Separation of late mitotic functions of polo-like kinase 1 with chemical genetics

描述(申请人提供)：Polo-like kinase1(Plk1)是驱动人类细胞分裂的机制的中心组件。它的功能是防止细胞获得或丢失染色体，这可能会导致癌症。相反，它也是癌症治疗的一个有希望的靶点。PLK1通过将磷酸盐结合并转移到底物蛋白来执行人类细胞分裂中的多个事件。然而，所需的准确的磷酸化事件仍然不清楚，因为它们发生在人类有丝分裂的60分钟内。识别这些事件可以阐明Plk1靶向药物对正常细胞的细胞毒性、耐药性和意外影响的机制。创新：传统的基因工具可以观察Plk1缺失对所有底物的总影响。相比之下，化学遗传学允许通过取消底物子集的磷酸化来解剖功能横截面。这一途径揭示了Plk1在人类细胞分裂中的新功能。本文设计的工具将有助于理解其他多功能激酶的功能。方法：本项目的目标1试图阐明Plk1在染色体分离中的功能。首先，通过互补分析来检验Plk1 C-末端Polo-box结构域在分离中的功能意义。下一步，将确定造成错误分离的直接基质。这些发现将显示部分抑制Plk1将如何影响细胞分裂。AIM 2将阐明启动胞质分裂所需的Plk1依赖事件。最初，这将集中在中心纺锤体的一个组成部分Cyk4/RACGAP1的磷酸化上。这些磷酸化的功能意义将在生化分析和抢救实验中得到检验。在本研究的第二部分，将测试其他底物Plk1磷酸化对胞质分裂的影响的功能意义。展望：总体目标是全面了解Plk1的功能。这将为临床Plk1抑制剂的细胞毒性机制提供重要的见解，识别其抑制的敏感生物标记物，并开辟新的方法学来剖析其他蛋白激酶的多种功能--其他蛋白激酶是最重要的癌症药物靶点之一。

DESCRIPTION (provided by applicant): Polo-like kinase 1 (Plk1) is a central component of the machinery that drives human cell division. Its function is required to prevent cells from gaining or losing chromosomes, which can lead to cancer. Conversely, it is also a promising target for cancer therapy. Plk1 executes multiple events in human cell division by binding and transferring a phosphate to substrate proteins. However, the precise phosphorylation events that are required remain obscure because they occur close together within the 60 minutes of human mitosis. Identification of these events can elucidate mechanisms of cytotoxicity, resistance, and unintended effects of Plk1-targeted drugs on normal cells. Innovation: Traditional genetic tools allow observation of the total effect of Plk1 loss on all substrates. In contrast, chemical genetics allows dissection of functional cross sections by abrogating phosphorylations of a subset of substrates. This approach has revealed new functions of Plk1 in division of human cells. The tools devised here will be useful for understanding functions of other multifunctional kinases. Approach: AIM 1 of this project seeks to elucidate Plk1 function in chromosome segregation. First, the functional significance of the Plk1 C-terminal polo-box domain in segregation will be tested by complementation assays. Next, the direct substrate responsible for missegregation will be identified. These findings will show how partial inhibition of Plk1 will affect dividing cells. AIM 2 will clarify Plk1-dependent events required to initiate cytokinesis. Initially this will focus on phosphorylation of Cyk4/RACGAP1, a component of the central spindle. The functional significance of these phosphorylations will be tested in biochemical assays and rescue experiments. In the second part of this AIM, the functional significance of additional Plk1 phosphorylations of other substrates will be tested for effect on cytokinesis. Outlook: The overall goal is to provide a comprehensive understanding of Plk1 function. This will provide crucial insight into the mechanisms of cytotoxicity for clinical Plk1 inhibitors, identify sensitive biomarkers of its inhibition, and pioneer new methodology to dissect multiple functions of other protein kinases-one of the most important classes of cancer drug targets.