Cdc20 Mutants Research Articles

The Bub1-Plk1 kinase complex promotes spindle checkpoint signalling through Cdc20 phosphorylation.

The spindle checkpoint senses unattached kinetochores and inhibits the Cdc20-bound anaphase-promoting complex or cyclosome (APC/C), to delay anaphase, thereby preventing aneuploidy. A critical checkpoint inhibitor of APC/CCdc20 is the mitotic checkpoint complex (MCC). It is unclear whether MCC suffices to inhibit all cellular APC/C. Here we show that human checkpoint kinase Bub1 not only directly phosphorylates Cdc20, but also scaffolds Plk1-mediated phosphorylation of Cdc20. Phosphorylation of Cdc20 by Bub1–Plk1 inhibits APC/CCdc20 in vitro and is required for checkpoint signalling in human cells. Bub1–Plk1-dependent Cdc20 phosphorylation is regulated by upstream checkpoint signals and is dispensable for MCC assembly. A phospho-mimicking Cdc20 mutant restores nocodazole-induced mitotic arrest in cells depleted of Mad2 or BubR1. Thus, Bub1–Plk1-mediated phosphorylation of Cdc20 constitutes an APC/C-inhibitory mechanism that is parallel, but not redundant, to MCC formation. Both mechanisms are required to sustain mitotic arrest in response to spindle defects.

DNA damage response and spindle assembly checkpoint function throughout the cell cycle to ensure genomic integrity.

Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved.

Mad2 and the APC/C compete for the same site on Cdc20 to ensure proper chromosome segregation

The spindle assembly checkpoint (SAC) is essential to ensure proper chromosome segregation and thereby maintain genomic stability. The SAC monitors chromosome attachment, and any unattached chromosomes generate a "wait anaphase" signal that blocks chromosome segregation. The target of the SAC is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C) that triggers anaphase and mitotic exit by ubiquitylating securin and cyclin B1. The inhibitory complex formed by the SAC has recently been shown to inhibit Cdc20 by acting as a pseudosubstrate inhibitor, but in this paper, we show that Mad2 also inhibits Cdc20 by binding directly to a site required to bind the APC/C. Mad2 and the APC/C competed for Cdc20 in vitro, and a Cdc20 mutant that does not bind stably to Mad2 abrogated the SAC in vivo. Thus, we provide insights into how Cdc20 binds the APC/C and uncover a second mechanism by which the SAC inhibits the APC/C.

Functional redundancy between Cdc20 ubiquitination and p31comet

The spindle checkpoint senses unattached kinetochores in a mitotic cell, and inhibits the function of Cdc20, the mitotic activator of the anaphase‐promoting complex or cyclosome (APC/C), thus delaying anaphase onset. Upon checkpoint activation, unattached kinetochores recruit checkpoint proteins and produce diffusible APC/C‐inhibitory signals. The mitotic checkpoint complex (MCC) consisting of BubR1 (Mad3 in yeast), Bub3, Mad2, and Cdc20 is a critical checkpoint inhibitor of APC/C. When all kinetochores achieve proper microtubule attachment and are under tension, the spindle checkpoint turns off and MCC disassembles to allow APC/C activation. Several mechanisms have been implicated in MCC disassembly, including p31comet‐and UbcH10‐dependent Cdc20 autoubiquitination and proteasomal degradation. However, the role of Cdc20 autoubiquitination in checkpoint inactivation has remained controversial, because preventing Cdc20 ubiquitination by mutating all lysines in Cdc20 to arginines does not prevent MCC disassembly.In this study, we have further investigated the roles and coordination of p31comet, proteasomal degradation, and Cdc20 ubiquitination in MCC disassembly. We show that both the proteasome and p31comet are required for timely MCC disassembly in HeLa cells by treatment of proteasome inhibitor MG132 or RNA interference (RNAi). Depletion of p31comet further delays MCC disassembly in cells treated with MG132, suggesting that p31comet has a proteasome‐independent role. Consistently, p31comet depletion delays Mad2–Cdc20 dissociation and mitotic exit in cells expressing Cdc20 mutants deficient for autoubiquitination. Taken together, our results reconcile earlier conflicting data, and suggest p31comet and Cdc20 ubiquitination act in somewhat redundant pathways to promote MCC disassembly.

Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31comet

The spindle checkpoint senses unattached or improperly attached kinetochores during mitosis, inhibits the anaphase-promoting complex or cyclosome (APC/C), and delays anaphase onset to prevent aneuploidy. The mitotic checkpoint complex (MCC) consisting of BubR1, Bub3, Mad2, and Cdc20 is a critical APC/C-inhibitory checkpoint complex in human cells. At the metaphase-anaphase transition, the spindle checkpoint turns off, and MCC disassembles to allow anaphase onset. The molecular mechanisms of checkpoint inactivation are poorly understood. A major unresolved issue is the role of Cdc20 autoubiquitination in this process. Although Cdc20 autoubiquitination can promote Mad2 dissociation from Cdc20, a nonubiquitinatable Cdc20 mutant still dissociates from Mad2 during checkpoint inactivation. Here, we show that depletion of p31(comet) delays Mad2 dissociation from Cdc20 mutants that cannot undergo autoubiquitination. Thus both p31(comet) and ubiquitination of Cdc20 are critical mechanisms of checkpoint inactivation. They act redundantly to promote Mad2 dissociation from Cdc20.

The role of aneuploidy in promoting and suppressing tumors

Impaired mitotic checkpoint signaling can both promote and suppress tumors. The mitotic checkpoint targets Cdc20, the specificity factor of the ubiquitin ligase that promotes anaphase by targeting cyclin B and securin for destruction. In this issue, Li et al. (2009. J. Cell Biol. doi:10.1083/jcb.200904020) use gene replacement to produce mice expressing a Cdc20 mutant that cannot be inhibited by the mitotic checkpoint. In addition to the expected aneuploidy, these animals have a high tumor incidence that is likely caused by persistent aneuploidy coupled with nonmitotic functions of mutant Cdc20.

APC/CCdc20 Controls the Ubiquitin-Mediated Degradation of p21 in Prometaphase

During the G1/S transition, p21 proteolysis is mediated by Skp2; however, p21 reaccumulates in G2 and is degraded again in prometaphase. How p21 degradation is controlled in mitosis remains unexplored. We found that Cdc20 (an activator of the ubiquitin ligase APC/C) binds p21 in cultured cells and identified a D box motif in p21 necessary for APC/C(Cdc20)-mediated ubiquitylation of p21. Overexpression of Cdc20 or Skp2 destabilized wild-type p21; however, only Skp2, but not Cdc20, was able to destabilize a p21(D box) mutant. Silencing of Cdc20 induced an accumulation of p21, increased the fraction of p21 bound to Cdk1, and inhibited Cdk1 activity in p21(+/+) prometaphase cells, but not in p21(-/-) cells. Thus, in prometaphase Cdc20 positively regulates Cdk1 by mediating the degradation of p21. We propose that the APC/C(Cdc20)-mediated degradation of p21 contributes to the full activation of Cdk1 necessary for mitotic events and prevents mitotic slippage during spindle checkpoint activation.

Phosphorylation of Cdc20 by Bub1 Provides a Catalytic Mechanism for APC/C Inhibition by the Spindle Checkpoint

