Institute of Technology, University of Tartu, Tartu 50411, Estonia.
Nature. 2011 Oct 12;480(7375):128-31. doi: 10.1038/nature10560.
Multisite phosphorylation of proteins has been proposed to transform a graded protein kinase signal into an ultrasensitive switch-like response. Although many multiphosphorylated targets have been identified, the dynamics and sequence of individual phosphorylation events within the multisite phosphorylation process have never been thoroughly studied. In Saccharomyces cerevisiae, the initiation of S phase is thought to be governed by complexes of Cdk1 and Cln cyclins that phosphorylate six or more sites on the Clb5-Cdk1 inhibitor Sic1, directing it to SCF-mediated destruction. The resulting Sic1-free Clb5-Cdk1 complex triggers S phase. Here, we demonstrate that Sic1 destruction depends on a more complex process in which both Cln2-Cdk1 and Clb5-Cdk1 act in processive multiphosphorylation cascades leading to the phosphorylation of a small number of specific phosphodegrons. The routes of these phosphorylation cascades are shaped by precisely oriented docking interactions mediated by cyclin-specific docking motifs in Sic1 and by Cks1, the phospho-adaptor subunit of Cdk1. Our results indicate that Clb5-Cdk1-dependent phosphorylation generates positive feedback that is required for switch-like Sic1 destruction. Our evidence for a docking network within clusters of phosphorylation sites uncovers a new level of complexity in Cdk1-dependent regulation of cell cycle transitions, and has general implications for the regulation of cellular processes by multisite phosphorylation.
蛋白质的多部位磷酸化被认为可以将逐渐增强的蛋白激酶信号转化为超灵敏的开关样反应。尽管已经鉴定出许多多磷酸化的靶标，但在多部位磷酸化过程中各个磷酸化事件的动态和顺序从未被彻底研究过。在酿酒酵母中，S 期的启动被认为是由 Cdk1 和 Cln 周期蛋白复合物控制的，这些复合物在 Clb5-Cdk1 抑制剂 Sic1 上磷酸化六个或更多位点，使其定向到 SCF 介导的破坏。由此产生的无 Sic1 的 Clb5-Cdk1 复合物触发 S 期。在这里，我们证明 Sic1 的破坏取决于一个更复杂的过程，其中 Cln2-Cdk1 和 Clb5-Cdk1 都在连续的多磷酸化级联反应中起作用，导致少数特定磷酸化降解区的磷酸化。这些磷酸化级联的途径由 Sic1 中周期蛋白特异性对接基序和 Cdk1 的磷酸适配器亚基 Cks1 介导的精确定向对接相互作用所塑造。我们的结果表明，Clb5-Cdk1 依赖性磷酸化产生了正反馈，这是开关样 Sic1 破坏所必需的。我们在磷酸化位点簇内的对接网络的证据揭示了 Cdk1 依赖性细胞周期转换调控的新复杂性水平，并且对通过多部位磷酸化调节细胞过程具有普遍意义。
