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

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.

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