基于Phos-tag凝胶电泳技术的肿瘤细胞增殖过程中Aurora B激酶活性的调控机制研究

Cell proliferation is largely regulated by the network driven by cell cycle dependent protein kinases. A deep comprehension of this network is of vital importance to develop drugs for cancer therapy. Aurora B is a widely studied mitotic kinase and it has been found to be overexpressed in many cancers, such as lung cancer, prostate cancer and ovarian cancer. However, it has been unclear that how the kinase activity of Aurora B is regulated during the tumor cell proliferation. In this study, the phosphorylation status of Aurora B during cell proliferation cycle will be identified, and the relationship between the phosphorylation and kinase activity of Aurora B in tumor cells will be carefully examined. To analyze the phosphorylation sites of Aurora B more efficiently and accurately, a new tool for protein phosphorylation analysis, Phos-tag containing SDS-PAGE, will be introduced. In addition, the phospho-specific antibodies will be generated to further confirm the phosphorylation status of Aurora B. Afterwards, a theoretical model for Aurora B kinase activity regulation by phosphorylation will be established. Furthermore, we will investigate the role of phosphorylation in chromosome alignment, spindle checkpoint assembly. It will be interesting to find out the relationship among the phosphorylation, kinase activity regulation and cell proliferation, which is important to understand the mechanism of tumorigenesis.

Aurora B激酶在多种癌症中高表达，正确理解Aurora B激酶在肿瘤发生发展中的功能和活性调控机制对于增加对肿瘤发病机制的认识、加快新型小分子药物开发都有重要意义。本项目围绕肿瘤细胞增殖过程中Aurora B激酶活性调控这个关键问题，在蛋白质和基因水平上，通过综合运用Phos-tag凝胶电泳这项新技术和磷酸化抗体制备、放射自显影等成熟方法，系统地揭示肿瘤细胞增殖过程中Aurora B的磷酸化修饰与Aurora B激酶活性之间的关系，建立Aurora B激酶活性调控的理论模型，以期阐明Aurora B在肿瘤细胞G2/M期起始活化等活性调控中的重要问题；在细胞水平上，通过免疫荧光、活细胞成像等技术深入研究Aurora B磷酸化修饰对染色体列队、纺锤体装配检验点组装等重大事件的影响，从而揭示磷酸化修饰-激酶活性-肿瘤细胞增殖三者之间的关系，为阐明肿瘤发生发展的机制提供有益的线索。

Aurora B激酶的高表达与人类多种癌症的发生、发展密切相关，深入阐明Aurora B激酶在肿瘤发生发展过程中的活性调控机制，有助于加深人们对肿瘤发病机理的认识，加快新型抗肿瘤药物的开发。本项目围绕肿瘤细胞增殖过程中Aurora B激酶的活化机制这个核心问题展开研究，通过Phos-tag凝胶电泳技术发现Aurora B在有丝分裂期被特异性磷酸化，通过磷酸化质谱、磷酸化抗体制备结合生物信息学分析方法，鉴定出了T236 、T64 、S62等磷酸化位点。T236位点磷酸化修饰调控Aurora B的细胞定位和激酶活性。针对T236位点的特异性磷酸化抗体显示T236磷酸化特异性地发生在有丝分裂期，且有一部分T236位点磷酸化的Aurora B定位在动粒上。非磷酸化突变体GFP-Aurora B T236A在有丝分裂期的着丝粒定位减弱，且该突变体的T232位点无法被磷酸化，在HeLa细胞中过表达GFP-Aurora B T236A导致有丝分裂异常，并产生大量的多核和细胞核形态异常的细胞，活细胞动态观察显示GFP-Aurora B T236A导致细胞有丝分裂退出异常。体外磷酸化实验结果显示，Chk2激酶可以在体外磷酸化T236位点，然而尚无证据支持Chk2在细胞内负责Aurora B T236的磷酸化。此外，我们也发现CDK1在体内和体外磷酸化Aurora B的第64位苏氨酸，非磷酸化突变体GFP-Aurora B T64A的激酶活性明显降低，说明CDK1对Aurora B的T64磷酸化是Aurora B完全发挥激酶活性所必需的。进一步的研究还发现T236、T64位点的磷酸化调控Aurora B与Survivin等因子的结合能力。通过运用PAML4.9e等工具，我们重构了Aurora祖先蛋白序列，从进化的角度更进一步揭示了Aurora B激酶活性调控的保守位点和保守机制。综上所述，在有丝分裂期，CDK1及其他激酶通过磷酸化T236、T64等位点促进Aurora B的活化，从而调控有丝分裂进程，揭示了Aurora B活性调控的新机制。