A Wee1 checkpoint inhibits anaphase onset

Cdk1 drives both mitotic entry and the metaphase-to-anaphase transition. Past work has shown that Wee1 inhibition of Cdk1 blocks mitotic entry. Here we show that the budding yeast Wee1 kinase, Swe1, also restrains the metaphase-to-anaphase transition by preventing Cdk1 phosphorylation and activation of the mitotic form of the anaphase-promoting complex/cyclosome (APC^Cdc20). Deletion of SWE1 or its opposing phosphatase MIH1 (the budding yeast cdc25⁺) altered the timing of anaphase onset, and activation of the Swe1-dependent morphogenesis checkpoint or overexpression of Swe1 blocked cells in metaphase with reduced APC activity in vivo and in vitro. The morphogenesis checkpoint also depended on Cdc55, a regulatory subunit of protein phosphatase 2A (PP2A). cdc55Δ checkpoint defects were rescued by mutating 12 Cdk1 phosphorylation sites on the APC, demonstrating that the APC is a target of this checkpoint. These data suggest a model in which stepwise activation of Cdk1 and inhibition of PP2A^Cdc55 triggers anaphase onset.     

Rca1 inhibits APC-Cdh1(Fzr) and is required to prevent cyclin degradation in G2.

We demonstrate that Rca1 is an essential inhibitor of the anaphase-promoting complex/cyclosome (APC) in Drosophila. APC activity is restricted to mitotic stages and G1 by its activators Cdc20-Fizzy (Cdc20(Fzy)) and Cdh1-Fizzy-related (Cdh1(Fzr)), respectively. In rca1 mutants, cyclins are degraded prematurely in G2 by APC-Cdh1(Fzr)-dependent proteolysis, and cells fail to execute mitosis. Overexpression of Cdh1(Fzr) mimics the rca1 phenotype, and coexpression of Rca1 blocks this Cdh1(Fzr) function. We show that Rca1 and Cdh1(Fzr) are in a complex that also includes the APC component Cdc27. Previous studies have shown that phosphorylation of Cdh1 prevents its interaction with the APC. Our data reveal a different mode of APC regulation by Rca1 at the G2 stage, when low Cdk activity is unable to inhibit Cdh1(Fzr) interaction.     

Mes1 controls the meiosis I to meiosis II transition by distinctly regulating the anaphase-promoting complex/cyclosome coactivators Fzr1/Mfr1 and Slp1 in fission yeast

Meiosis is a specialized form of cell division generating haploid gametes and is dependent upon protein ubiquitylation by the anaphase-promoting complex/cyclosome (APC/C). Accurate control of the APC/C during meiosis is important in all eukaryotic cells and is in part regulated by the association of coactivators and inhibitors. We previously showed that the fission yeast meiosis-specific protein Mes1 binds to a coactivator and inhibits APC/C; however, regulation of the Mes1-mediated APC/C inhibition remains elusive. Here we show how Mes1 distinctively regulates different forms of the APC/C. We study all the coactivators present in the yeast genome and find that only Slp1/Cdc20 is essential for meiosis I progression. However, Fzr1/Mfr1 is a critical target for Mes1 inhibition because fzr1Δ completely rescues the defect on the meiosis II entry in mes1Δ cells. Furthermore, cell-free studies suggest that Mes1 behaves as a pseudosubstrate for Fzr1/Mfr1 but works as a competitive substrate for Slp1. Intriguingly, mutations in the D-box or KEN-box of Mes1 increase its recognition as a substrate by Fzr1, but not by Slp1. Thus Mes1 interacts with two coactivators in a different way to control the activity of the APC/C required for the meiosis I/meiosis II transition.     

Cdk1 promotes kinetochore bi-orientation and regulates Cdc20 expression during recovery from spindle checkpoint arrest

The spindle assembly checkpoint (SAC), an evolutionarily conserved surveillance pathway, prevents chromosome segregation in response to conditions that disrupt the kinetochore-microtubule attachment. Removal of the checkpoint-activating stimulus initiates recovery during which spindle integrity is restored, kinetochores become bi-oriented, and cells initiate anaphase. Whether recovery ensues passively after the removal of checkpoint stimulus, or requires mediation by specific effectors remains uncertain. Here, we report two unrecognized functions of yeast Cdk1 required for efficient recovery from SAC-induced arrest. We show that Cdk1 promotes kinetochore bi-orientation during recovery by restraining premature spindle elongation thereby extinguishing SAC signalling. Moreover, Cdk1 is essential for sustaining the expression of Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) required for anaphase progression. We suggest a model in which Cdk1 activity promotes recovery from SAC-induced mitotic arrest by regulating bi-orientation and APC/C activity. Our findings provide fresh insights into the regulation of mitosis and have implications for the therapeutic efficacy of anti-mitotic drugs.     

