All eukaryotic cells must exit mitosis to divide. Exit from mitosis is achieved by inactivation of the same mitotic cyclin/cyclin-dependent kinase (CDK) enzyme that drives cells into mitosis. Mitotic cyclin/CDK is down-regulated at the end of mitosis by two different mechanisms in budding yeast: induction of the CDK inhibitor Sic1 by Swi5 and the turnover of mitotic cyclin promoted by Hctl (aka Cdhl) and the anaphasepromoting complex-cyclosome (APC) ubiquitin ligase. Early in mitosis, mitotic cyclin/CDK keeps its antagonists at bay by attaching inhibitory phosphates on Swi5 and Hctl. During anaphase-telophase, the protein phosphatase Cdc14 enables the exit from mitosis by reversing these inhibitory phosphorylations. A watershed event in the exit from mitosis is the activation of Cdc14, which is achieved by its release from the nucleolar anchor protein Net1. Thus, a key to understanding how cells exit mitosis is to understand how Cdc14 is mobilized from the nucleolus at the end of mitosis. This mobilization involves two signaling pathways: the Cdc Fourteen Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN). The FEAR network instigates the transient release of Cdc14 from the nucleolus during early anaphase, whereas the MEN somehow transforms this brief nuclear furlough into a more sustained dispersal of Cdc14 throughout the cell that is able to drive exit from mitosis. The goal of the 3 specific aims described in this application is to understand how the FEAR network and the MEN bring about the activation of Cdc14. In the first aim we will seek to determine how the FEAR network and phosphorylation of Net1 by mitotic cyclin/CDK collaborate to bring about FEAR. In the second aim we will address the hypothesis that a key function of the MEN is to alter the nucleocytoplasmic distribution of Cdc14 released from the nucleolus through the action of the FEAR network. In the third aim, we will seek proteins that serve as substrates of the MEN component Dbf2 to understand how this protein kinase brings about sustained dispersal of Cdc14 throughout the cell. Given the conservation of the FEAR network and MEN proteins, the work described here has excellent potential to shed insight into the growth and division of normal and cancerous human cells, and thereby highlight new candidate targets for anti-cancer drugs. Our work may also provide a useful paradigm for understanding how other pathways that employ reversible nucleolar sequestra - such as the ARF-Mdm2- p53 circuit - are regulated.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM059940-08S1
Application #
7886112
Study Section
Cell Development and Function Integrated Review Group (CDF)
Program Officer
Zatz, Marion M
Project Start
2009-07-20
Project End
2010-03-31
Budget Start
2009-07-20
Budget End
2010-03-31
Support Year
8
Fiscal Year
2009
Total Cost
$90,165
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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Azzam, Ramzi; Chen, Susan L; Shou, Wenying et al. (2004) Phosphorylation by cyclin B-Cdk underlies release of mitotic exit activator Cdc14 from the nucleolus. Science 305:516-9
Shou, W; Sakamoto, K M; Keener, J et al. (2001) Net1 stimulates RNA polymerase I transcription and regulates nucleolar structure independently of controlling mitotic exit. Mol Cell 8:45-55
Mah, A S; Jang, J; Deshaies, R J (2001) Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex. Proc Natl Acad Sci U S A 98:7325-30