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.
Entry into M phase of mitosis and meiosis requires phosphorylations of sites on many proteins by kinases including the major driver Cdk1/Cyclin B. To ensure that these phosphorylations are not prematurely removed, a system involving a second kinase called Great wall and its substrate Endosulfine is turned on during M phase to inactivate the phosphatase (PP2A/B55) that targets these phosphosites. The major goal of this project is to define the biochemical events that reverse this system later during M phase exit, allowing PP2A/B55 to remove these many phosphorylations so that cells can reset themselves for the round of division.
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