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. ? ?
|Mohl, Dane A; Huddleston, Michael J; Collingwood, Therese S et al. (2009) Dbf2-Mob1 drives relocalization of protein phosphatase Cdc14 to the cytoplasm during exit from mitosis. J Cell Biol 184:527-39|
|Mah, Angie S; Elia, Andrew E H; Devgan, Geeta et al. (2005) Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening. BMC Biochem 6:22|
|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|