A Bub2p-dependent spindle checkpoint pathway regulates the Dbf2p kinase in budding yeast

Exit from mitosis in all eukaroytes requires inactivation of the mitotic kinase. This occurs principally by ubiquitin-mediated proteolysis of the cyclin subunit controlled by the anaphase-promoting complex (APC). However, an abnormal spindle and/or unattached kinetochores activates a conserved spindle checkpoint that blocks APC function. This leads to high mitotic kinase activity and prevents mitotic exit. DBF2 belongs to a group of budding yeast cell cycle genes that when mutated prevent cyclin degradation and block exit from mitosis. DBF2 encodes a protein kinase which is cell cycle regulated, peaking in metaphase-anaphase B/telophase, but its function remains unknown. Here, we show the Dbf2p kinase activity to be a target of the spindle checkpoint. It is controlled specifically by Bub2p, one of the checkpoint components that is conserved in fission yeast and higher eukaroytic cells. Significantly, in budding yeast, Bub2p shows few genetic or biochemical interactions with other members of the spindle checkpoint. Our data now point to the protein kinase Mps1p triggering a new parallel branch of the spindle checkpoint in which Bub2p blocks Dbf2p function.     

Dual inhibition of Cdc20 by the spindle checkpoint

The metaphase-to-anaphase transition is triggered by the Anaphase-Promoting Complex (APC), an E3 ubiquitin ligase that targets proteins for degradation, leading to sister chromatid separation and mitotic exit. The function of APC is controlled by the spindle checkpoint that delays anaphase onset in the presence of any chromosome that has not established bipolar attachment to the mitotic spindle. In this way, the checkpoint ensures accurate chromosome segregation. The spindle checkpoint is mostly activated from kinetochores that are not attached to microtubules or not under tension that is normally generated from bipolar attachment. These kinetochores recruit several spindle checkpoint proteins to assemble an inhibitory complex composed of checkpoint proteins Mad2, Bub3, and Mad3/BubR1. This complex binds and inhibits Cdc20, an activator and substrate adaptor for APC. In addition, the checkpoint complex promotes Cdc20 degradation, thus lowering Cdc20 protein level upon checkpoint activation. This dual inhibition on Cdc20 likely ensures that the spindle checkpoint is sustained even when the cell contains only a single unattached kinetochore.     

The role of Cdh1p in maintaining genomic stability in budding yeast

Cdh1p, a substrate specificity factor for the cell cycle-regulated ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), promotes exit from mitosis by directing the degradation of a number of proteins, including the mitotic cyclins. Here we present evidence that Cdh1p activity at the M/G(1) transition is important not only for mitotic exit but also for high-fidelity chromosome segregation in the subsequent cell cycle. CDH1 showed genetic interactions with MAD2 and PDS1, genes encoding components of the mitotic spindle assembly checkpoint that acts at metaphase to prevent premature chromosome segregation. Unlike cdh1delta and mad2delta single mutants, the mad2delta cdh1delta double mutant grew slowly and exhibited high rates of chromosome and plasmid loss. Simultaneous deletion of PDS1 and CDH1 caused extensive chromosome missegregation and cell death. Our data suggest that at least part of the chromosome loss can be attributed to kinetochore/spindle problems. Our data further suggest that Cdh1p and Sic1p, a Cdc28p/Clb inhibitor, have overlapping as well as nonoverlapping roles in ensuring proper chromosome segregation. The severe growth defects of both mad2delta cdh1delta and pds1delta cdh1dDelta strains were rescued by overexpressing Swe1p, a G(2)/M inhibitor of the cyclin-dependent kinase, Cdc28p/Clb. We propose that the failure to degrade cyclins at the end of mitosis leaves cdh1delta mutant strains with abnormal Cdc28p/Clb activity that interferes with proper chromosome segregation.     

Mutual regulation between the spindle checkpoint and APC/C

Accurate chromosome segregation during mitosis is critical for maintaining genomic stability. The spindle checkpoint is a cellular surveillance system that ensures the fidelity of chromosome segregation. In response to sister chromatids not properly captured by spindle microtubules, the spindle checkpoint interferes with the functions of Cdc20, the mitotic activator of the anaphase-promoting complex or cyclosome (APC/C), thereby blocking APC/C-mediated degradation of securin and cyclin B to delay anaphase onset. This review summarizes the recent progress on the mechanisms by which checkpoint proteins inhibit APC/C, the conformational and enzymatic activation of checkpoint proteins, and the emerging roles of APC/C-dependent ubiquitination in checkpoint inactivation.     

