Regulation of cell division by mitotic kinases

有丝分裂激酶对细胞分裂的调节

• 批准号: 9230854
• 负责人: Ekaterina L Grishchuk
• 金额: $ 31.82万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-05-01 至 2018-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9230854
• 关键词: Affect; Behavior; Binding; Binding Sites; Biochemical; Biophysics; Cell division; Cells; Centromere; Chemicals; Chromosome Segregation; Chromosomes; Complex; Defect; Development; Diffusion; Dimerization; Elements; Ensure; Exhibits; Feedback; Future; Genetic; Genetic Materials; Goals; Human; In Vitro; Inherited; Intuition; Kinetics; Kinetochores; Lead; Link; Malignant Neoplasms; Measures; Mechanics; Microspheres; Microtubules; Mitotic; Modeling; Molecular; Nonlinear Dynamics; Pathway interactions; Pattern; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Play; Pregnancy loss; Process; Reaction; Recombinants; Regulation; Role; Signal Transduction; Sister; System; Testing; aurora B kinase; daughter cell; defined contribution; density; dimer; experimental study; genetic regulatory protein; in vivo; predictive modeling; public health relevance; reconstitution; targeted cancer therapy

DESCRIPTION (provided by applicant): Accurate chromosome segregation depends on bi-orientation (i.e., sister kinetochores attaching to spindle microtubules (MTs) from opposite poles), which relies on tension-dependent stabilization of kinetochore MTs. Multiple lines of evidence suggest that Aurora B kinase, the enzymatic component of the chromosome passenger complex (CPC), is a key element of this mechanism. Strikingly, CPC binding sites are enriched at the inner centromere, not at the outer kinetochore where Aurora B substrates bind MT ends. Moreover, phosphorylation of these kinetochore substrates, which reduces MT binding, decreases as the distance from the centromere to the kinetochore increases with tension. These observations established a correlative link between Aurora B signaling and tension and led to an intuitively attractive "spatial separation" model, in which distance-dependent phosphorylation plays a crucial role in bi-orientation. However, the mechanisms underlying distance-dependent phosphorylation by Aurora B are unknown. Furthermore, whether tension can be sensed by Aurora B-independent pathways in vivo, as suggested by in vitro experiments has not been tested. Aurora B is known to auto-activate by auto-phosphorylation in trans, so we developed a quantitative model to describe the resulting non-intuitive spatial nonlinear dynamics. Our model predicts that distance- dependent phosphorylation by Aurora B is established by a reaction-diffusion mechanism. The essential features of this mechanism are: 1) Aurora B auto-activates at centromeres due to a high density of CPC binding sites (i.e., clustering), and 2) this activity propagates to kinetochores by unbinding of active kinase, diffusion, and kinase activation/inactivation reactions in solution, which depend on the soluble kinase/phosphatase ratio. Aim 1 will test the hypothesis that Aurora B regulates kinetochore-MT interactions through a reaction-diffusion process in vivo. We will manipulate CPC clustering at centromeres, the kinetics of centromere unbinding, and the soluble kinase/phosphatase ratio, and measure the effects of these perturbations on kinetochore function. Aim 2 will reconstitute Aurora B phosphorylation dynamics and spatially-regulated MT binding in vitro. The in vivo situation is complex, and in vitro reconstitution will allow us to establish direct, quantitative relationships between the inputs and the behavior of the system. This aim builds on an in vitro system that we developed using recombinant Aurora B and phosphatase and fluorescent substrates. Aim 3 will define the contributions of tension and Aurora B to regulating kinetochore MTs in vivo. To uncouple tension from Aurora B, we developed a chemically-induced dimerization strategy that allows us to directly manipulate Aurora B activity at kinetochores and independent of tension. We will (1) determine how controlled changes in Aurora B activity at kinetochores affect MT dynamics, and (2) dissect the direct effects of tension and indirect effects via Aurora B. The result of these experiments will b detailed understanding of how both biochemical and mechanical changes at kinetochores control interactions with MTs.

描述（由申请人提供）：准确的染色体分离依赖于双向性（即，姐妹着丝点从相反的极点附着在纺锤体微管（MTs）上），这依赖于着丝点MTs的张力依赖性稳定。多项证据表明，染色体乘客复合物（CPC）的酶成分Aurora B激酶是该机制的关键因素。引人注目的是，CPC结合位点富集于内着丝粒，而不是在Aurora B底物结合MT末端的外着丝粒。此外，随着从着丝粒到着丝粒的距离随着张力的增加而增加，这些着丝粒底物的磷酸化会减少MT的结合。这些观察结果建立了极光B信号和张力之间的相关联系，并导致了一个直观的有吸引力的“空间分离”模型，其中距离依赖性磷酸化在双向定向中起着至关重要的作用。然而，极光B的距离依赖性磷酸化机制尚不清楚。此外，体外实验表明，在体内是否可以通过不依赖Aurora b的途径感知张力尚未得到验证。已知Aurora B通过反式中的自磷酸化自动激活，因此我们开发了一个定量模型来描述由此产生的非直观的空间非线性动力学。我们的模型预测极光B的距离依赖性磷酸化是通过反应扩散机制建立的。该机制的基本特征是：1)由于CPC结合位点的高密度（即聚类），Aurora B在着丝粒上自动激活；2)这种活性通过活性激酶的解结合、扩散和溶液中激酶的激活/失活反应传播到着丝粒，这些反应取决于可溶性激酶/磷酸酶的比例。目的1将验证Aurora B通过体内反应-扩散过程调节着丝酶- mt相互作用的假设。我们将控制着丝粒的CPC聚类、着丝粒解结合动力学和可溶性激酶/磷酸酶比率，并测量这些扰动对着丝粒功能的影响。目的2将重建Aurora B磷酸化动力学和体外空间调节的MT结合。体内的情况是复杂的，体外重构将允许我们建立直接的，定量的输入和行为之间的关系