Entry into mitosis catalyzed by the Cdc2/Cyclin B kinase, which phosphorylates multiple proteins to induce the dramatic cellular rearrangements characteristic of mitosis. When DNA replication is inhibited, a cell cycle checkpoint pathway prevents mitosis through inhibitory phosphorylation of Cdc2 at Y15 and T14. This inhibition involves sustained activity of the Cdc2 Y15-directed kinase, Wee1, as well as suppression of the Cdc2 Y15 phosphatase, Cdc25. We have discovered that a novel G2/M regulator, Hsl7, promotes intranuclear Wee1 degradation, and that the DNA replication checkpoint disrupts Hsl7-Wee1 interactions. In parallel, Cdc25 is inhibited through binding to 14-3-3 protein. Removal of 14-3-3 from Cdc25 is required for its mitotic activation, and we have found that dissociation of the 14-3-3-Cdc25 complex requires phosphorylation of both Cdc25 T138 (which reduces the affinity of 14-3-3 for Cdc25) and intermediate filament proteins (which then bind 14-3-3, thereby serving as an abundant 14-3-3 """"""""sink""""""""). Additionally, we have discovered that a previously described apoptotic inhibitor, Aven, can also regulate mitotic entry and may be important for DNA-responsive checkpoint operation. The objective of this proposal to elucidate both the Wee1 and Cdc25-modulatory arms of DNA-responsive checkpoint pathways with the long term goal of fully understanding the control of M phase entry. Towards this end, we propose to I) Delineate the role of Hsl7 in checkpoint-mediated Wee1 regulation II) Elucidate the mechanism(s) which regulate 14-3-3 release from Cdc25 and III) Determine how Aven regulates mitotic entry. ? ? ?

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM067225-06
Application #
7482372
Study Section
Cellular Signaling and Dynamics Study Section (CSD)
Program Officer
Zatz, Marion M
Project Start
2003-09-01
Project End
2011-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
6
Fiscal Year
2008
Total Cost
$273,000
Indirect Cost
Name
Duke University
Department
Pharmacology
Type
Schools of Medicine
DUNS #
044387793
City
Durham
State
NC
Country
United States
Zip Code
27705
Tang, Wanli; Wu, Judy Qiju; Chen, Chen et al. (2010) Emi2-mediated inhibition of E2-substrate ubiquitin transfer by the anaphase-promoting complex/cyclosome through a D-box-independent mechanism. Mol Biol Cell 21:2589-97
Wu, Judy Qiju; Guo, Jessie Yanxiang; Tang, Wanli et al. (2009) PP1-mediated dephosphorylation of phosphoproteins at mitotic exit is controlled by inhibitor-1 and PP1 phosphorylation. Nat Cell Biol 11:644-51
Tang, Wanli; Wu, Judy Qiju; Guo, Yanxiang et al. (2008) Cdc2 and Mos regulate Emi2 stability to promote the meiosis I-meiosis II transition. Mol Biol Cell 19:3536-43
Guo, Jessie Yanxiang; Yamada, Ayumi; Kajino, Taisuke et al. (2008) Aven-dependent activation of ATM following DNA damage. Curr Biol 18:933-42
Wu, Qiju; Guo, Yanxiang; Yamada, Ayumi et al. (2007) A role for Cdc2- and PP2A-mediated regulation of Emi2 in the maintenance of CSF arrest. Curr Biol 17:213-24
Wu, Judy Qiju; Hansen, David V; Guo, Yanxiang et al. (2007) Control of Emi2 activity and stability through Mos-mediated recruitment of PP2A. Proc Natl Acad Sci U S A 104:16564-9
Zhu, Wenge; Ukomadu, Chinweike; Jha, Sudhakar et al. (2007) Mcm10 and And-1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. Genes Dev 21:2288-99
Margolis, Seth S; Perry, Jennifer A; Forester, Craig M et al. (2006) Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. Cell 127:759-73
Margolis, Seth S; Perry, Jennifer A; Weitzel, Douglas H et al. (2006) A role for PP1 in the Cdc2/Cyclin B-mediated positive feedback activation of Cdc25. Mol Biol Cell 17:1779-89
Casaletto, Jessica B; Nutt, Leta K; Wu, Qiju et al. (2005) Inhibition of the anaphase-promoting complex by the Xnf7 ubiquitin ligase. J Cell Biol 169:61-71

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