The role of multisite phosphorylation in establishing temporal order during mitosis

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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.