Mitotic Exit Regulation through Distinct Domains within the Protein Kinase Cdc15

Exit from mitosis is the final cell cycle transition that leads to the production of two genetically identical daughter cells. Proper execution of this transition is essential for each of the two daughter cells to receive a complete complement of the genome and, thus, to maintain ploidy. In all eukaryotes examined to date, exit from mitosis is brought about by the inactivation of mitotic cyclin-dependent kinase (mitotic CDK) activity (reviewed in reference 26). In budding yeast (Saccharomyces cerevisiae), the protein phosphatase Cdc14 induces the inactivation of mitotic CDKs by reversing CDK-dependent phosphorylation on proteins important for promoting mitotic CDK inactivation (14, 15, 41; reviewed in reference 2). Cdc14 is regulated by an inhibitor, Cfi1/Net1. Cfi1/Net1 inhibits and sequesters Cdc14 in the nucleolus throughout most of the cell cycle (34, 42). Only during anaphase and telophase is Cdc14 released from its inhibitor (34, 36, 42). To date, two pathways have been identified that control Cdc14 localization: the FEAR network (Cdc 14 early anaphase release) (30, 35, 37, 44) and the mitotic exit network (MEN) (reviewed in references 2, 17, and 24). The FEAR network appears to be activated at the metaphase-anaphase transition and promotes Cdc14 release from the nucleolus during early anaphase. MEN is thought to be activated when the nucleus migrates into the daughter cell during anaphase (3, 29) and is essential for promoting and sustaining release of Cdc14 from the nucleolus during late stages of anaphase. Temperature-sensitive mutations in MEN components cause cells to arrest in telophase with Cdc14 sequestered in the nucleolus and high levels of mitotic CDK activity (15, 32, 34, 38, 41). MEN is a signal transduction pathway comprised of a GTPase, Tem1; the putative guanine nucleotide exchange factor Lte1; the two-component GTPase-activating protein complex Bub2-Bfa1; the protein kinases Cdc15, Dbf2, and Cdc5; a Dbf2-associated factor, Mob1; and a scaffold protein, Nud1 (reviewed in reference 2). Localization of MEN components to spindle pole bodies (SPBs) and in vitro assays for Dbf2 kinase activation have been used to order MEN components into a pathway (3, 4, 10, 19, 22, 23, 40, 43, 45). These analyses revealed that Tem1 binds to and functions upstream of Cdc15, which activates Dbf2 in a Mob1-dependent manner. The polo kinase Cdc5 regulates the activity of several MEN components (13, 19, 40). Most MEN components have been shown to localize to SPBs (reviewed in reference 2). Tem1, Bub2, and Bfa1 predominantly localize to the SPB that migrates into the daughter cell during anaphase (3, 6, 9, 29). Cdc5 localizes to both SPBs during metaphase and anaphase (33), whereas Cdc15 and Dbf2 localize to both SPBs only during anaphase (4, 10, 25, 40, 43). Localization of Dbf2 to SPBs coincides with activation of Dbf2 kinase activity, suggesting that SPB association of Dbf2 is a prerequisite for Dbf2 activation (8, 40). Localization of most MEN components to SPBs requires the SPB component Nud1. Cells carrying a temperature-sensitive allele in NUD1 arrest in telophase with Tem1, Cdc15, and Dbf2 mislocalized, suggesting that SPB localization of MEN components is important for promoting mitotic exit (3, 11, 40). Three cellular events have been identified that control MEN activation: spindle position, mitotic spindle damage, and DNA damage. All are thought to regulate Tem1 through Lte1 and Bub2-Bfa1 (reviewed in references 2, 12, and 24). However, although Bub2-Bfa1 and Lte1 are important for controlling MEN activity, several lines of evidence suggest that MEN is regulated at additional levels. Neither LTE1 nor BUB2 nor BFA1 is essential for viability. Furthermore, in an unperturbed cell cycle, neither deletion of BUB2 or BFA1 nor overexpression of LTE1 causes a cell cycle defect or a premature release of Cdc14 from the nucleolus (3). Thus, other components of MEN are likely subject to regulation. One MEN component known to be a target of regulation is the protein kinase Cdc15. Cdc14 released from the nucleolus by the FEAR network during early anaphase causes dephosphorylation of Cdc15, allowing it to become a more potent activator of mitotic exit (16, 25, 35, 43). In order to identify if additional modes of Cdc15 regulation exist, we have determined how different domains of Cdc15 function to regulate mitotic exit. We mapped the region of Cdc15 required for association with SPBs to a domain adjacent to the kinase domain. Our analyses also show that Cdc15 self-associates and that this self-association is mediated through a region that overlaps with the SPB localization domain of Cdc15. This domain is essential for viability, suggesting that SPB association and/or self-association is required for CDC15 function. Finally, we find that overexpression of CDC15 lacking the C-terminal 224 amino acids (aa), but not overexpression of full-length CDC15, causes hyperactivation of MEN as judged by elevated levels of Dbf2 kinase activity and premature release of Cdc14 from the nucleolus. Our results suggest the presence of at least three functional domains within Cdc15 aside from the kinase domain: an SPB association domain, a self-association domain, and a domain that inhibits MEN signaling.