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(由申请人提供)：有丝分裂的进入是由激酶MPF(M期促进因子；CDK1/Cyclin B)的爆炸性、自动调节激活驱动的，它使各种有丝分裂磷酸蛋白中的S/TP位点磷酸化。我们的实验室已经发现了一种名为长城的新的激酶，它也是G2/M转换所必需的，部分是通过影响激活MPF的自动调节环路来实现的。然而，长城还必须在细胞周期中扮演其他关键角色。这一结论源于以下事实：来自非洲爪哇M相阻滞脑脊液卵提取液的长城免疫耗竭导致一种形式的M期退出，而相反，一旦建立了脑脊液状态，MPF对于维持M期是必不可少的。我们最近的结果表明：(1)MPF在M期磷酸化并帮助激活长城，以及(2)一旦激活，长城功能就会导致与调节亚基B55Delta相关的一种特定形式的磷酸酶PP2A失活。通过这种方式，长城保护了MPF添加到MPF底物上的S/TP位点的磷酸化，包括自动调节环路的组件。在没有长城的情况下，PP2A/B55Delta会立即从有丝分裂磷酸蛋白中去除这些磷酸化；结果，在间期耗尽长城的细胞或提取物不能进入M期，而在M期耗尽长城的细胞或提取物迅速退出这种状态进入间期。在第一个具体目标中，我们建议剖析从长城激活到PP2A/B55Delta失活的途径。为了实现这一目标，我们将确定长城蛋白磷酸化的相关底物(S)，并定义阻断磷酸酶功能的途径末端的生化变化。在第二个特定目标中，我们将把从长城到PP2A/B55Delta的途径放在更大的细胞周期转变的背景下，允许进入和退出M期。我们将确定在M期激活长城的关键磷酸化，并确定是否有任何这些磷酸化是通过MPF以外的激酶添加到长城的。这项研究的目标之一是产生一种具有结构性活性的长城蛋白，该蛋白可以在细菌细胞中表达，并用于长城结构的研究。接下来，我们将确定在M阶段退出时，如何去除长城上激活的磷酸化，以及长城添加到其衬底上的磷酸化。最后，我们将检查一大批亚磷酸盐提取物中的去磷酸化速率。通过这种方式，我们希望找到控制PP2A/B55Delta磷酸酶底物专一性的规则，并在这样做的过程中，我们将识别最需要长城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：我们之前已经证明，长城激活剂能促进非洲爪哇卵提取物进入M期并维持M期。这个项目的主要目标是确定生化途径，通过该途径，长城在M期的激活导致一种形式的磷酸酶PP2A失活，否则将消除有丝分裂状态所需的磷酸化。