To ensure the fidelity of chromosome segregation, the spindle checkpoint blocks the ubiquitin ligase activity of APC/C Cdc20 in response to a single chromatid not properly attached to the mitotic spindle. Here we show that HeLa cells depleted for Bub1 by RNA interference are defective in checkpoint signaling. Bub1 directly phosphorylates Cdc20 in vitro and inhibits the ubiquitin ligase activity of APC/C Cdc20 catalytically. A Cdc20 mutant with all six Bub1 phosphorylation sites removed is refractory to Bub1-mediated phosphorylation and inhibition in vitro. Upon checkpoint activation, Bub1 itself is hyperphosphorylated and its kinase activity toward Cdc20 is stimulated. Ectopic expression of the nonphosphorylatable Cdc20 mutant allows HeLa cells to escape from mitosis in the presence of spindle damage. Therefore, Bub1-mediated phosphorylation of Cdc20 is required for proper checkpoint signaling. We speculate that inhibition of APC/C Cdc20 by Bub1 in a catalytic fashion may partly account for the exquisite sensitivity of the spindle checkpoint.

Phosphorylation of Cdc20 is required for its inhibition by the spindle checkpoint.

The spindle checkpoint delays anaphase until all chromosomes are properly attached to spindle microtubules. When the spindle checkpoint is activated at unattached kinetochores, the checkpoint proteins BubR1, Bub3 and Mad2 bind and inhibit Cdc20, an activator of the anaphase-promoting complex (APC). Here, we show that Xenopus laevis Cdc20 is phosphorylated at Ser 50, Thr 64, Thr 68 and Thr 79 during mitosis and that mitogen-activated protein kinase (MAPK) contributes to the phosphorylation at Thr 64 or Thr 68. Cdc20 mutants that are phosphorylation-deficient are able to activate the APC in X. laevis egg extracts. However, Cdc20 mutants in which any of the four phosphorylation sites were altered to Ala or Val failed to respond to the spindle checkpoint signal, owing to their reduced affinity for the spindle checkpoint proteins. This study demonstrates that the spindle checkpoint stops anaphase by inhibiting fully-phosphorylated Cdc20. Our results also have implications for the spindle checkpoint silencing mechanism.

Cortex, a Drosophila gene required to complete oocyte meiosis, is a member of the Cdc20/fizzy protein family

Mutations in cortex and grauzone cause abnormal arrest in Drosophila female meiosis. cortex was mapped to a 14 kb interval in 26F-27A by the male recombination mapping method. While these experiments mapped the gene accurately, they also illustrated some complexities of this method. Rescue results showed that a 2.8 kb genomic fragment from this interval was able to fully rescue the cortex phenotype. The 2.8 kb rescuing fragment contains a single open reading frame. The predicted amino acid sequence indicates that cortex encodes a WD-repeat protein and is a distant member of the Cdc20 protein family. Results from a developmental Northern analysis showed that the cortex transcript is expressed at high levels during oogenesis and early embryogenesis. Interestingly, the meiotic metaphase-anaphase II arrest defect in embryos laid by cortex homozygous females resembles the mitotic metaphase-anaphase defects observed in yeast cdc20 mutants. The predicted nature of the Cortex protein, together with the observed meiotic phenotype in cortex mutants, suggest that a similar pathway to the cdc20 dependent APC-mediated proteolysis pathway, which governs the metaphase-anaphase transition in mitosis, is also important in regulating oocyte meiosis.

Cortex, a Drosophila gene required to complete oocyte meiosis, is a member of the Cdc20/fizzy protein family

Summary: Mutations in cortex and grauzone cause abnormal arrest in Drosophila female meiosis. cortex was mapped to a 14 kb interval in 26F–27A by the male recombination mapping method. While these experiments mapped the gene accurately, they also illustrated some complexities of this method. Rescue results showed that a 2.8 kb genomic fragment from this interval was able to fully rescue the cortex phenotype. The 2.8 kb rescuing fragment contains a single open reading frame. The predicted amino acid sequence indicates that cortex encodes a WD-repeat protein and is a distant member of the Cdc20 protein family. Results from a developmental Northern analysis showed that the cortex transcript is expressed at high levels during oogenesis and early embryogenesis. Interestingly, the meiotic metaphase-anaphase II arrest defect in embryos laid by cortex homozygous females resembles the mitotic metaphase-anaphase defects observed in yeast cdc20 mutants. The predicted nature of the Cortex protein, together with the observed meiotic phenotype in cortex mutants, suggest that a similar pathway to the cdc20 dependent APC-mediated proteolysis pathway, which governs the metaphase-anaphase transition in mitosis, is also important in regulating oocyte meiosis. genesis 29:141–152, 2001. © 2001 Wiley-Liss, Inc.

