Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics
Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics
批准号:
10321536
负责人:
Gina M Tomarchio
金额:
$4.68万
依托单位国家:
美国
项目类别:
财政年份:
2020
资助国家:
美国
项目状态:
已结题
起止时间:
2020-12-11 至 2023-12-10
关键词:
AddressAllelesAneuploidyBindingBiological AssayCRISPR/Cas technologyCellsCentrosomeChromosome SegregationChromosomesComplexDataDaughterDefectDevelopmentDimerizationDiploid CellsDynein ATPaseEnsureGenomic InstabilityHematologic NeoplasmsHematologyHumanImpairmentIn VitroInfluentialsKinesinLeadLiteratureMalignant NeoplasmsMasksMediatingMicroscopyMicrotubule-Organizing CenterMicrotubulesMitosisMitoticMitotic Spindle ApparatusMitotic spindleMotorMovementMutationPathway interactionsPatientsPhenotypePhosphoric Monoester HydrolasesPhosphorylationPhosphorylation SitePhosphotransferasesPlayPositioning AttributePrognosisProteinsPublishingRoleSirolimusSister ChromatidSiteSolidSomatic CellSystemTestingTotal Internal Reflection FluorescentTransgenic OrganismsWorkanalogattenuationaurora kinase Abasecancer cellcancer survivalcancer therapycell cortexcell motilitychemical geneticschromosome missegregationdaughter celldesigndynactinexperimental studyinhibitorinsightinterestmutantoverexpressionphosphoproteomicspreventscaffoldsmall molecule inhibitorspatiotemporaltargeted cancer therapytooltumorigenesis
中文摘要
摘要摘要
有丝分裂酶和磷酸酶对于防止染色体分离中的错误是必不可少的,这种错误可能导致
非整倍体和癌症。Aurora Kinase A(AURA)是一种重要的有丝分裂酶,通常在
人类癌症。AURA在有丝分裂纺锤体(一种复杂的微管)的组装中具有广泛研究的作用。
负责将姐妹染色单体分离成两个相同的子细胞的阵列。研究气场是具有挑战性的。
在纺锤体组装之外发挥作用,因为抑制会导致有丝分裂纺锤体的破坏,而有丝分裂纺锤体可以掩盖
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,通过允许特定的和有条件的抑制Aura来绕过这些问题
活动。使用培养的人类细胞,我们用CRISPR-Cas9灭活了内源性AURA并挽救了它
具有转基因AURA等位基因(AURA-AS)的活性。基因的改变使这个等位基因
对不能抑制任何其他细胞激酶的修饰的ATP竞争性抑制剂特别敏感。我们的
目的是利用这一工具研究先兆与癌症之间的联系。具体来说,我们感兴趣的是
灵气和中心体数字放大之间的联系,这在固体和
恶性血液病。额外的中心体可以产生具有两个以上极的有丝分裂纺锤体,因此
造成毁灭性的染色体错误分离。气场抑制与形成
文献中的多极纺锤形。我们的初步数据表明,光环活动阻止了这种多极
纺锤体表型通过促进额外的中心体“聚集”成两个纺锤体极来实现。这让我们找到了我们的
中枢假说:AURA的磷酸化是在额外存在的情况下形成双极纺锤体的关键
中心体。基于文献中的证据,我们假设AURA通过
HSET中纺锤极聚焦运动的磷酸化及促进有丝分裂纺锤体的定位
定位因子NUMA到细胞皮质(目标2)。确定HSET的AURA-磷酸化是否重要
为了提高中心体聚集的效率,我们将使用体外激酶检测来显示直接磷酸化(1.1)。
我们将使用HSET磷酸化位点突变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分析,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先兆的丧失是否会损害有丝分裂纺锤体的定位或
NUMA定位于细胞皮质(2.1),如果改变NUMA定位会影响中心体的效率
集群(2.2)。所有提议的实验都使用我们建立的光环研究系统。这项工作具有特殊的意义
感兴趣,因为实体和血液系统的恶性肿瘤通常都会增加中心体的数量;
将它们聚集成两个纺锤体极,可以防止它们的染色体分裂成多个不能存活的染色体
子代细胞。这项工作将描述先兆和独特的癌症生存机制之间的联系,以及
因此,将为AURA特定癌症疗法的设计和实施提供信息。
英文摘要
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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Identifying Mechanisms of Aurora Kinase A in Centrosome Clustering Using Chemical Genetics
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批准号:10538615
-
项目类别:
-
资助金额:$4.77万
-
财政年份:2020
-
负责人:Gina M Tomarchio
-
依托单位:
海外基金