Multisite Phosphorylation and M-Phase Regulation

 描述（由申请人提供）：有丝分裂是由细胞周期蛋白B1-Cdk 1的激活和活性复合物易位到细胞核引发的。有丝分裂的全或无、不可逆的性质来自于由相互连接的正反馈和双负反馈回路组成的触发电路，其中活性细胞周期蛋白B1-Cdk 1促进细胞周期蛋白B1-Cdk 1活化，并且核细胞周期蛋白B1-Cdk 1促进细胞周期蛋白B1-Cdk 1易位。这种系统级的行为取决于反馈回路各个组成部分的动力学特性，包括它们的响应函数的形状。在上一个资助期间，我们发现Wee 1A和Cdc 25 C的多位点磷酸化使它们能够产生高度超灵敏的反应，这反过来又使有丝分裂触发的双稳态成为可能。在这里，我们建议建立在这些发现，以确定如何多位点磷酸化导致的超灵敏度调节的关键Cdk 1底物和调节Wee 1A，并检查蛋白质去磷酸化的时间顺序在有丝分裂退出。有三个具体目标：目标1。Cks蛋白在Cdk磷酸化调节中的作用。有丝分裂Cdk 1复合物由三种蛋白质组成：Cdk 1蛋白激酶、变构激活剂细胞周期蛋白B和第三种小蛋白，在S. pombe，Cks 1在S.酿酒酵母和Cks 1或2在脊椎动物中，其功能更神秘。我们最近发现，非洲爪蟾Cks 2作为一个二价适配器，独立地结合到Cdk 1和磷酸Wee 1A，并建立一个实质性的阈值到开关样响应Wee 1A Cdk 1。在这里，我们的目的是探索这种现象的普遍性，并检查磷酸化的调节CKS 2。目标2. Wee 1A失活的机制我们已经表明，Wee 1A在T53磷酸化引发蛋白质随后在T104和T150磷酸化，这些磷酸化导致Wee 1A失活。在这里，我们提出了一系列的生物物理和结构研究，旨在了解T104/T150磷酸化如何影响Wee 1A激酶结构域。目标3.有丝分裂退出的时间组织机制。有丝分裂退出是一个高度组织化的细胞生物学过程。在这里，我们建议描述有丝分裂磷蛋白的去磷酸化是如何组织的时间，并确定什么机制允许去磷酸化进行有序的方式。

 DESCRIPTION (provided by applicant): Mitosis is initiated by the activation of cyclin B1-Cdk1 and the translocation of the active complexes to the nucleus. The all-or-none, irreversible nature of mitosis arises from a bistable trigger circuit composed of interlinked positive and double-negative feedback loops where active cyclin B1-Cdk1 promotes cyclin B1-Cdk1 activation, and nuclear cyclin B1-Cdk1 promotes cyclin B1-Cdk1 translocation. This systems-level behavior depends upon the kinetic properties of the individual components of the feedback loops, including the shapes of their response functions. During the last funding period, we showed that the multisite phosphorylation of Wee1A and Cdc25C allows them to generate highly ultrasensitive response, which in turn makes the bistability of the mitotic trigger possible. Here we propose to build upon these discoveries to determine how multisite phosphorylation leads to the ultrasensitive regulation of the key Cdk1 substrate and regulator Wee1A, and to examine the temporal order of protein dephosphorylation during mitotic exit. There are three Specific Aims: Aim 1. The role of Cks proteins in the regulation of Cdk phosphorylation. Mitotic Cdk1 complexes consist of three proteins: the Cdk1 protein kinase, the allosteric activator cyclin B, and a third small protein referred to as Suc1 in S. pombe, Cks1 in S. cerevisiae, and Cks1 or 2 in vertebrates, whose function is more enigmatic. We have recently found that Xenopus Cks2 acts as a bivalent adaptor, binds independently to both Cdk1 and phospho-Wee1A, and builds a substantial threshold into the switch-like response of Wee1A to Cdk1. Here we aim to explore the generality of this phenomenon and examine the regulation of Cks2 by phosphorylation. Aim 2. The mechanism of Wee1A inactivation. We have shown that the phosphorylation of Wee1A at T53 primes the protein for subsequent phosphorylations at T104 and T150, and that these phosphorylations result in Wee1A inactivation. Here we propose a series of biophysical and structural studies aimed at understanding how T104/T150 phosphorylation affects the Wee1A kinase domain. Aim 3. Mechanisms of temporal organization in mitotic exit. Mitotic exit is a highly organized cell biological process. Here we propose to describe how the dephosphorylation of mitotic phosphoproteins is organized temporally, and determine what mechanisms allow the dephosphorylation to proceed in an orderly fashion.