Cell cycle arrest in cdc20 mutants of Saccharomyces cerevisiae is independent of Ndc10p and kinetochore function but requires a subset of spindle checkpoint genes.

The spindle checkpoint ensures accurate chromosome segregation by inhibiting anaphase onset in response to altered microtubule function and impaired kinetochore function. In this study, we report that the ability of the anti-microtubule drug nocodazole to inhibit cell cycle progression in Saccharomyces cerevisiae depends on the function of the kinetochore protein encoded by NDC10. We examined the role of the spindle checkpoint in the arrest in cdc20 mutants that arrest prior to anaphase with an aberrant spindle. The arrest in cdc20 defective cells is dependent on the BUB2 checkpoint and independent of the BUB1, BUB3, and MAD spindle checkpoint genes. We show that the lesion recognized by Bub2p is not excess microtubules, and the cdc20 arrest is independent of kinetochore function. We show that Cdc20p is not required for cyclin proteolysis at two points in the cell cycle, suggesting that CDC20 is distinct from genes encoding integral proteins of the anaphase promoting complex.

DNA polymerase epsilon encoded by cdc20+ is required for chromosomal DNA replication in the fission yeast Schizosaccharomyces pombe.

DNA polymerase II (PolII), the homologue of mammalian DNA polymerase epsilon, is essential for chromosomal DNA replication in the budding yeast Saccharomyces cerevisiae and also participates in S-phase checkpoint control. An important issue is whether chromosomal DNA replication in other eukaryotes, including the fission yeast Schizosaccharomyces pombe--in which the characteristics of replication origins are poorly defined--also requires DNA polymerase epsilon. It has been shown that DNA polymerase epsilon is not required for the in vitro replication of SV40 DNA by human cell extracts. We have cloned and sequenced S. pombe pol2+, which is identical to the cell-cycle gene cdc20+, encoding the catalytic polypeptide of DNA polymerase epsilon (Pol epsilon). The predicted amino acid sequence of Pol epsilon is highly homologous to that of S. cerevisiae PolII and human Pol epsilon. Consistent with this, the Pol epsilon polypeptide was recognized by polyclonal antibodies against S. cerevisiae PolII holoenzyme (PolII*). The terminal morphology of cells containing the disrupted pol2 gene was similar to that of DNA replication mutant cells and cdc20 mutant cells. Furthermore, the Pol epsilon activity from temperature-sensitive S. pombe cdc20 mutant cells was temperature-sensitive, and chromosomal DNA replication in the mutant cells was inhibited at the restrictive temperatures. These data strongly suggest that Pol epsilon is required for normal chromosomal DNA replication in S. pombe, as is PolII in S. cerevisiae. Thus, eukaryotic chromosomal DNA is replicated differently from that of viral SV40 DNA.

Three-dimensional analysis and ultrastructural design of mitotic spindles from the cdc20 mutant of Saccharomyces cerevisiae.

