Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37212, USA.
Proc Natl Acad Sci U S A. 2012 Jun 19;109(25):9899-904. doi: 10.1073/pnas.1201366109. Epub 2012 Jun 4.
Cyclin-dependent kinase 1 (Cdk1) kinase dephosphorylation and activation by Cdc25 phosphatase are essential for mitotic entry. Activated Cdk1 phosphorylates Cdc25 and other substrates, further activating Cdc25 to form a positive feedback loop that drives the abrupt G2/mitosis switch. Conversely, mitotic exit requires Cdk1 inactivation and reversal of Cdk1 substrate phosphorylation. This dephosphorylation is mediated, in part, by Clp1/Cdc14, a Cdk1-antagonizing phosphatase, which reverses Cdk1 phosphorylation of itself, Cdc25, and other Cdk1 substrates. Thus, Cdc25 phosphoregulation is essential for proper G2-M transition, and its contributions to cell cycle control have been modeled based on studies using Xenopus and human cell extracts. Because cell extract systems only approximate in vivo conditions where proteins interact within dynamic cellular environments, here, we use Schizosaccharomyces pombe to characterize, both experimentally and mathematically, the in vivo contributions of Cdk1-mediated phosphorylation of Cdc25 to the mitotic transition. Through comprehensive mapping of Cdk1 phosphosites on Cdc25 and characterization of phosphomutants, we show that Cdc25 hyperphosphorylation by Cdk1 governs Cdc25 catalytic activation, the precision of mitotic entry, and unvarying cell length but not Cdc25 localization or abundance. We propose a mathematical model that explains Cdc25 regulation by Cdk1 through a distributive and disordered phosphorylation mechanism that ultrasensitively activates Cdc25. We also show that Clp1/Cdc14 dephosphorylation of Cdk1 sites on Cdc25 controls the proper timing of cell division, a mechanism that is likely due to the double negative feedback loop between Clp1/Cdc14 and Cdc25 that controls the abruptness of the mitotic exit switch.
周期蛋白依赖性激酶 1（Cdk1）激酶的去磷酸化和 Cdc25 磷酸酶的激活对于有丝分裂进入是必不可少的。激活的 Cdk1 磷酸化 Cdc25 和其他底物，进一步激活 Cdc25 形成正反馈环，驱动 G2/有丝分裂转换的突然发生。相反，有丝分裂退出需要 Cdk1 失活和 Cdk1 底物磷酸化的逆转。这种去磷酸化部分由 Clp1/Cdc14 介导，Clp1/Cdc14 是一种与 Cdk1 拮抗的磷酸酶，它逆转 Cdk1 对自身、Cdc25 和其他 Cdk1 底物的磷酸化。因此，Cdc25 的磷酸化调节对于正确的 G2-M 转换至关重要，其对细胞周期控制的贡献已基于使用非洲爪蟾和人细胞提取物的研究进行了建模。由于细胞提取物系统仅近似于体内条件，其中蛋白质在动态细胞环境中相互作用，因此，我们使用酿酒酵母来描述 Cdk1 介导的 Cdc25 磷酸化对有丝分裂转换的体内贡献，无论是通过实验还是数学方法。通过对 Cdc25 上 Cdk1 磷酸化位点的全面映射和磷酸突变体的表征，我们表明 Cdk1 对 Cdc25 的过度磷酸化控制了 Cdc25 的催化激活、有丝分裂进入的精度和不变的细胞长度，但不控制 Cdc25 的定位或丰度。我们提出了一个数学模型，该模型通过一种分布式和无序的磷酸化机制来解释 Cdk1 通过 Cdk1 对 Cdc25 的调节，该机制超敏地激活了 Cdc25。我们还表明，Cdc25 上 Cdk1 位点的 Clp1/Cdc14 去磷酸化控制了细胞分裂的适当时间，这种机制可能是由于 Clp1/Cdc14 和 Cdc25 之间的双负反馈环控制了有丝分裂退出开关的突然性。