Role of the kinetochore protein Ndc10 in mitotic checkpoint activation in Saccharomyces cerevisiae

Mitotic checkpoints delay cell cycle progression in response to alterations in the mitotic apparatus, thus ensuring correct chromosome segregation. While improper spindle orientation activates the Bub2/Bfa1-dependent checkpoint in budding yeast, delaying exit from mitosis, lack of bipolar kinetochore-microtubule attachment activates a signal transduction cascade that prevents both anaphase onset and exit from mitosis by inhibiting the Cdc20/APC (Anaphase Promoting Complex)-mediated proteolysis of securin and inactivation of mitotic cyclin-dependent kinases (CDKs), respectively. Proteolysis of the securin Pdsl is necessary to liberate the separase Esp1, which then triggers sister chromatid separation, whereas inactivation of mitotic CDKs is a prerequisite for exit from mitosis and for starting a new round of DNA replication in the next cell cycle. In budding yeast, this latter checkpoint response involves the proteins Mad1, 2, 3, Bub1 and Bub3, whose vertebrate counterparts localize to unattached kinetochores. Mutations that alter other kinetochore proteins result in mitotic checkpoint activation, while the ndc10-1 mutation not only impairs kinetochore function, but also disrupts the checkpoint response, indicating a role for Ndc10 in this process. Here we present evidence that Ndc10 is not part of the Bub2/Bfa1-dependent pathway, and its role in the checkpoint response might also be different from that of the other Mad and Bub proteins. Indeed, Ndc10, unlike other mitotic checkpoint proteins, is not required for the mitotic block induced by overexpression of the Mpsl protein kinase, which is implicated in mitotic checkpoint control. Furthermore, the delay in mitotic exit caused by non-degradable Pds1, which does not require Mad and Bub proteins, depends on Ndc10 function. We propose that a pathway involving Ndc10 might monitor defects in the mitotic apparatus independently of the Mad and Bub proteins. Since the Espl separase is required for exit from mitosis in both ndc10-1 and nocodazole-treated mad2delta cells, the two signal transduction cascades might ultimately converge on the inactivation of Esp1.     

Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint

Mitotic progression is controlled by proteolytic destruction of securin and cyclin. The mitotic E3 ubiquitin ligase, known as the anaphase promoting complex or cyclosome (APC/C), in partnership with its activators Cdc20p and Cdh1p, targets these proteins for degradation. In the presence of defective kinetochore-microtubule interactions, APC/C(Cdc20) is inhibited by the spindle checkpoint, thereby delaying anaphase onset and providing more time for spindle assembly. Cdc20p interacts directly with Mad2p, and its levels are subject to careful regulation, but the precise mode(s) of APC/C( Cdc20) inhibition remain unclear. The mitotic checkpoint complex (MCC, consisting of Mad3p, Mad2p, Bub3p and Cdc20p in budding yeast) is a potent APC/C inhibitor. Here we focus on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. We identify and analyse the function of two motifs in Mad3p, KEN30 and KEN296, which are conserved from yeast Mad3p to human BubR1. These KEN amino acid sequences resemble 'degron' signals that confer interaction with APC/C activators and target proteins for degradation. We show that both Mad3p KEN boxes are necessary for spindle checkpoint function. Mutation of KEN30 abolished MCC formation and stabilised Cdc20p in mitosis. In addition, mutation of Mad3-KEN30, APC/C subunits, or Cdh1p, stabilised Mad3p in G1, indicating that the N-terminal KEN box could be a Mad3p degron. To determine the significance of Mad3p turnover, we analysed the consequences of MAD3 overexpression and found that four-fold overproduction of Mad3p led to chromosome bi-orientation defects and significant chromosome loss during recovery from anti-microtubule drug induced checkpoint arrest. In conclusion, Mad3p KEN30 mediates interactions that regulate the proteolytic turnover of Cdc20p and Mad3p, and the levels of both of these proteins are critical for spindle checkpoint signaling and high fidelity chromosome segregation.     

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.     

