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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Cellular Signaling and Regulatory Systems Study Section (CSRS)
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Hamlet, Michelle R
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Cornell University
Schools of Earth Sciences/Natur
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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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