Dual-mode regulation of the APC/C by CDK1 and MAPK controls meiosis I progression and fidelity

Female meiosis is driven by the activities of two major kinases, cyclin-dependent kinase 1 (Cdk1) and mitogen-activated protein kinase (MAPK). To date, the role of MAPK in control of meiosis is thought to be restricted to maintaining metaphase II arrest through stabilizing Cdk1 activity. In this paper, we find that MAPK and Cdk1 play compensatory roles to suppress the anaphase-promoting complex/cyclosome (APC/C) activity early in prometaphase, thereby allowing accumulation of APC/C substrates essential for meiosis I. Furthermore, inhibition of MAPK around the onset of APC/C activity at the transition from meiosis I to meiosis II led to accelerated completion of meiosis I and an increase in aneuploidy at metaphase II. These effects appear to be mediated via a Cdk1/MAPK-dependent stabilization of the spindle assembly checkpoint, which when inhibited leads to increased APC/C activity. These findings demonstrate new roles for MAPK in the regulation of meiosis in mammalian oocytes.     

Spindle checkpoint activation at meiosis I advances anaphase II onset via meiosis-specific APC/C regulation

During mitosis, the spindle assembly checkpoint (SAC) inhibits the Cdc20-activated anaphase-promoting complex/cyclosome (APC/C(Cdc20)), which promotes protein degradation, and delays anaphase onset to ensure accurate chromosome segregation. However, the SAC function in meiotic anaphase regulation is poorly understood. Here, we examined the SAC function in fission yeast meiosis. As in mitosis, a SAC factor, Mad2, delayed anaphase onset via Slp1 (fission yeast Cdc20) when chromosomes attach to the spindle improperly. However, when the SAC delayed anaphase I, the interval between meiosis I and II shortened. Furthermore, anaphase onset was advanced and the SAC effect was reduced at meiosis II. The advancement of anaphase onset depended on a meiosis-specific, Cdc20-related factor, Fzr1/Mfr1, which contributed to anaphase cyclin decline and anaphase onset and was inefficiently inhibited by the SAC. Our findings show that impacts of SAC activation are not confined to a single division at meiosis due to meiosis-specific APC/C regulation, which has probably been evolved for execution of two meiotic divisions.     

The Bfa1/Bub2 GAP complex comprises a universal checkpoint required to prevent mitotic exit

At the end of the cell cycle, cyclin-dependent kinase (CDK) activity is inactivated to allow mitotic exit [1]. A protein phosphatase, Cdc14, plays a key role during mitotic exit in budding yeast by activating the Cdh1 component of the anaphase-promoting complex to degrade cyclin B (Clb) and inducing the CDK inhibitor Sic1 to inactivate Cdk1 [2]. To prevent mitotic exit when the cell cycle is arrested at G2/M, cells must prevent CDK inactivation. In the spindle checkpoint pathway, this is accomplished through Bfa1/Bub2, a heteromeric GTPase-activating protein (GAP) that inhibits Clb degradation by keeping the G protein Tem1 inactive [3-5]. Tem1 is required for Cdc14 activation. Here we show that in budding yeast, BUB2 and BFA1 are also required for the maintenance of G2/M arrest in response to DNA damage and to spindle misorientation. cdc13-1 bub2 and cdc13-1 bfa1 but not cdc13-1 mad2 double mutants rebud and reduplicate their DNA at the restrictive temperature. We also found that the delay in mitotic exit in mutants with misoriented spindles depended on BUB2 and BFA1, but not on MAD2. We propose that Bfa1/Bub2 checkpoint pathway functions as a universal checkpoint in G2/M that prevents CDK inactivation in response to cell-cycle delay in G2/M.     

