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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM048430-19
Application #
8211025
Study Section
Cellular Signaling and Regulatory Systems Study Section (CSRS)
Program Officer
Hamlet, Michelle R
Project Start
1992-09-30
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2012-12-31
Support Year
19
Fiscal Year
2012
Total Cost
$381,925
Indirect Cost
$133,922
Name
Cornell University
Department
Biochemistry
Type
Schools of Earth Sciences/Natur
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Défachelles, Lénaïg; Raich, Natacha; Terracol, Régine et al. (2015) RZZ and Mad1 dynamics in Drosophila mitosis. Chromosome Res 23:333-42
Williams, Byron C; Filter, Joshua J; Blake-Hodek, Kristina A et al. (2014) Greatwall-phosphorylated Endosulfine is both an inhibitor and a substrate of PP2A-B55 heterotrimers. Elife 3:e01695
Wainman, Alan; Giansanti, Maria Grazia; Goldberg, Michael L et al. (2012) The Drosophila RZZ complex - roles in membrane trafficking and cytokinesis. J Cell Sci 125:4014-25
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
Kim, Min-Young; Bucciarelli, Elisabetta; Morton, Diane G et al. (2012) Bypassing the Greatwall-Endosulfine pathway: plasticity of a pivotal cell-cycle regulatory module in Drosophila melanogaster and Caenorhabditis elegans. Genetics 191:1181-97
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
Goldberg, Michael L (2010) Greatwall kinase protects mitotic phosphosites from barbarian phosphatases. Proc Natl Acad Sci U S A 107:12409-10
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

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