The role of multisite phosphorylation in establishing temporal order during mitosis

多位点磷酸化在有丝分裂过程中建立时间顺序中的作用

• 批准号: 321115587
• 负责人: Dr. Julia Kamenz
• 金额: --
• 依托单位: Department of Chemical and Systems Biology
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2016
• 资助国家: 德国
• 起止时间: 2015-12-31 至 2017-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/321115587?language=en
• 关键词: role; multisite; phosphorylation; establishing; temporal

In eukaryotes the transition between interphase and mitosis is characterized by extensive cellular reorganization. During mitotic exit sister chromatids separate, chromatin decondenses and the nuclear envelope reassembles. Failure to maintain the specific order of these events can result in genomic instability or cell death. Mitotic entry and exit are driven by the changing activity of the cyclin-dependent kinase (Cdk1), which phosphorylates hundreds of substrates, most of them at multiple sites. Particularly in vertebrates, little is known about how the change in kinase activity translates into distinct substrate phosphorylation dynamics and eventually results in a precise temporal order of events. Multisite phosphorylation is hypothesized to constitute an effective mechanism to determine the order of events during mitotic entry and exit. I will test this hypothesis experimentally using Xenopus laevis egg extracts: I have recently identified the nucleoporin Nup53 (MP44) as a model substrate for studying Cdk1 multisite phosphorylation in vitro and in its native environment using nuclear magnetic resonance spectroscopy (NMR). Combining NMR experiments and biochemical assays I will study the mechanism of multisite phosphorylation with high temporal and single amino acid resolution during the mitotic transitions. I will further characterize the phosphorylation and dephosphorylation dynamics of various other Cdk1 substrates and investigate how multisite phosphorylation contributes to determining their different temporal kinetics. By supporting the experimental results with mathematical models and computer simulations I aim to identify common principles of temporal coordination via multisite phosphorylation - principles that might be applicable to numerous other signaling pathways.

在真核生物中，间期和有丝分裂之间的过渡以广泛的细胞重组为特征。在有丝分裂退出时，姐妹染色单体分离，染色质去致密化，核被膜重组。不能维持这些事件的特定顺序可能导致基因组不稳定或细胞死亡。有丝分裂的进入和退出是由细胞周期蛋白依赖性激酶（Cdk 1）的活性变化驱动的，Cdk 1磷酸化数百种底物，其中大多数在多个位点。特别是在脊椎动物中，很少有人知道激酶活性的变化如何转化为不同的底物磷酸化动力学，并最终导致事件的精确时间顺序。多位点磷酸化被假设为构成一种有效的机制，以确定在有丝分裂的进入和退出的事件的顺序。我将测试这个假设实验使用非洲爪蟾卵提取物：我最近确定了核孔蛋白Nup 53（MP44）作为模型基板研究Cdk 1多位点磷酸化在体外和在其原生环境中使用核磁共振光谱（NMR）。结合核磁共振实验和生化分析，我将研究多位点磷酸化的机制与高时间和单个氨基酸的分辨率在有丝分裂转换。我将进一步表征各种其他Cdk 1底物的磷酸化和去磷酸化动力学，并研究多位点磷酸化如何有助于确定其不同的时间动力学。通过数学模型和计算机模拟来支持实验结果，我的目标是通过多位点磷酸化确定时间协调的共同原则-这些原则可能适用于许多其他信号通路。