Anaphase-promoting complex/cyclosome controls the stability of TPX2 during mitotic exit

TPX2, a microtubule-associated protein, is required downstream of Ran-GTP to induce spindle assembly. TPX2 activity appears to be tightly regulated during the cell cycle, and we report here one molecular mechanism for this regulation. We found that TPX2 protein levels are cell cycle regulated, peaking in mitosis and declining sharply during mitotic exit. TPX2 is degraded in mitotic extracts, as well as in HeLa cells exiting from mitosis. This instability depends, both in vitro and in vivo, on the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that controls mitotic progression. In a reconstituted system, TPX2 is efficiently ubiquitinated by APC/C that has been activated by Cdh1. Two discrete elements in TPX2 are required for recognition by APC/C^Cdh1: a KEN box and a novel element in amino acids 1 to 86. Interestingly, the latter element, which has no known APC/C recognition motifs, is required for the ubiquitination of TPX2 by APC/C^Cdh1 in vitro and for its degradation in vivo. We conclude that APC/C^Cdh1 controls the stability of TPX2, thereby ensuring accurate regulation of the spindle assembly in the cell cycle.     

Bub1 and BubR1: at the interface between chromosome attachment and the spindle checkpoint

The spindle checkpoint ensures genome fidelity by temporarily halting chromosome segregation and the ensuing mitotic exit until the last kinetochore is productively attached to the mitotic spindle. At the interface between proper chromosome attachment and the metaphase-to-anaphase transition are the mammalian spindle checkpoint kinases. Compelling evidence indicates that the checkpoint kinases Bub1 and BubR1 have the added task of regulating kinetochore-microtubule attachments. However, the debate on the requirement of kinase activity is in full swing. This minireview summarizes recent advances in our understanding of the core spindle checkpoint kinases Bub1 and BubR1 and considers evidence that supports and opposes the role of kinase activity in regulating their functions during mitosis.     

Mitotic phosphatase activity is required for MCC maintenance during the spindle checkpoint

The spindle checkpoint prevents activation of the anaphase-promoting complex (APC/C) until all chromosomes are correctly attached to the mitotic spindle. Early in mitosis, the mitotic checkpoint complex (MCC) inactivates the APC/C by binding the APC/C activating protein CDC20 until the chromosomes are properly aligned and attached to the mitotic spindle, at which point MCC disassembly releases CDC20 to activate the APC/C. Once the APC/C is activated, it targets cyclin B and securin for degradation, and the cell progresses into anaphase. While phosphorylation is known to drive many of the events during the checkpoint, the precise molecular mechanisms regulating spindle checkpoint maintenance and inactivation are still poorly understood. We sought to determine the role of mitotic phosphatases during the spindle checkpoint. To address this question, we treated spindle checkpoint-arrested cells with various phosphatase inhibitors and examined the effect on the MCC and APC/C activation. Using this approach we found that 2 phosphatase inhibitors, calyculin A and okadaic acid (1 μM), caused MCC dissociation and APC/C activation leading to cyclin A and B degradation in spindle checkpoint-arrested cells. Although the cells were able to degrade cyclin B, they did not exit mitosis as evidenced by high levels of Cdk1 substrate phosphorylation and chromosome condensation. Our results provide the first evidence that phosphatases are essential for maintenance of the MCC during operation of the spindle checkpoint.     

Multiple Anaphase-promoting Complex/Cyclosome Degrons Mediate the Degradation of Human Sgo1

