The role of multisite phosphorylation in establishing temporal order during mitosis

真核生物间期和有丝分裂的过渡以广泛的细胞重组为特征。在有丝分裂结束时，姐妹染色单体分离，染色质去浓缩，核膜重新组装。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周期蛋白依赖性激酶（Cdk1）活性的变化驱动的，该激酶磷酸化数百种底物，其中大多数在多个位点磷酸化。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。多位点磷酸化被认为是决定有丝分裂进入和退出过程中事件顺序的有效机制。我将用非洲蟾卵提取物对这一假设进行实验验证：我最近发现核孔蛋白Nup53 （MP44）作为模型底物，用于在体外和自然环境中使用核磁共振波谱（NMR）研究Cdk1多位点磷酸化。结合核磁共振实验和生化分析，我将研究在有丝分裂转变过程中具有高时间和单氨基酸分辨率的多位点磷酸化的机制。我将进一步表征各种其他Cdk1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。通过用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来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。

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.