Greatwall Kinase and the Mitotic Control of Phosphatase Activity

描述(由申请人提供)：有丝分裂进入是由激酶MPF(M期促进因子；CDK1/Cyclin B)的爆炸性激活推动的，它在数千个磷酸位点上磷酸化数百个目标蛋白。当细胞随后退出有丝分裂时，许多这些磷酸化被与调节亚基B55(PP2A/B55)相关的磷酸酶PP2A去除。为了保护MPF介导的磷酸化不被过早去磷酸化，PP2A/B55在M期被称为长城(Gw1)的激酶(Gw1)及其效应因子Endosulfinin(Endos)的通路关闭。MPF磷酸化从而激活Gw1；Gw1进而磷酸化从而激活Endos，而磷酸化Endos(PEndos)与PP2A/B55结合并因此失活。拟议的研究将解决这一系统如何在M阶段退出时发生逆转。前两个特定的目标将在初步结果的基础上展开，这些结果表明：(1)消除GwL驱动的pEndos上磷酸化的主要磷酸酶出人意料地是PP2A/B55本身；(2)我们称之为不公平竞争抑制的简单机制可以解释pEndos如何同时作为PP2A/B55的抑制剂和底物。第一个特定目的中描述的实验将剖析不公平竞争机制的分子基础，调查pEndos的作用可能受到长城磷酸化以外的事件影响的方式，并在体外用纯化的成分和在更复杂的细胞环境中对M期退出的这一方面进行数学建模。在第二个特定目标中，我们将探索不正当竞争抑制是否反映了一个古老的调节模块，即属于AGC家族(如长城)的激酶通过小中间分子(如Endo)的磷酸化来调节属于PPP家族的磷酸酶(如PP2A/B55)。我们将测试已知的磷酸酶PP1的磷酸蛋白调节因子是否通过不公平竞争机制起作用，并将根据我们从pEndos和PP2A/B55获得的见解寻找其他PPP磷酸酶的新调节因子。在第三个特定目标中，我们将鉴定当细胞退出M期时使Gwl激酶失活的磷酸酶(S)。初步结果表明，这些GWL失活的磷酸酶中至少有一个对药物冈田酸不敏感，因此不可能是PP2A/B55。我们将询问这种冈田酸不敏感的磷酸酶是否对应于一种已知的参与M期退出的酶，或者相反，Gwl失活酶是否定义了一种新的磷酸酶活性。这项建议中描述的工作有可能加深我们对关键细胞周期转变和磷酸酶生物学的理解。

DESCRIPTION (provided by applicant): Mitotic entry is driven by the explosive activation of the kinase MPF (M phase promoting factor; Cdk1/cyclin B), which phosphorylates hundreds of target proteins at thousands of phosphosites. When cells subsequently exit mitosis, many of these phosphorylations are removed by the phosphatase PP2A associated with the regulatory subunit B55 (PP2A/B55). In order to protect the MPF-mediated phosphorylations from premature dephosphorylation, PP2A/B55 is shut off during M phase by a pathway involving a kinase called Greatwall (Gwl) and its effector Endosulfine (Endos). MPF phosphorylates and thus activates Gwl; Gwl in turn phosphorylates and thus activates Endos, and phosphorylated Endos (pEndos) binds to and thus inactivates PP2A/B55. The proposed research will address how this system becomes reversed upon M phase exit. The first two Specific Aims will expand upon preliminary results indicating that: (1) the major phosphatase that removes the Gwl-driven phosphorylation on pEndos is surprisingly PP2A/B55 itself; and (2) a simple mechanism we call inhibition by unfair competition can explain how pEndos can simultaneously act as an inhibitor and a substrate of PP2A/B55. The experiments described in the first Specific Aim will dissect the molecular basis for the unfair competition mechanism, investigate the means by which pEndos action may be influenced by events other than Greatwall phosphorylation, and model this aspect of M phase exit both in vitro with purified components and mathematically in the more complex environment of the cell. In the second Specific Aim, we will explore the possibility that inhibition by unfair competition reflects an ancient regulatory module through which kinases belonging to the AGC family (like Greatwall) regulate phosphatases belonging to the PPP family (like PP2A/B55) via the phosphorylation of small intermediary molecules (such as Endos). We will test whether known phosphoprotein regulators of the phosphatase PP1 operate through the unfair competition mechanism, and we will search for novel regulators of other PPP phosphatases using a strategy based on our insights from pEndos and PP2A/B55. In the third Specific Aim, we will identify the phosphatase(s) that inactive Gwl kinase when cells exit M phase. Preliminary results indicate that at least one of these Gwl- inactivating phosphatases is insensitive to the drug okadaic acid, and thus cannot be PP2A/B55. We will ask if this okadaic acid-insensitive phosphatase corresponds to an enzyme already known to be involved in M phase exit, or instead whether the Gwl-inactivating enzyme defines a novel phosphatase activity. The work described in this proposal has the potential to deepen our understanding of key cell cycle transitions and the biology of phosphatases.