Greatwall Kinase and the Mitotic Control of Phosphatase Activity

长城激酶和磷酸酶活性的有丝分裂控制

• 批准号: 8016013
• 负责人: MICHAEL L GOLDBERG
• 金额: $ 38.19万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2013-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8016013
• 关键词: Biochemical; Biochemical Pathway; Biological Assay; Cell Cycle; Cell Cycle Regulation; Cells; Cyclin B; Down-Regulation; Ensure; Event; Family; G2/M Transition; Generations; Goals; In Vitro; Interphase; Investigation; Laboratories; M Phase Arrest; Maintenance; Maturation-Promoting Factor; Mediating; Mediator of activation protein; Methods; Mitosis; Mitotic; Oocytes; Pathway interactions; Phosphoproteins; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Play; Protein Dephosphorylation; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Regulation; Role; Site; Specificity; Structure; Substrate Specificity; Testing; Xenopus; base; egg; novel; prevent; public health relevance

DESCRIPTION (provided by applicant): Mitotic entry is driven by the explosive, autoregulatory activation of the kinase MPF (M phase promoting factor; Cdk1/cyclin B), which phosphorylates S/TP sites in a variety of mitotic phosphoproteins. Our laboratory has identified a novel kinase called Greatwall that is also required for the G2/M transition, in part by influencing the autoregulatory loop that activates MPF. However, Greatwall must also have other critical roles in the cell cycle. This conclusion arises from the fact that immunodepletion of Greatwall from Xenopus M phase-arrested CSF egg extracts causes a form of M phase exit, while in contrast, MPF is largely dispensable for maintaining M phase once the CSF state has been established. Our recent results indicate that: (1) MPF phosphorylates and helps activate Greatwall during M phase, and (2) Once activated, Greatwall function leads to the inactivation of a particular form of the phosphatase PP2A associated with the regulatory subunit B55delta. In this way, Greatwall protects the phosphorylations added by MPF to the S/TP sites on MPF substrates, including components of the autoregulatory loop. In the absence of Greatwall, PP2A/B55delta would immediately remove these phosphorylations from mitotic phosphoproteins; as a result, cells or extracts depleted for Greatwall in interphase cannot enter M phase, while cells or extracts depleted for Greatwall during M phase rapidly exit this state to interphase. In the first specific aim, we propose to dissect the pathway leading from Greatwall activation to PP2A/B55delta inactivation. To achieve this goal, we will identify the relevant substrate(s) phosphorylated by Greatwall, and we will also define the biochemical changes at the end of the pathway that block function of the phosphatase. In the second specific aim, we will place the pathway from Greatwall to PP2A/B55delta in the larger context of the cell cycle transitions that allow entry into, and exit from, M phase. We will identify the critical phosphorylations that activate Greatwall during M phase, and determine whether any of these are added to Greatwall by kinases other than MPF. One goal of this line of investigation is the generation of a constitutively active Greatwall protein that can be expressed in bacterial cells and used for studies of Greatwall structure. We will next determine how the activating phosphorylations on Greatwall, as well as the phosphorylations Greatwall adds to its substrates, are removed upon M phase exit. Finally, we will examine the rates of dephosphorylation in extracts of a large panel of phosphosites. In this way, we hope to find the rules governing the substrate specificity of PP2A/B55delta phosphatase, and in doing so, we will identify the phosphorylations that most require Greatwall-mediated protection from the phosphatase so as to ensure M phase entry and maintenance. PUBLIC HEALTH RELEVANCE: We have previously shown that Greatwall kinase promotes M phase entry and maintenance in Xenopus egg extracts. The major goal of this project is to define the biochemical pathway through which the activation of Greatwall during M phase leads to the inactivation of a form of the phosphatase PP2A that would otherwise remove phosphorylations required for the mitotic state.

描述（由申请方提供）：有丝分裂进入由激酶MPF（M期促进因子; Cdk 1/细胞周期蛋白B）的爆发性自动调节激活驱动，该激活可磷酸化多种有丝分裂磷蛋白中的S/TP位点。我们的实验室已经发现了一种名为Greatwall的新型激酶，它也是G2/M转变所需的，部分原因是影响激活MPF的自动调节回路。然而，长城在细胞周期中还必须发挥其他关键作用。这一结论源于以下事实：来自非洲爪蟾M期停滞的CSF卵提取物的Greatwall的免疫耗竭导致M期退出的形式，而相反，一旦CSF状态已经建立，MPF在很大程度上是为了维持M期。我们最近的研究结果表明：（1）MPF磷酸化并帮助激活M期的Greatwall，（2）一旦激活，Greatwall功能导致与调节亚基B55 δ相关的磷酸酶PP2A的特定形式失活。通过这种方式，Greatwall保护MPF添加到MPF底物上的S/TP位点的磷酸化，包括自动调节环的组分。在没有Greatwall的情况下，PP2A/B55 delta将立即从有丝分裂磷蛋白中去除这些磷酸化;因此，在间期中耗尽Greatwall的细胞或提取物不能进入M期，而在M期期间耗尽Greatwall的细胞或提取物迅速离开这种状态进入间期。在第一个具体的目标，我们建议解剖的途径，导致从长城激活PP2A/B55 δ失活。为了实现这一目标，我们将确定Greatwall磷酸化的相关底物，我们还将确定阻断磷酸酶功能的途径末端的生化变化。在第二个具体目标中，我们将把从Greatwall到PP2A/B55 delta的途径置于允许进入和退出M期的细胞周期转换的更大背景中。我们将确定在M期激活Greatwall的关键磷酸化，并确定这些磷酸化是否由MPF以外的激酶添加到Greatwall。这一系列研究的一个目标是产生组成型活性的Greatwall蛋白，其可以在细菌细胞中表达并用于Greatwall结构的研究。接下来我们将确定Greatwall上的激活磷酸化以及Greatwall添加到其底物上的磷酸化如何在M相退出时被去除。最后，我们将研究一个大的磷酸化位点的提取物的脱磷酸化率。通过这种方式，我们希望找到控制PP2A/B55 δ磷酸酶底物特异性的规则，并且在这样做的过程中，我们将确定最需要Greatwall介导的磷酸酶保护的磷酸化，以确保M期进入和维持。 公共卫生相关性：我们以前已经表明，长城激酶促进非洲爪蟾卵提取物中的M期进入和维持。本项目的主要目标是确定M期期间Greatwall激活导致磷酸酶PP2A失活的生化途径，否则磷酸酶PP2A将去除有丝分裂状态所需的磷酸化。