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

利用化学遗传学鉴定中心体聚类中 Aurora 激酶 A 的机制

• 批准号: 10538615
• 负责人: Gina M Tomarchio
• 金额: $ 4.77万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-12-11 至 2023-12-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10538615
• 关键词: Address; Alleles; Aneuploidy; Binding; Biological Assay; CRISPR/Cas technology; Cells; Centrosome; Chromosome Segregation; Chromosomes; Complex; Data; Defect; Development; Dimerization; Diploid Cells; Dynein ATPase; Ensure; Genomic Instability; Hematologic Neoplasms; Human; Impairment; In Vitro; Influentials; Kinesin; Literature; Malignant Neoplasms; Masks; Mediating; Microscopy; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic Spindle Apparatus; Mitotic spindle; Motor; Movement; Mutation; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Positioning Attribute; Prognosis; Proteins; Publishing; Role; Sirolimus; Sister Chromatid; Solid; Somatic Cell; System; Testing; Total Internal Reflection Fluorescent; Transgenic Organisms; Work; analog; attenuation; aurora kinase A; cancer cell; cancer survival; cancer therapy; cell cortex; cell motility; chemical genetics; chromosome missegregation; daughter cell; design; dynactin; experimental study; inhibitor; insight; interest; mutant; overexpression; patient prognosis; phosphoproteomics; prevent; scaffold; small molecule inhibitor; spatiotemporal; targeted cancer therapy; tool; tumorigenesis; Address; Alleles; Aneuploidy; Binding; Biological Assay; CRISPR/Cas technology; Cells; Centrosome; Chromosome Segregation; Chromosomes; Complex; Data; Defect; Development; Dimerization; Diploid Cells; Dynein ATPase; Ensure; Genomic Instability; Hematologic Neoplasms; Human; Impairment; In Vitro; Influentials; Kinesin; Literature; Malignant Neoplasms; Masks; Mediating; Microscopy; Microtubule-Organizing Center; Microtubules; Mitosis; Mitotic; Mitotic Spindle Apparatus; Mitotic spindle; Motor; Movement; Mutation; Pathway interactions; Phenotype; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphorylation Site; Phosphotransferases; Positioning Attribute; Prognosis; Proteins; Publishing; Role; Sirolimus; Sister Chromatid; Solid; Somatic Cell; System; Testing; Total Internal Reflection Fluorescent; Transgenic Organisms; Work; analog; attenuation; aurora kinase A; cancer cell; cancer survival; cancer therapy; cell cortex; cell motility; chemical genetics; chromosome missegregation; daughter cell; design; dynactin; experimental study; inhibitor; insight; interest; mutant; overexpression; patient prognosis; phosphoproteomics; prevent; scaffold; small molecule inhibitor; spatiotemporal; targeted cancer therapy; tool; tumorigenesis

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.

摘要摘要 有丝分裂激酶和磷酸酶对于预防染色体隔离的错误至关重要 到非整倍性和癌症。 Aurora激酶A（AURA）是一种必需的有丝分裂激酶，通常在 人类癌。光环在有丝分裂纺锤体设备的组装中具有良好的作用，这是一个复杂的微管 阵列负责将姐妹染色单体分为两个相同的子细胞。研究光环是挑战 在主轴组件之外的功能，因为抑制作用会导致有丝分裂纺锤体的破坏 其他相关表型。此外，大多数光环小分子抑制剂可能是混杂的。我们有 开发了一个通过允许特定和有条件抑制光环来绕过这些问题的系统 活动。使用培养的人类细胞，我们用CRISPR-CAS9灭活了内源性Aura并营救了它 具有转基因的转基因Aura等位基因（Aura-as）的活性。遗传改变使这个等位基因 对无法抑制任何其他细胞激酶的改良ATP竞争抑制剂特别敏感。我们的 目的是使用此工具来研究光环与癌症之间的联系。具体来说，我们对 光环和中心体数量扩增之间的连接，这在固体和固体和 血液系统恶性肿瘤。额外的中心体可以与两个以上的两极形成有丝分裂的纺锤体 引起毁灭性的染色体错误分离。灵气抑制与形成有关 文献中的多极纺锤体。我们的初步数据表明，Aura活动可防止这种多极 纺锤表型通过将额外的中心体的“聚类”促进到两个主轴杆中。这使我们进入了我们的 中心假设是，在额外 中心体。根据文献中的证据，我们假设光环通过 主轴螺旋聚焦驱动蛋白HSET（AIM 1）的磷酸化并通过促进有丝分裂的定位 将因子定位为NUMA到细胞皮层（AIM 2）。确定HSES的光环磷酸化是否很重要 为了提高中心体聚类的效率，我们将使用体外激酶刺阿萨斯显示直接的磷酸化（1.1）。 我们将使用HSES磷酸位点突变体来确定这种磷酸化是否与中心体聚类有关 （1.2）并执行体外微管运动评估以确定过敏磷酸化如何影响HSES的磷酸化 活动（1.3）。此外，我们将使用显微镜来确定AURA的损失是否损害有丝分裂的纺锤体定位或 NUMA定位到细胞皮层（2.1），如果改变了NUMA定位效应中心体的效率 聚类（2.2）。所有提出的实验都使用我们既定的系统来研究灵气。这项工作特别 固体和血液系统恶性肿瘤的兴趣通常会增加中心体的数量。经过 将它们聚集成两个主轴杆，它们可以防止其染色体分为多个不可或缺的 子细胞。这项工作将描述光环与独特的癌症生存机制之间的联系 因此，对于AURA特异性癌症疗法的设计和实施，将是有益的。