Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics

摘要 有丝分裂激酶和磷酸酶对于防止染色体分离错误是必不可少的， 非整倍体和癌症的关系。极光激酶A（AurA）是一种必需的有丝分裂激酶，在细胞分裂中经常上调。 人类癌症AurA在有丝分裂纺锤体（一种复杂的微管）的组装中有着广泛的研究 负责将姐妹染色单体分离成两个相同的子细胞的阵列。研究AurA具有挑战性 在纺锤体组装外起作用，因为抑制导致有丝分裂纺锤体的破坏， 其他相关表型。此外，大多数AurA小分子抑制剂可能是混杂的。我们有 开发了一个系统，通过允许特异性和条件性抑制AurA来规避这些问题 活动使用培养的人类细胞，我们已经用CRISPR-Cas9灭活了内源性AurA，并挽救了它的生命。 基因修饰的转基因AurA等位基因（AurA-AS）的活性。基因改变使得这个等位基因 对不能抑制任何其他细胞激酶的修饰的ATP竞争性抑制剂特异性敏感。我们 目的是利用这个工具来研究AurA与癌症之间的联系。具体来说，我们感兴趣的是 AurA和中心体数目扩增之间的联系，这在固体和 血液恶性肿瘤额外的中心体可以产生两极以上的有丝分裂纺锤体， 导致毁灭性的染色体错误分离。AurA抑制与 多极纺锤体。我们的初步数据表明，AurA活动阻止了这种多极化， 纺锤体表型通过促进额外的中心体“聚集”成两个纺锤体极。这使我们 中心假设，AurA磷酸化是必不可少的双极纺锤体的形成，在存在额外的 中心体基于文献中的证据，我们假设AurA通过以下途径介导中心体聚集： HSET（Aim 1）纺锤极聚焦驱动蛋白的磷酸化和通过促进有丝分裂纺锤体的定位 将因子NuMA定位到细胞皮质（Aim 2）。确定HSET的AurA磷酸化是否重要 对于中心体聚集的效率，我们将使用体外激酶测定来显示直接磷酸化（1.1）。 我们将使用HSET磷酸化位点突变体来确定这种磷酸化是否与中心体聚集有关 (1.2)并进行体外微管运动试验以确定AurA磷酸化如何影响HSET 活动（1.3）。此外，我们将使用显微镜来确定Aura的缺失是否会损害有丝分裂纺锤体的定位， NuMA定位于细胞皮质（2.1），如果改变NuMA定位，则影响中心体的效率 聚类（2.2）。所有拟议的实验使用我们建立的系统研究Aura。这项工作特别 令人感兴趣的是，实体和血液恶性肿瘤通常具有增加的中心体数目; 将它们聚集成两个纺锤极，它们可以阻止它们的染色体分裂成多个， 子细胞这项工作将描述AurA和一种独特的癌症生存机制之间的联系， 因此，将为AurA特异性癌症疗法的设计和实施提供信息。

SUMMARY ABSTRACT Mitotic kinases and phosphatases are essential for preventing errors in chromosome segregation that can lead to aneuploidy and cancer. Aurora Kinase A (AurA) is an essential mitotic kinase that is often upregulated in human cancers. AurA has a well-studied role in assembly of the mitotic spindle apparatus, a complex microtubule array responsible for separating sister chromatids into two identical daughter cells. It is challenging to study AurA function outside of spindle assembly because inhibition causes disruption of the mitotic spindle which can mask other relevant phenotypes. Further, most AurA small molecule inhibitors can be promiscuous. We have developed a system that circumvents these issues by allowing for specific and conditional inhibition of AurA activity. Using cultured human cells, we have inactivated endogenous AurA with CRISPR-Cas9 and rescued its activity with a genetically modified, transgenic AurA allele (AurA-AS). The genetic alteration makes this allele specifically sensitive to modified ATP competitive inhibitors that cannot inhibit any other cellular kinases. Our objective is to use this tool to study the connection between AurA and cancer. Specifically, we are interested in the connection between AurA and centrosome number amplifications, which are common in both solid and hematological malignancies. Extra centrosomes can create mitotic spindles with more than two poles thus causing devastating chromosome mis-segregation. AurA inhibition has been associated with formation of multipolar spindles in the literature. Our preliminary data suggests that AurA activity prevents this multipolar spindle phenotype by promoting the “clustering” of extra centrosomes into two spindle poles. This led us to our central hypothesis that AurA phosphorylation is essential for bi-polar spindle formation in the presence of extra centrosomes. Based on evidence in the literature, we hypothesize that AurA mediates centrosome clustering via phosphorylation of spindle-pole focusing kinesin HSET (Aim 1) and via promoting localization of mitotic spindle positioning factor NuMA to the cell cortex (Aim 2). To determine if AurA-phosphorylation of HSET is important for efficiency of centrosome clustering, we will use in vitro kinase assays to show direct phosphorylation (1.1). We will use HSET phospho-site mutants to determine if this phosphorylation is relevant to centrosome clustering (1.2) and to perform in vitro microtubule motility assays to determine how AurA phosphorylation effects HSET’s activity (1.3). Further, we will use microscopy to determine if loss of AurA impairs mitotic spindle positioning or NuMA localization to the cell cortex (2.1) and if altered NuMA localization effects efficiency of centrosome clustering (2.2). All proposed experiments use our established system for studying AurA. This work is of particular interest as both solid and hematological malignancies often have increased number of centrosomes; by clustering them into two spindle poles, they can prevent division of their chromosomes into multiple, inviable daughter cells. This work will describe a connection between AurA and a unique cancer survival mechanism and will therefore be informative for design and implementation of AurA-specific cancer therapies.