Adenovirus E4orf4 protein induces PP2A-dependent growth arrest in Saccharomyces cerevisiae and interacts with the anaphase-promoting complex/cyclosome

Adenovirus early region 4 open reading frame 4 (E4orf4) protein has been reported to induce p53-independent, protein phosphatase 2A (PP2A)-dependent apoptosis in transformed mammalian cells. In this report, we show that E4orf4 induces an irreversible growth arrest in Saccharomyces cerevisiae at the G2/M phase of the cell cycle. Growth inhibition requires the presence of yeast PP2A-Cdc55, and is accompanied by accumulation of reactive oxygen species. E4orf4 expression is synthetically lethal with mutants defective in mitosis, including Cdc28/Cdk1 and anaphase-promoting complex/cyclosome (APC/C) mutants. Although APC/C activity is inhibited in the presence of E4orf4, Cdc28/Cdk1 is activated and partially counteracts the E4orf4-induced cell cycle arrest. The E4orf4-PP2A complex physically interacts with the APC/C, suggesting that E4orf4 functions by directly targeting PP2A to the APC/C, thereby leading to its inactivation. Finally, we show that E4orf4 can induce G2/M arrest in mammalian cells before apoptosis, indicating that E4orf4-induced events in yeast and mammalian cells are highly conserved.     

Behind the wheel and under the hood: Functions of cyclin-dependent kinases in response to DNA damage

Cell division and the response to genotoxic stress are intimately connected in eukaryotes, for example, by checkpoint pathways that signal the presence of DNA damage or its ongoing repair to the cell cycle machinery, leading to reversible arrest or apoptosis. Recent studies reveal another connection: the cyclin-dependent kinases (CDKs) that govern both DNA synthesis (S) phase and mitosis directly coordinate DNA repair processes with progression through the cell cycle. In both mammalian cells and yeast, the two major modes of double strand break (DSB) repair – homologous recombination (HR) and non-homologous end joining (NHEJ) – are reciprocally regulated during the cell cycle. In yeast, the cell cycle kinase Cdk1 directly promotes DSB repair by HR during the G2 phase. In mammalian cells, loss of Cdk2, which is active throughout S and G2 phases, results in defective DNA damage repair and checkpoint signaling. Here we provide an overview of data that implicate CDKs in the regulation of DNA damage responses in yeast and metazoans. In yeast, CDK activity is required at multiple points in the HR pathway; the precise roles of CDKs in mammalian HR have yet to be determined. Finally, we consider how the two different, and in some cases opposing, roles of CDKs – as targets of negative regulation by checkpoint signaling and as positive effectors of repair pathway selection and function – could be balanced to produce a coordinated and effective response to DNA damage.     

Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint

The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.     

Regulation of glia number in Drosophila by Rap/Fzr, an activator of the anaphase-promoting complex, and Loco, an RGS protein

Glia mediate a vast array of cellular processes and are critical for nervous system development and function. Despite their immense importance in neurobiology, glia remain understudied and the molecular mechanisms that direct their differentiation are poorly understood. Rap/Fzr is the Drosophila homolog of the mammalian Cdh1, a regulatory subunit of the anaphase-promoting complex/cyclosome (APC/C). APC/C is an E3 ubiquitin ligase complex well characterized for its role in cell cycle progression. In this study, we have uncovered a novel cellular role for Rap/Fzr. Loss of rap/fzr function leads to a marked increase in the number of glia in the nervous system of third instar larvae. Conversely, ectopic expression of UAS-rap/fzr, driven by repo-GAL4, results in the drastic reduction of glia. Data from clonal analyses using the MARCM technique show that Rap/Fzr regulates the differentiation of surface glia in the developing larval nervous system. Our genetic and biochemical data further indicate that Rap/Fzr regulates glial differentiation through its interaction with Loco, a regulator of G-protein signaling (RGS) protein and a known effector of glia specification. We propose that Rap/Fzr targets Loco for ubiquitination, thereby regulating glial differentiation in the developing nervous system.     

The APC/C recruits cyclin B1–Cdk1–Cks in prometaphase before D box recognition to control mitotic exit

The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1–Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to APC/C^Cdc20 makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control.     

The APC/C activator FZR1 coordinates the timing of meiotic resumption during prophase I arrest in mammalian oocytes

FZR1, an activator of the anaphase-promoting complex/cyclosome (APC/C), is recognized for its roles in the mitotic cell cycle. To examine its meiotic function in females we generated an oocyte-specific knockout of the Fzr1 gene (Fzr1(Δ/Δ)). The total number of fully grown oocytes enclosed in cumulus complexes was 35-40% lower in oocytes from Fzr1(Δ/Δ) mice and there was a commensurate rise in denuded, meiotically advanced and/or fragmented oocytes. The ability of Fzr1(Δ/Δ) oocytes to remain prophase I/germinal vesicle (GV) arrested in vitro was also compromised, despite the addition of the phosphodiesterase milrinone. Meiotic competency of smaller diameter oocytes was also accelerated by Fzr1 loss. Cyclin B1 levels were elevated ~5-fold in Fzr1(Δ/Δ) oocytes, whereas securin and CDC25B, two other APC/C(FZR1) substrates, were unchanged. Cyclin B1 overexpression can mimic the effects of Fzr1 loss on GV arrest and here we show that cyclin B1 knockdown in Fzr1(Δ/Δ) oocytes affects the timing of meiotic resumption. Therefore, the effects of Fzr1 loss are mediated, at least in part, by raised cyclin B1. Thus, APC/C(FZR1) activity is required to repress cyclin B1 levels in oocytes during prophase I arrest in the ovary, thereby maintaining meiotic quiescence until hormonal cues trigger resumption.     

