Aurora-A激酶在G2后期调控有丝分裂检查点建立和激活的机制研究

Aneuploidy caused by chromosome mis-segregation in mitosis may induce malignant transformation and tumorigenesis. The Spindle Assembly Checkpoint (SAC) is the key surveillance mechanism to ensure the accurate separation of duplicated genome. Despite extensive studies, how SAC is controlled, especially how SAC is initially established and activated at early mitosis is largely unknown. Aurora-A is one of the major kinase in mitosis. It is involved in centrosome maturation and separation before mitosis, and bi-polar spindle formation as well as cytokinesis in mitosis. Aurora-A over-expression is found in a number of tumor subtypes and is a bio-marker of poor prognosis in some tumors. It was suggested that high level of Aurora-A could impair the function of SAC to allow tumor cells go through mitosis with errors. However, whether and how Aurora-A regulate SAC is not known. Using MEF cells from an Aurora-A conditional knock-out mice, we found for the first time that Aurora-A was required for initial establishment of SAC during early mitosis. In addition, using live cell imaging we confirmed that Aurora-A inhibition delayed the recruitment of SAC to kinetochore in prophase. Thus, in this study we propose to further elucidate the mechanism underlying of the regulation of initial establishment of SAC by Aurora-A. The downstream regulator and new pathways in SAC regulation in prophase will be explored. This study will help us reveal the network orchestrates mitotic entry and SAC activation, which will provide valuable insights to understand and fight against cancer.

分裂期染色体错误分离造成的非整倍体细胞会促进细胞的恶性转化并诱发癌症。纺锤体检查点是细胞内监控染色体分离前动粒与微管准确结合的关键机制。目前对检查点精细的调控，尤其对核膜破裂前，检查点信号的最初建立和激活的过程了解得还很少。Aurora-A是分裂期的关键激酶，在中心体成熟分离，两极纺锤体的建立中都起重要的作用。Aurora-A在多个肿瘤亚型中过表达，虽然有证据暗示过量的Aurora-A会破坏纺锤体检查点的功能，但机制还不清楚。利用条件性敲除Aurora-A小鼠的MEF细胞，我们的前期工作首次发现Aurora-A参与纺锤体检查点的初期建立，活细胞成像确证了抑制Aurora-A可以使检查点的建立显著延后。本课题中我们计划近一步阐明Aurora-A参与检查点建立的方式，信号通路及时空关系。探索其在G2期新的上下游信号，深入理解分裂期促进信号与检查点建立信号相互交流的调控网络。

细胞在分裂初期核膜破裂前，检查点信号需要及时的建立和激活，目前对这一过程的调控了解得还很少。Aurora-A是分裂期的关键激酶，在中心体成熟分离，两极纺锤体的建立中都起重要的作用。有证据暗示Aurora-A的失调会破坏纺锤体检查点的功能，但机制还不清楚。本项目我们计划阐明激酶Aurora-A在时空上参与有丝分裂检查点复合体建立的机制；寻找并验证Aurora-A在G2中后期到分裂前期（prophase），细胞核内的新底物和结合蛋白分子，揭示其调控分裂期检查点快速激活的方式及上下游通路。.本项目执行的一年时间里，按照计划我们利用条件性敲除Aurora-A小鼠的MEF细胞，首次发现Aurora-A参与纺锤体检查点的初期建立，活细胞成像确证了抑制Aurora-A可以使检查点的建立显著延后。进一步的机制研究发现Aurora-A通过调节组蛋白H3T3的磷酸化状态来调控CPC复合体在着丝粒上的组装过程，从而激活检查点蛋白的及时组装。通过序列分析和生物信息学的筛选，我们发现H3T3的激酶Haspin上有多个保守的Aurora-A磷酸化位点，暗示了Aurora-A可能直接调控Haspin的活性来参与组蛋白的修饰以及下游的着丝粒的组装。Aurora-A与Haspin的结合和磷酸化验证正在进行中，这些结果将进一步阐明Aurora-A在有丝分裂早期激活检查点组装通路的分子机制。