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）：准确的染色体分离取决于双向（即，从相反的两极连接到纺锤体微管（MT）的姐妹动粒），其依赖于动粒MT的张力依赖性稳定。多条证据表明，极光B激酶，染色体乘客复合物（CPC）的酶组分，是这一机制的关键要素。引人注目的是，CPC结合位点富集在内部着丝粒，而不是在极光B底物结合MT末端的外部动粒。此外，磷酸化的这些动粒基板，这减少MT结合，减少从着丝粒的动粒的距离增加与张力。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位中起着至关重要的作用。然而，极光B的距离依赖性磷酸化的机制尚不清楚。此外，张力是否可以感知极光B-独立的途径在体内，在体外实验中所建议的尚未测试。已知极光B通过反式自磷酸化自激活，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，Aurora B在着丝粒处自动激活（即，聚集），和2）该活性通过活性激酶的解结合、扩散和溶液中的激酶活化/失活反应传播到动粒，这取决于可溶性激酶/磷酸酶比率。目的1将测试的假设，极光B调节kinetochore-MT的相互作用，通过反应扩散过程在体内。我们将操纵CPC在着丝粒的聚类，着丝粒解结合的动力学，和可溶性激酶/磷酸酶的比例，并测量这些扰动对动粒功能的影响。目的2将在体外重建Aurora B磷酸化动力学和空间调节的MT结合。体内情况是复杂的，体外重建将使我们能够建立输入和细胞行为之间的直接定量关系。 系统这一目标建立在我们使用重组极光B和磷酸酶和荧光底物开发的体外系统上。目的3将定义张力和极光B对体内调节动粒MT的贡献。为了解除极光B的张力，我们开发了一种化学诱导的二聚化策略，使我们能够直接操纵极光B在着丝粒的活性和独立的张力。我们将（1）确定如何控制极光B活动在着丝粒的变化影响MT动力学，（2）剖析张力的直接影响和间接影响，通过极光B。这些实验的结果将B详细了解着丝粒的生化和机械变化如何控制与MT的相互作用。