Greatwall Kinase and the Mitotic Control of Phosphatase Activity

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）的爆发性激活驱动，其在数千个磷酸化位点磷酸化数百种靶蛋白。当细胞随后退出有丝分裂时，这些磷酸化中的许多被与调节亚基B55（PP 2A/B55）相关的磷酸酶PP 2A去除。为了保护MPF介导的磷酸化免于过早去磷酸化，PP 2A/B55在M期期间通过涉及称为Greatwall（Gwl）的激酶及其效应物Endosulfine（Endos）的途径关闭。MPF磷酸化并因此激活Gwl; Gwl继而磷酸化并因此激活Endos，并且磷酸化的Endos（pEndos）结合并因此失活PP 2A/B55。拟议的研究将解决这个系统如何在M相退出时逆转。前两个特定目的将扩展初步结果，表明：（1）去除pEndos上Gwl驱动的磷酸化的主要磷酸酶令人惊讶地是PP 2A/B55本身;和（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PP 2A/B55的抑制剂和底物。在第一个具体目标中描述的实验将剖析不公平竞争机制的分子基础，研究pEndos作用可能受到Greatwall磷酸化以外的事件影响的方式，并在体外用纯化组分和在细胞的更复杂环境中数学建模M期退出的这一方面。在第二个具体目标中，我们将探讨不公平竞争抑制反映了一个古老的调节模块的可能性，通过该模块，AGC家族的激酶（如长城）通过小的中间分子（如Endos）的磷酸化调节PPP家族的磷酸酶（如PP 2A/B55）。我们将测试磷酸酶PP 1的已知磷蛋白调节剂是否通过不公平竞争机制进行操作，并且我们将使用基于我们对pEndos和PP 2A/B55的见解的策略来寻找其他PPP磷酸酶的新型调节剂。在第三个具体目标中，我们将鉴定当细胞退出M期时使Gwl激酶失活的磷酸酶。初步结果表明，这些Gwl失活磷酸酶中的至少一种对药物冈田酸不敏感，因此不可能是PP 2A/B55。我们将询问这种冈田酸不敏感的磷酸酶是否对应于已知参与M期退出的酶，或者Gwl失活酶是否定义了新的磷酸酶活性。本提案中描述的工作有可能加深我们对关键细胞周期转换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.