The Role of Cdc55 in the Spindle Checkpoint Is through Regulation of Mitotic Exit inSaccharomyces cerevisiae

Abstract

Cdc55, a B-type regulatory subunit of protein phosphatase 2A, has been implicated in mitotic spindle checkpoint activity and maintenance of sister chromatid cohesion during metaphase. The spindle checkpoint is composed of two independent pathways, one leading to inhibition of the metaphase-to-anaphase transition by checkpoint proteins, including Mad2, and the other to inhibition of mitotic exit by Bub2. We show that Cdc55 is a negative regulator of mitotic exit. A cdc55 mutant, like a bub2 mutant, prematurely releases Cdc14 phosphatase from the nucleolus during spindle checkpoint activation, and premature exit from mitosis indirectly leads to loss of sister chromatid cohesion and inviability in nocodazole. The role of Cdc55 is separable from Bub2 and inhibits release of Cdc14 through a mechanism independent of the known negative regulators of mitotic exit. Epistasis experiments indicate Cdc55 acts either downstream or independent of the mitotic exit network kinase Cdc15. Interestingly, the B-type cyclin Clb2 is partially stable during premature activation of mitotic exit in a cdc55 mutant, indicating mitotic exit is incomplete. INTRODUCTIONThe critical task of mitosis is equal segregation of sister chromatids to daughter cells. Faithful chromosome transmission is required for maintenance of genomic integrity, and failures in this process lead to aneuploid states characteristic of cancer (Nowak et al., 2002). Checkpoints are mechanisms that inhibit cell division in the presence of damage, allowing time for repair and subsequent reentry into the cell cycle (Hartwell and Weinert, 1989). The spindle checkpoint arrests cells in mitosis when spindle function is inadequate for faithful chromosome segregation (Lew and Burke, 2003). This checkpoint can be induced by benzimidazole drugs, which cause microtubule depolymerization and compromised attachment to kinetochores. To arrest in mitosis, cells must stabilize sister chromatid cohesion to prevent premature separation of chromosomes and the mitotic cyclin Clb2 to maintain mitotic cyclin-dependent kinase activity (Lew and Burke, 2003). Maintenance of both cohesion and mitotic cyclin-dependent kinase (CDK) activity under these conditions requires the cell to stabilize proteins whose destruction is normally initiated by an E3 ubiquitin ligase known as the anaphase-promoting complex (APC) (Harper et al., 2002;Vodermaier, 2004).The activity and substrate specificity of the APC is a key point of regulation in the cell cycle. Two distinct APC activators, Cdc20 and Cdh1, act at metaphase and mitotic exit respectively (Visintin et al., 1997). These proteins direct degradation of different, but partially overlapping, substrates to promote the irreversible passage through these cell cycle stages. APC Cdc20 targets Pds1 "securin", a protein required for metaphase sister chromatid cohesion, and the S-phase cyclin Clb5, while APC Cdh1 targets Ase1, Cdc20 and others. Degradation of Clb2 is biphasic, with a subset of the protein being degraded by APC Cdc20 , and the remainder by APCCdh...

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).…”

A Redundant Function for the N-Terminal Tail of Ndc80 in Kinetochore–Microtubule Interaction in Saccharomyces cerevisiae

“…Isogenic MATa cells were synchronized with a-factor and released to the cell cycle in the absence (A) or presence (C) of 10 mg/ml of benomyl and 25 mg/ml carbendezim (BEN). Cells were sampled every 15 min and prepared for flow cytometry and Western blots (Yellman and Burke 2006). The untreated cells from A were analyzed by fluorescence microscopy for nuclear division (B).…”

PP2ACdc55is required for multiple events during meiosis I

“…30 cdc55 cells are unable to establish a mitotic spindle damage checkpoint and rapidly lose viability when exposed to non-toxic doses of the microtubule destabilizing drugs nocodazole or benomyl. 33,34 In the presence of nocodazole, cdc55 cells maintain levels of the anaphase inhibitor Pds1 (securin). 35 However, overexpression of a nondegradable Pds1 does not fully suppress chromatid segregation, suggesting Cdc55 regulates sister chromatid separation independent of Pds1.…”

“…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.…”