Phosphatase 2A Negatively Regulates Mitotic Exit inSaccharomyces cerevisiae

Abstract

In budding yeast Saccharomyces cerevisiae, Cdc5 kinase is a component of mitotic exit network (MEN), which inactivates cyclin-dependent kinase (CDK) after chromosome segregation. cdc5-1 mutants arrest at telophase at the nonpermissive temperature due to the failure of CDK inactivation. To identify more negative regulators of MEN, we carried out a genetic screen for genes that are toxic to cdc5-1 mutants when overexpressed. Genes that encode the B-regulatory subunit (Cdc55) and the three catalytic subunits (Pph21, Pph22, and Pph3) of phosphatase 2A (PP2A) were isolated. In addition to cdc5-1, overexpression of CDC55, PPH21, or PPH22 is also toxic to other temperature-sensitive mutants that display defects in mitotic exit. Consistently, deletion of CDC55 partially suppresses the temperature sensitivity of these mutants. Moreover, in the presence of spindle damage, PP2A mutants display nuclear localized Cdc14, the key player in MEN pathway, indicative of MEN activation. All the evidence suggests the negative role of PP2A in mitotic exit. Finally, our genetic and biochemical data suggest that PP2A regulates the phosphorylation of Tem1, which acts at the very top of MEN pathway. INTRODUCTIONThe key driving force for cell division in all eukaryotic cells is the conserved cyclin-dependent kinase (CDK). CDK activity fluctuates during the cell cycle, peaking at the S and M phases and dropping at the G1 phase. Although the high CDK activity is required for DNA duplication and chromosome segregation, the low CDK activity at late M and G1 phase is essential for the loading of DNA replication complexes onto replication origins (Noton and Diffley, 2000;Lengronne and Schwob, 2002). Thus, cells have developed a signal transduction pathway, named the mitotic exit network (MEN), to inactivate CDK after mitosis (Morgan, 1999). The components of the MEN pathway include protein kinases Cdc5, Cdc15, Dbf2, a GTPase Tem1, a phosphatase Cdc14, and a Dbf2 binding protein Mob1 Luca and Winey, 1998;Lee et al., 2001a). Phosphatase Cdc14, the key player in the MEN pathway, dephosphorylates Cdh1, an activator for APC (anaphase promoting complex), and subsequently activates the degradation of a Btype cyclin, Clb2. Cdc14 also enhances the stability of Sic1 protein, which acts as a CDK inhibitor (Visintin et al., 1998). During most of the cell cycle, Cdc14 is localized in the nucleolus, but its translocation out of the nucleolus in anaphase and telophase allows it to encounter its substrates, such as Cdh1 and Sic1. All the components in the MEN pathway are required for the release of Cdc14 from the nucleolus, suggesting that Cdc14 acts downstream of MEN pathway (Shou et al., 1999;Visintin et al., 1999).The regulation of MEN activity is achieved through the multilayer control of Tem1, a small GTPase that localizes at the spindle pole body (SPB) and acts on the very top of the MEN pathway. Tem1's activator, the GTPase exchange factor (GEF) Lte1, exhibits daughter-cell specific localization. Thus, SPB-localized Tem1 is activated after it encounters...

Characterization of ypa1 and ypa2, the Schizosaccharomyces pombe Orthologs of the Peptidyl Proyl Isomerases That Activate PP2A, Reveals a Role for Ypa2p in the Regulation of Cytokinesis

“…In budding yeast, PP2A has also been implicated in regulating mitotic exit and the spindle orientation checkpoint (Queralt et al 2006;Wang and Ng 2006;Chan and Amon 2009). Studies in other systems have reported that some forms of PP2A are inactivated to permit entry into mitosis and that these are important subsequently for mitotic exit (Barr et al 2011).…”

Temporal control of the dephosphorylation of Cdk substrates by mitotic exit pathways in budding yeast

“…Because the FEAR controls Cdc14 release by inhibiting PP2A Cdc55 (9), the FEAR pathway is hyperactive in cdc55⌬ mutants, wherein the B-regulatory subunit of PP2A is deleted (7,8). To further confirm that CLB5 overexpression is toxic to FEAR mutants, we examined the growth of FEAR mutants overexpressing CLB5 in the absence of Cdc55.…”

“…Nocodazole is a microtubule poison that arrests cells at metaphase with nucleolar localized Cdc14. Deletion of CDC55 results in Cdc14 release in nocodazole-treated cells due to the activation of FEAR (7,8). Cells lacking Bfa1, the negative regulator of MEN, also show released Cdc14 in the presence of nocodazole (17).…”

PP2ACdc55is required for multiple events during meiosis I

“…35 However, overexpression of a nondegradable Pds1 does not fully suppress chromatid segregation, suggesting Cdc55 regulates sister chromatid separation independent of Pds1. Cdc55 has been identified as a negative regulator of mitotic exit, 34,36 as it inhibits the release of Cdc14 from the nucleolus into the nucleus, a hallmark event occurring upon activation of the mitotic exit network (MEN). 34,37,38 Cdc55 is required for sister chromatid cohesion in the presence of DNA damage 35 and is involved in Sgo1-dependent meiotic cohesin cleavage through separase regulation.…”