The three-dimensional organization of mitotic microtubules in a mutant strain of Saccharomyces cerevisiae has been studied by computer-assisted serial reconstruction. At the nonpermissive temperature, cdc20 cells arrested with a spindle length of approximately 2.5 microns. These spindles contained a mean of 81 microtubules (range, 56-100) compared with 23 in wild-type spindles of comparable length. This increase in spindle microtubule number resulted in a total polymer length up to four times that of wild-type spindles. The spindle pole bodies in the cdc20 cells were approximately 2.3 times the size of wild-type, thereby accommodating the abnormally large number of spindle microtubules. The cdc20 spindles contained a large number of interpolar microtubules organized in a "core bundle." A neighbor density analysis of this bundle at the spindle midzone showed a preferred spacing of approximately 35 nm center-to-center between microtubules of opposite polarity. Although this is evidence of specific interaction between antiparallel microtubules, mutant spindles were less ordered than the spindle of wild-type cells. The number of noncore microtubules was significantly higher than that reported for wild-type, and these microtubules did not display a characteristic metaphase configuration. cdc20 spindles showed significantly more cross-bridges between spindle microtubules than were seen in the wild type. The cross-bridge density was highest between antiparallel microtubules. These data suggest that spindle microtubules are stabilized in cdc20 cells and that the CDC20 gene product may be involved in cell cycle processes that promote spindle microtubule disassembly.

The CDC20 gene product of Saccharomyces cerevisiae, a beta-transducin homolog, is required for a subset of microtubule-dependent cellular processes.

Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.

The CDC20 Gene Product of Saccharomyces cerevisiae, a β-Transducin Homolog, Is Required for a Subset of Microtubule-Dependent Cellular Processes

Previous analysis of cdc20 mutants of the yeast Saccharomyces cerevisiae suggests that the CDC20 gene product (Cdc20p) is required for two microtubule-dependent processes, nuclear movements prior to anaphase and chromosome separation. Here we report that cdc20 mutants are defective for a third microtubule-mediated event, nuclear fusion during mating of G1 cells, but appear normal for a fourth microtubule-dependent process, nuclear migration after DNA replication. Therefore, Cdc20p is required for a subset of microtubule-dependent processes and functions at multiple stages in the life cycle. Consistent with this interpretation, we find that cdc20 cells arrested by alpha-factor or at the restrictive temperature accumulate anomalous microtubule structures, as detected by indirect immunofluorescence. The anomalous microtubule staining patterns are due to cdc20 because intragenic revertants that revert the temperature sensitivity have normal microtubule morphologies. cdc20 mutants have a sevenfold increase in the intensity of antitubulin fluorescence in intranuclear spindles compared with spindles from wild-type cells, yet the total amount of tubulin is indistinguishable by Western immunoblot analysis. This result suggests that Cdc20p modulates microtubule structure in wild-type cells either by promoting microtubule disassembly or by altering the surface of the microtubules. Finally, we cloned and sequenced CDC20 and show that it encodes a member of a family of proteins that share homology to the beta subunit of transducin.

Preliminary characterization of maturation-promoting factor from yeast Saccharomyces cerevisiae *

It has been known for some time that maturation-promoting factor (MPF) appears in a wide variety of eukaryotic cells at M phase and exerts equal M-phase-promoting activity in both meiotic cells and mitotic cells in a non-specific manner. MPF was extracted from cdc20 mutant cells of the yeast Saccharomyces cerevisiae synchronized at M phase by incubation at the restrictive temperature. When injected into immature oocytes of Xenopus laevis, yeast MPF caused meiosis reinitiation in a dose-dependent manner and even in the presence of cycloheximide. Yeast MPF exerted its activity in starfish oocytes as well. MPF activity was obtained only from cells in M phase and not from G1, S or G2 phase cells, indicating cyclical changes during the yeast mitotic cell cycle. Preliminary characterization of yeast MPF revealed that its activity was associated with a heat-labile protein having a sedimentation coefficient value of 6 S. In contrast to the current assumption that MPF is a Ca-sensitive phosphoprotein stabilized by phosphorylated small molecules, such as ATP and Na-beta-glycerophosphate, the present study revealed that yeast MPF was still active even after treatment with either Ca2+ or alkaline phosphatase. Furthermore, it was found that yeast MPF and these phosphorylated small molecules were complementary in inducing reinitiation of meiosis, since the meiosis-reinitiating activity was detected only when both were present simultaneously and almost undetectable when either of them was present alone.(ABSTRACT TRUNCATED AT 250 WORDS)