Dynamics of re-constitution of the human nuclear proteome after cell division is regulated by NLS-adjacent phosphorylation

Phosphorylation by the cyclin-dependent kinase 1 (Cdk1) adjacent to nuclear localization signals (NLSs) is an important mechanism of regulation of nucleocytoplasmic transport. However, no systematic survey has yet been performed in human cells to analyze this regulatory process, and the corresponding cell-cycle dynamics have not yet been investigated. Here, we focused on the human proteome and found that numerous proteins, previously not identified in this context, are associated with Cdk1-dependent phosphorylation sites adjacent to their NLSs. Interestingly, these proteins are involved in key regulatory events of DNA repair, epigenetics, or RNA editing and splicing. This finding indicates that cell-cycle dependent events of genome editing and gene expression profiling may be controlled by nucleocytoplasmic trafficking. For in-depth investigations, we selected a number of these proteins and analyzed how point mutations, expected to modify the phosphorylation ability of the NLS segments, perturb nucleocytoplasmic localization. In each case, we found that mutations mimicking hyper-phosphorylation abolish nuclear import processes. To understand the mechanism underlying these phenomena, we performed a video microscopy-based kinetic analysis to obtain information on cell-cycle dynamics on a model protein, dUTPase. We show that the NLS-adjacent phosphorylation by Cdk1 of human dUTPase, an enzyme essential for genomic integrity, results in dynamic cell cycle-dependent distribution of the protein. Non-phosphorylatable mutants have drastically altered protein re-import characteristics into the nucleus during the G1 phase. Our results suggest a dynamic Cdk1-driven mechanism of regulation of the nuclear proteome composition during the cell cycle.

细胞周期蛋白依赖性激酶1（cyclin-dependent kinase 1, Cdk1）在核定位信号（nuclear localization signals, NLSs）邻近位点介导的磷酸化修饰，是调控核质运输的重要机制。然而，目前尚未在人类细胞中针对该调控过程开展系统性研究，其对应的细胞周期动态变化亦未被探明。本研究聚焦人类蛋白质组，发现大量此前未被关联至该调控场景的蛋白质，其核定位信号邻近位点存在Cdk1依赖性磷酸化位点。有趣的是，这些蛋白质参与DNA修复、表观遗传学、RNA编辑与剪接等关键调控事件。该发现提示，细胞周期依赖的基因组编辑与基因表达谱分析事件，或可通过核质运输实现调控。为开展深入机制研究，我们筛选了一批此类蛋白质，并分析了预期可改变核定位信号区段磷酸化能力的点突变，如何扰动核质定位过程。所有实验均显示，模拟过度磷酸化的突变会阻断蛋白质的核导入过程。为解析上述现象的分子机制，我们以脱氧尿苷三磷酸酶（dUTPase）为模型蛋白，通过基于视频显微镜的动力学分析，获取其细胞周期动态相关信息。研究表明，人类脱氧尿苷三磷酸酶——一种对基因组完整性至关重要的酶——的核定位信号邻近位点被Cdk1磷酸化后，该蛋白会呈现动态的细胞周期依赖性分布。在G1期，无法发生磷酸化的突变体的核重新导入特性发生了显著改变。本研究结果提示，存在一种由Cdk1介导的动态调控机制，可在细胞周期进程中调控核蛋白质组的组成。