The END network couples spindle pole assembly to inhibition of the anaphase-promoting complex/cyclosome in early mitosis

Cyclin-dependent kinase 1 (Cdk1) initiates mitosis and later activates the anaphase-promoting complex/cyclosome (APC/C) to destroy cyclins. Kinetochore-derived checkpoint signaling delays APC/C-dependent cyclin B destruction, and checkpoint-independent mechanisms cooperate to limit APC/C activity when kinetochores lack checkpoint components in early mitosis. The APC/C and cyclin B localize to the spindle and poles, but the significance and regulation of these populations remain unclear. Here we describe a critical spindle pole-associated mechanism, called the END (Emi1/NuMA/dynein-dynactin) network, that spatially restricts APC/C activity in early mitosis. The APC/C inhibitor Emi1 binds the spindle-organizing NuMA/dynein-dynactin complex to anchor and inhibit the APC/C at spindle poles, and thereby limits destruction of spindle-associated cyclin B. Cyclin B/Cdk1 activity recruits the END network and establishes a positive feedback loop to stabilize spindle-associated cyclin B critical for spindle assembly. The organization of the APC/C on the spindle also provides a framework for understanding microtubule-dependent organization of protein destruction.     

Identification of Residues in Fission Yeast and Human P34(cdc2) Required for S-M Checkpoint Control

In fission yeast, regulation of p34(cdc2) plays an important role in the checkpoint coupling mitosis to completion of DNA replication. The cdc2 mutations cdc2-3w (C67Y) and cdc2-4w (C67F) abolish checkpoint control without seriously affecting normal cell proliferation. However the molecular basis of this phenotype is not known. To better understand the role of p34(cdc2) in checkpoint control, we have screened for more mutations in Schizosaccharomyces pombe cdc2 with this phenotype. We have isolated cdc2-3w and cdc2-4w, as well as three new cdc2 alleles: cdc2-6w (N66I), cdc2-7w (E8V) and cdc2-8w (K9E). The altered residues map to two different regions on opposite faces of the protein, suggesting that the interaction between p34(cdc2) and components of the checkpoint pathway may be complex. In contrast to cdc2-3w and cdc2-4w, the new mutations alter residues that are conserved between the fission yeast cdc2(+) and other cdks, including the human CDC2 protein. Expression of the equivalent human CDC2 mutants in fission yeast abolishes checkpoint control, suggesting that these residues could be involved in checkpoint-dependent regulation of other eukaryotic cdks.     

The cdc25 phosphatase is essential for the G2/M phase transition in the basidiomycete yeast Ustilago maydis

Cdc25-related phosphatases reverse the inhibitory phosphorylation of mitotic Cyclin-dependent kinases mediated by Wee1-related kinases, thereby promoting entry into mitosis. In the fission yeast, Schizosaccharomyces pombe, Cdc25 is required for entry into mitosis, while in the budding yeast Saccharomyces cerevisiae, Mih1 (the homologue of Cdc25) is not required for entry into mitosis or for viability. As these differences were linked to the different cell division and growth mechanism of these species, we sought to analyse the roles of Cdc25 in Ustilago maydis, which as S. cerevisiae divides by budding, but relies in a polar growth. This basidiomycete yeast is perfectly suited to analyse the relationships between cell cycle and morphogenesis. We show that U. maydis contains a single Cdc25-related protein, which is essential for growth. Loss of Cdc25 function results in a specific G2 arrest that correlated with high level of Tyr15 phosphorylation of Cdk1. Moreover, we show genetic interactions of cdc25 with wee1 and clb2 that support the notion that in U. maydis Cdc25 counteracts the Wee1-mediated inhibitory phosphorylation of Cdk1-Clb2 complex. Our results supports a model in which inhibitory phosphorylation of Cdk1 is a primary mechanism operating at G2/M transition in this fungus.