Molecular dissection of mitotic kinase functions using chemogenomics

任何有机体的发育、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，胞质分裂时细胞的分裂。这一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。在这项研究计划中，我们将阐明一组名为激酶的酶的生化机制和细胞功能，该模块在细胞周期中控制目标蛋白的磷酸化。蛋白质上磷酸基团的可逆加成常常导致其活性的变化。当细胞进入有丝分裂时，几种蛋白质被激酶酶磷酸化，从而引发细胞转化。然而，与细胞分裂相关的激酶的确切功能仍然不完全清楚。因为每个有丝分裂激酶控制着几种底物蛋白和事件，所以破译它们的确切功能一直是非常困难的。为此，拟议的研究将利用新技术的发展。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，将使我们能够通过识别当激酶活性严重受损时变得至关重要的基因来绘制有丝分裂酶的功能图谱。我们还将确定当一种激酶被部分抑制时，可以加速增殖的基因突变。以这种方式确定的修饰基因可能与该激酶密切协作或相互拮抗，从而揭示新的功能。我们已经开始对PLK1、CDK1和Wee1这三个关键的有丝分裂酶采取这种策略，已经发现了这些激酶之间的新的功能联系和有丝分裂、胞质分裂和DNA复制的特定分子机制。这项研究计划将剖析这些联系，以加强我们对细胞分裂中最关键的有丝分裂酶功能的理解。将结合分子遗传学、显微镜、生物化学和蛋白质组学进行研究。从长远来看，这一策略将扩展到对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细胞生物学基本方面的理解，这些细胞生物学是受保护的人类和其他真核物种。这一多学科项目还将提供一个极好的机会，培养一名博士后、一名博士生和本科生进行研究。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有利地位。

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.