MOLECULAR MECHANISMS UNDERLYING CDC14 ACTIVATION DURING MITOTIC EXIT IN SACCHAROMYCES CEREVISIAE

In budding yeast, progression through anaphase and exit from mitosis are controlled by the conserved protein phosphatase Cdc14. The activity of Cdc14 is regulated in space and time by changes in its subcellular localization. For most of the cell cycle up to metaphase, the phosphatase is sequestered in the nucleolus, by binding to a competitive inhibitor called Cfi1 (also known as Net1) (Shou et al., 1999; Straight et al., 1999; Visintin et al., 1999). During anaphase, Cdc14 is released from its inhibitor by the sequential activation of two signaling cascades, the Cdc Fourteen Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN). Once released Cdc14 spreads throughout the nucleus and the cytoplasm, where it reaches its targets and promotes progression through and exit from mitosis (Pereira et al., 2002; Shou et al., 1999; Stegmeier et al., 2002; Visintin et al., 1999; Yoshida et al., 2002). Several in vivo and in vitro observations suggest that phosphorylation of Cdc14 and/or Cfi1 is responsible for the dissociation of Cdc14 from its inhibitor. Three kinases have been implicated in the process: the polo-like kinase Cdc5, the Clb2-Cdk complex and the MEN kinase Dbf2 (Azzam et al., 2004; Geymonat et al., 2003; Hu and Elledge, 2002; Hu et al., 2001; Mohl et al., 2009; Pereira et al., 2002; Queralt et al., 2006; Stegmeier et al., 2002; Visintin et al., 2003; Yoshida and Toh-e, 2002). The aim of my project was to assess the contribution of the above-mentioned kinases and to identify the molecular mechanisms by which these kinases mediate the release of Cdc14 from its inhibitor. By modulating the kinases of interest alone or in mutual combination we found that Cdc14 is released from the nucleolus by the combined activity of two kinases, Cdc5 always and either Clb-Cdks or Dbf2. Once active, Cdc14 triggers a negative feedback loop that, in the presence of stable levels of mitotic cyclins, generates periodic cycles of Cdc14 release and sequestration. Similar phenotypes have been described for yeast bud formation and centrosome duplication. A common theme emerges where events that must happen only once per cycle, although intrinsically capable of oscillations, are limited to one occurrence by their coupling with the cyclin-Cdk cell cycle engine.