Molecular dissection of mitotic kinase functions using chemogenomics

任何生物的发育、生存和繁殖都需要其细胞正确分裂的能力。细胞分裂需要染色体的复制、有丝分裂时染色体的分离和细胞质分裂时细胞的分裂。这个复杂的过程涉及到重要的调节酶对多种细胞内事件的复杂协调。在这个研究项目中，我们将阐明在细胞周期中控制靶蛋白磷酸化的激酶模块的生化机制和细胞功能。在蛋白质上可逆地加入磷酸基团往往会导致其活性的变化。当细胞进入有丝分裂时，一些蛋白质被激酶磷酸化，引发细胞转化。然而，涉及激酶在细胞分裂中的确切功能仍不完全清楚。因为每个有丝分裂激酶控制几个底物蛋白和事件，破译它们的精确功能是非常困难的。为此目的，拟议的研究将利用新的技术发展。使用CRISPR和新开发的选择性激酶抑制剂化合物的基因编辑方法的出现，将使我们能够通过鉴定当激酶活性严重受损时必不可少的基因来绘制有丝分裂激酶的功能景观。我们还将确定当激酶被部分抑制时可以加速增殖的基因突变。以这种方式鉴定的修饰基因可能与激酶密切合作或拮抗，揭示新的功能。我们已经开始在三种关键的有丝分裂激酶PLK1、CDK1和Wee1上应用这种策略，这已经揭示了这些激酶与有丝分裂、细胞分裂和DNA复制中特定分子机制之间的新功能联系。本研究计划将剖析这些联系，以增强我们对细胞分裂中有丝分裂激酶最关键功能的理解。将使用分子遗传学、显微镜学、生物化学和蛋白质组学的结合。从长远来看，这一策略将扩展到其他调节有丝分裂的酶家族的研究。这项研究产生的新知识将增加我们对保守的人类和其他真核生物物种的细胞生物学基本方面的理解。该多学科项目还将为培养一名博士后、一名博士和一名本科生提供良好的机会。这些受训人员将获得科学和技术能力以及横向技能，这将使他们在未来的职业生涯中处于良好的位置。

The development, survival and reproduction of any organism necessitate the ability of its cells to divide correctly. Cell division requires the replication of chromosomes, their segregation in mitosis and the cell's splitting in cytokinesis. This intricate process involves the complex coordination of multiple intracellular events by important regulatory enzymes. In this research program, we will elucidate biochemical mechanisms and cellular functions of a module of enzymes named kinases that controls the phosphorylation of target proteins during the cell cycle. The reversible addition of a phosphate group on a protein often results in changes in its activities. When cells enter mitosis, several proteins become phosphorylated by kinase enzymes to elicit cellular transformations. However, the precise functions involving kinases in cell division are still incompletely understood. Because each mitotic kinase controls several substrate proteins and events, deciphering their precise functions has been very difficult. To this end, the proposed research will take advantage of new technological developments. The advent of gene editing methods using CRISPR and newly developed selective kinase inhibitor compounds will allow us to map the functional landscape of mitotic kinases by the identification of genes that become essential when the activity of a kinase is critically compromised. We will also identify gene mutations that can accelerate proliferation when a kinase is partially inhibited. Modifier genes identified in this manner may function in close collaboration or antagonism with the kinase, revealing new functions. We have begun to deploy this strategy for three crucial mitotic kinases named PLK1, CDK1 and Wee1, which has already uncovered novel functional connections between these kinases and specific molecular mechanisms in mitosis, cytokinesis and DNA replication. This research program will dissect these connections to enhance our understanding of the most crucial functions mitotic kinases in cell division. A combination of molecular genetics, microscopy, biochemistry and proteomics will be used. In the longer term, this strategy will be extended to the study of other families of enzymes regulating mitosis. The new knowledge generated by this research will increase our understanding of basic aspects of cell biology that are conserved humans and other eukaryotic species. This multidisciplinary program will also provide an excellent opportunity to train one postdoc, one PhD student and undergraduate students in research. These trainees will acquire scientific and technical competences as well as transversal skills that will place them in excellent positions for their future careers.