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

描述（由申请人提供）：polo样激酶1 （Plk1）是驱动人类细胞分裂机制的核心组成部分。它的功能是防止细胞获得或失去可能导致癌症的染色体。相反，它也是癌症治疗的一个有希望的靶点。Plk1通过结合并将磷酸盐转移到底物蛋白上，在人类细胞分裂过程中执行多个事件。然而，所需的精确磷酸化事件仍然不清楚，因为它们在人类有丝分裂的60分钟内紧密地发生。这些事件的鉴定可以阐明细胞毒性、耐药性和plk1靶向药物对正常细胞的非预期作用的机制。创新：传统的遗传工具允许观察Plk1损失对所有底物的总影响。相比之下，化学遗传学通过废除底物子集的磷酸化，允许解剖功能横截面。这种方法揭示了Plk1在人类细胞分裂中的新功能。这里设计的工具将有助于了解其他多功能激酶的功能。方法：本项目旨在阐明Plk1在染色体分离中的功能。首先，Plk1 c端polo-box结构域在分离中的功能意义将通过互补分析进行测试。接下来，将确定导致错分离的直接底物。这些发现将显示Plk1的部分抑制如何影响细胞分裂。AIM 2将阐明启动细胞分裂所需的plk1依赖性事件。最初，这将集中于Cyk4/RACGAP1的磷酸化，Cyk4/RACGAP1是中央纺锤体的一个组成部分。这些磷酸化的功能意义将在生化分析和抢救实验中进行测试。在本AIM的第二部分，将测试其他底物Plk1磷酸化的功能意义，以了解其对细胞分裂的影响。展望：总体目标是提供对Plk1功能的全面理解。这将为临床Plk1抑制剂的细胞毒性机制提供重要的见解，确定其抑制的敏感生物标志物，并开创新的方法来解剖其他蛋白激酶的多种功能-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.