染色体分离错误是流产的主要原因，在大多数人类肿瘤中都可以观察到。中心体的数量必须严格控制，因为正如在许多人类肿瘤中观察到的那样，过量的中心体会产生异常纺锤体，导致染色体分离错误。与许多重要的细胞周期事件一样，中心体的复制受到周期蛋白依赖性激酶活性（脊椎动物细胞中的Cdk2）的暂时限制。然而，时间调控和中心体复制本身的机制尚不清楚。我们的研究表明Mps1可能是这两种机制的关键。在缺乏Cdk2活性的情况下，Mps1从中心体中移除。这种去除需要蛋白酶体的活性，并由Mps1， T468内的单个Cdk2位点调节。将T468突变为丙氨酸可阻止Mps1在中心体积聚，但对全细胞Mps1水平没有影响。此外，Mps1T468A不能替代内源性Mps1在中心体复制中的功能。相反，阻止Mps1从中心体中移除会导致额外中心体的产生。我们在Mps1 n端发现了一个中心体定位结构域和一个围绕T468的降解信号，以及与这些结构域结合的中心体蛋白。我们还在中心体蛋白中发现了Mps1磷酸化位点，模仿该位点的磷酸化会导致中心体过度生产。因此，我们假设Mps1靶向中心体及其随后在中心体上的降解对于中心体复制的时间控制至关重要，并且Mps1底物的磷酸化调节了新中心体的组装。我们有三个目标，我们打算这样做；1)确定Mps1与中心体结合的特定残基，并测试这些残基在Mps1中心体定位域与其结合伙伴之间相互作用中的作用；2)确定中心体上负责Mps1降解的特定残基，并确定这些残基如何与T468协同调节Mps1降解信号与其结合伙伴之间的相互作用；3)确定Mps1磷酸化对其底物组装成中心体的贡献。我们的实验不仅将阐明Mps1的调控和功能，而且将解决中心体复制的时间控制和分子机制两个主要的生物学问题。

DESCRIPTION (provided by applicant): The mammalian Mps1 protein kinase is a key regulator of centrosome duplication and is the focus of our proposed project. Centrosomes are critical for mitotic spindle assembly, and excess centrosomes lead to abnormal spindles that cannot faithfully segregate chromosomes. Errors in chromosome segregation are the leading cause of miscarriage and are observed in the majority of human tumors. The number of centrosomes must be tightly controlled, because as observed in many human tumors excess centrosomes generate aberrant spindles that cause chromosome segregation errors. As with many important cell cycle events, the duplication of centrosomes is temporally restricted by cyclin-dependent kinase activity (Cdk2 in vertebrate cells). However, the mechanisms of temporal regulation and of centrosome duplication itself remain unknown. Our work suggests that Mps1 might be the key to both mechanisms. In the absence of Cdk2 activity Mps1 is removed from centrosomes. This removal requires proteasome activity and is regulated by a single Cdk2 site within Mps1, T468. Mutating T468 to alanine prevents Mps1 from accumulating at centrosomes, but has no effect on whole-cell Mps1 levels. Moreover, Mps1T468A cannot substitute for the function of endogenous Mps1 in centrosome duplication. In contrast, preventing the removal of Mps1 from centrosomes causes the production of extra centrosomes. We identified a centrosome localization domain in the Mps1 N-terminus and a degradation signal surrounding T468, as well as centrosomal proteins that bind to these domains. We also identified an Mps1 phosphorylation site within a centrosomal protein, and mimicking phosphorylation at this site causes centrosome overproduction. Accordingly, we hypothesize that both the targeting of Mps1 to centrosomes and its subsequent degradation at centrosomes are critical for the temporal control of centrosome duplication, and that phosphorylation of Mps1 substrates regulates the assembly of new centrosomes. We have three aims, and we propose to; 1) define specific residues responsible for binding of Mps1 to centrosomes, and to test the role of these residues in the interaction between the Mps1 centrosome localization domain and its binding partners; 2) define specific residues responsible for degradation of Mps1 at centrosomes, and determine how these residues cooperate with T468 to regulate interactions between the Mps1 degradation signal and its binding partners; and 3) determine the contribution of Mps1 phosphorylation to assembly of its substrates into centrosomes. Our experiments will not only elucidate the regulation and function of Mps1, a key centrosome regulator, but will also address two major unanswered biological problems, the temporal control and molecular mechanisms of centrosome duplication. PUBLIC HEALTH RELEVANCE: Chromosome segregation errors are the most common cause of miscarriage and are also observed early in tumorigenesis. This project seeks to characterize the regulation and function of the Mps1 protein kinase, whose misregulation causes the production of extra centrosomes that, like those found in many human tumors, perturb mitotic spindle assembly and cause errors in chromosome segregation. We propose to exploit domains within Mps1 that control its targeting and degradation, centrosomal proteins that bind to these domains, and Mps1 substrates to elucidate the mechanisms that control the production of centrosomes.