Shugoshin 1 (Sgo1) protects centromeric sister-chromatid cohesion in early mitosis and, thus, prevents premature sister-chromatid separation. The protein level of Sgo1 is regulated during the cell cycle; it peaks in mitosis and is down-regulated in G₁/S. Here we show that Sgo1 is degraded during the exit from mitosis, and its degradation depends on the anaphase-promoting complex/cyclosome (APC/C). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Sgo1 in human cells. Sgo1 is ubiquitinated by APC/C bound to Cdh1 (APC/C^Cdh1) in vitro. We have further identified two functional degradation motifs in Sgo1; that is, a KEN (Lys-Glu-Asn) box and a destruction box (D box). Although removal of either motif is not sufficient to stabilize Sgo1, Sgo1 with both KEN box and D box deleted is stable in cells. Surprisingly, mitosis progresses normally in the presence of non-degradable Sgo1, indicating that degradation of Sgo1 is not required for sister-chromatid separation or mitotic exit. Finally, we show that the spindle checkpoint kinase Bub1 contributes to the maintenance of Sgo1 steady-state protein levels in an APC/C-independent mechanism.Loss of sister-chromatid cohesion triggers chromosome segregation in mitosis and occurs in two steps in vertebrate cells (1-3). In prophase, cohesin is phosphorylated by mitotic kinases including Plk1 and removed from chromosome arms (1, 4). Then, cleavage of centromeric cohesin by separase takes place at the metaphase-to-anaphase transition to allow sister-chromatid separation (5). The shugoshin (Sgo) family of proteins plays an important role in the protection of centromeric cohesion (6, 7). Human cells depleted of Sgo1 by RNAi undergo massive chromosome missegregation (8-11). In cells with compromised Sgo1 function, centromeric cohesin is improperly phosphorylated and removed (4, 11), resulting in premature sister-chromatid separation. It has been shown recently that Sgo1 collaborates with PP2A to counteract the action of Plk1 and other mitotic kinases and to protect centromeric cohesin from premature removal (12-14). In addition, Sgo1 has also been shown to promote stable kinetochore-microtubule attachment and sense tension across sister kinetochores (8, 15). Thus, Sgo1 is crucial for mitotic progression and chromosome segregation.Orderly progression through mitosis is regulated by the anaphase-promoting complex/cyclosome (APC/C),² a large multiprotein ubiquitin ligase that targets key mitotic regulators for destruction by the proteasome (16). APC/C selects substrates for ubiquitination by using the Cdc20 or Cdh1 activator proteins to recognize specific sequences called APC/C degrons within target proteins (17). Several APC/C degrons have been characterized, including the destruction box (D box) and the Lys-Glu-Asn box (KEN box) (18, 19). The D box, with the consensus amino acid sequence of RXXLXXXN(X indicates any amino acid), are found in many APC/C substrates, including mitotic cyclins and are essential for their ubiquitin-mediated destruction. The KEN box, which contains a consensus KEN motif, is also found in several APC/C substrates and is preferentially but not exclusively recognized by APC/C^Cdh1. When APC/C is active, it directs progression through and exit from mitosis by catalyzing the ubiquitination and timely destruction of mitotic regulators, including cyclin A, cyclin B, and the separase inhibitor securin (16). The APC/C activity needs to be tightly controlled to prevent unscheduled substrate degradation. An important mechanism for APC/C regulation is the spindle checkpoint, which prevents the activation of APC/C and destruction of its substrates in response to kinetochores that have not properly attached to the mitotic spindle (20).Recent evidence shows that Sgo1 is a substrate of APC/C, and its protein levels oscillate during the cell cycle (8, 9). In this article we study the degradation of Sgo1 in human cells. We show that Sgo1 is degraded during mitotic exit, and this degradation depends on APC/C^Cdh1. We further show that both KEN and D boxes are required for Sgo1 degradation in vivo and ubiquitination in vitro. Removal of these motifs stabilizes Sgo1 in vivo. The prolonged presence of stable Sgo1 protein in human cells does not change the kinetics of chromosome segregation and mitotic exit. Therefore, a timely scheduled degradation of Sgo1 takes place but is not required for mitotic exit. Finally, we show that Bub1 regulates Sgo1 protein levels through a mechanism that does not involve APC/C-mediated degradation.     

Substrate degradation by the anaphase promoting complex occurs during mitotic slippage

Microtubule targeting drugs are successful in chemotherapy because they indefinitely activate the spindle assembly checkpoint. The spindle assembly checkpoint monitors proper attachment of all kinetochores to microtubules and tension between the kinetochores of sister chromatids to prevent premature anaphase entry. To this end, the activated spindle assembly checkpoint suppresses the E3 ubiquitin ligase activity of the anaphase-promoting complex (APC). In the continued presence of conditions that activate the spindle assembly checkpoint, cells eventually escape from mitosis by "slippage". It has not been directly tested whether APC activation accompanies slippage. Using cells blocked in mitosis with the microtubule assembly inhibitor nocodazole, we show that mitotic APC substrates are degraded upon mitotic slippage. To confirm that APC is normally activated upon mitotic slippage we have found that knockdown of Cdc20 and Cdh1, two mitotic activators of APC, prevents the degradation of APC substrates during mitotic slippage. Knockdown of Cdc20 and Cdh1 prevents the degradation of APC substrates during mitotic slippage. We provide the first direct demonstration that despite conditions that activate the spindle checkpoint, APC is indeed activated upon mitotic slippage of cells to interphase cells. Activation of the spindle checkpoint by microtubule targeting drugs used in chemotherapy may not indefinitely prevent APC activation.     

