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.
|Blake-Hodek, Kristina A; Williams, Byron C; Zhao, Yong et al. (2012) Determinants for activation of the atypical AGC kinase Greatwall during M phase entry. Mol Cell Biol 32:1337-53|
|Yamamoto, Tomomi M; Blake-Hodek, Kristina; Williams, Byron C et al. (2011) Regulation of Greatwall kinase during Xenopus oocyte maturation. Mol Biol Cell 22:2157-64|
|Peng, Aimin; Yamamoto, Tomomi M; Goldberg, Michael L et al. (2010) A novel role for greatwall kinase in recovery from DNA damage. Cell Cycle 9:4364-9|
|Wainman, Alan; Creque, Jacklyn; Williams, Byron et al. (2009) Roles of the Drosophila NudE protein in kinetochore function and centrosome migration. J Cell Sci 122:1747-58|
|Castilho, Priscila V; Williams, Byron C; Mochida, Satoru et al. (2009) The M phase kinase Greatwall (Gwl) promotes inactivation of PP2A/B55delta, a phosphatase directed against CDK phosphosites. Mol Biol Cell 20:4777-89|
|Zhao, Yong; Haccard, Olivier; Wang, Ruoning et al. (2008) Roles of Greatwall kinase in the regulation of cdc25 phosphatase. Mol Biol Cell 19:1317-27|
|Bonaccorsi, Silvia; Mottier, Violaine; Giansanti, Maria Grazia et al. (2007) The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division. Development 134:2183-93|
|Bolkan, Bonnie J; Booker, Ronald; Goldberg, Michael L et al. (2007) Developmental and cell cycle progression defects in Drosophila hybrid males. Genetics 177:2233-41|
|Lopes, Carla S; Sampaio, Paula; Williams, Byron et al. (2005) The Drosophila Bub3 protein is required for the mitotic checkpoint and for normal accumulation of cyclins during G2 and early stages of mitosis. J Cell Sci 118:187-98|
|Yu, Jiangtao; Fleming, Shawna L; Williams, Byron et al. (2004) Greatwall kinase: a nuclear protein required for proper chromosome condensation and mitotic progression in Drosophila. J Cell Biol 164:487-92|
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