The complexity of multisite phosphorylation mechanisms in regulating nuclear localization signals (NLSs) and nuclear export signals (NESs) is not understood, and its potential has not been used in synthetic biology. The nucleocytoplasmic shuttling of many proteins is regulated by cyclin-dependent kinases (CDKs) that rely on multisite phosphorylation patterns and short linear motifs (SLiMs) to dynamically control proteins in the cell cycle. We studied the role of motif patterns in nucleocytoplasmic shuttling using sensors based on the CDK targets Dna2, Psy4, and Mcm2/3 of Saccharomyces cerevisiae . We designed multisite phosphorylation modules by rearranging phosphorylation sites, cyclin-specific SLiMs, phospho-priming, phosphatase specificity, and NLS/NES phospho-regulation and obtained very different substrate localization dynamics. These included ultrasensitive responses with and without a delay, graded responses, and different homeostatic plateaus. Thus, CDK can do much more than trigger sequential switches during the cell cycle as it can drive complex patterns of protein localization and activity by using multisite phosphorylation networks.

中文翻译：

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一种合成生物学方法通过 NLS-NES 模块的多位点磷酸化揭示了多样化和动态的 CDK 响应谱

调节核定位信号 (NLS) 和核输出信号 (NES) 的多位点磷酸化机制的复杂性尚不清楚，其潜力尚未用于合成生物学。许多蛋白质的核质穿梭受细胞周期蛋白依赖性激酶 (CDK) 调节，CDK 依赖多位点磷酸化模式和短线性基序 (SLiM) 来动态控制细胞周期中的蛋白质。我们使用基于 CDK 目标 Dna2、Psy4 和 Mcm2/3 的传感器研究了基序模式在核质穿梭中的作用酿酒酵母. 我们通过重新排列磷酸化位点、细胞周期蛋白特异性 SLiM、磷酸引发、磷酸酶特异性和 NLS/NES 磷酸调节来设计多位点磷酸化模块，并获得了非常不同的底物定位动力学。这些包括有延迟和无延迟的超灵敏反应、分级反应和不同的稳态平台。因此，CDK 可以做的不仅仅是在细胞周期中触发顺序开关，因为它可以通过使用多位点磷酸化网络驱动复杂的蛋白质定位和活性模式。