Cdh1 controls the stability of TACC3

Transforming acidic coiled-coil protein 3 (TACC3) was reported to be important for regulating mitotic spindle assembly and chromosome segregation. While the protein level of TACC3 was shown to be altered during cell cycle progression, the molecular mechanism in controlling TACC3 level is unclear. Here, we show that TACC3 protein level can be regulated by Cdh1, a well known activator of anaphase-promoting complex/cyclosome. We identified Cdh1 as an interacting partner of TACC3 by a yeast array screen. Both in vitro and in vivo binding studies indicated that TACC3 can form complexes with Cdh1. Depletion of endogenous Cdh1 prolonged TACC3 protein level during mitotic exit. Alteration of Cdh1 level by ectopic overexpression or siRNA knockdown correlated well with an increase or decrease of ubiquitinated TACC3, respectively. Furthermore, the domain mapping studies of TACC3 revealed that multiple domains are involved in Cdh1-regulated degradation of TACC3. Altogether, our findings suggest that Cdh1 controls TACC3 protein stability during mitotic exit.     

Bub3 promotes Cdc20-dependent activation of the APC/C in S. cerevisiae

The spindle checkpoint ensures accurate chromosome segregation by sending a signal from an unattached kinetochore to inhibit anaphase onset. Numerous studies have described the role of Bub3 in checkpoint activation, but less is known about its functions apart from the spindle checkpoint. In this paper, we demonstrate that Bub3 has an unexpected role promoting metaphase progression in budding yeast. Loss of Bub3 resulted in a metaphase delay that was not a consequence of aneuploidy or the activation of a checkpoint. Instead, bub3Δ cells had impaired binding of the anaphase-promoting complex/cyclosome (APC/C) with its activator Cdc20, and the delay could be rescued by Cdc20 overexpression. Kinetochore localization of Bub3 was required for normal mitotic progression, and Bub3 and Cdc20 colocalized at the kinetochore. Although Bub1 binds Bub3 at the kinetochore, bub1Δ cells did not have compromised APC/C and Cdc20 binding. The results demonstrate that Bub3 has a previously unknown function at the kinetochore in activating APC/C-Cdc20 for normal mitotic progression.     

lemmingA encodes the Apc11 subunit of the APC/C in Drosophila melanogaster that forms a ternary complex with the E2-C type ubiquitin conjugating enzyme, Vihar and Morula/Apc2

Background

The spindle assembly checkpoint (SAC) inhibits anaphase progression in the presence of insufficient kinetochore-microtubule attachments, but cells can eventually override mitotic arrest by a process known as mitotic slippage or adaptation. This is a problem for cancer chemotherapy using microtubule poisons.

Results

Here we describe mitotic slippage in yeast bub2?? mutant cells that are defective in the repression of precocious telophase onset (mitotic exit). Precocious activation of anaphase promoting complex/cyclosome (APC/C)-Cdh1 caused mitotic slippage in the presence of nocodazole, while the SAC was still active. APC/C-Cdh1, but not APC/C-Cdc20, triggered anaphase progression (securin degradation, separase-mediated cohesin cleavage, sister-chromatid separation and chromosome missegregation), in addition to telophase onset (mitotic exit), during mitotic slippage. This demonstrates that an inhibitory system not only of APC/C-Cdc20 but also of APC/C-Cdh1 is critical for accurate chromosome segregation in the presence of insufficient kinetochore-microtubule attachments.

Conclusions

The sequential activation of APC/C-Cdc20 to APC/C-Cdh1 during mitosis is central to accurate mitosis. Precocious activation of APC/C-Cdh1 in metaphase (pre-anaphase) causes mitotic slippage in SAC-activated cells. For the prevention of mitotic slippage, concomitant inhibition of APC/C-Cdh1 may be effective for tumor therapy with mitotic spindle poisons in humans.     

Anaphase-promoting complex/cyclosome controls HEC1 stability

Objective: Chromosome segregation during mitosis requires a physically large proteinaceous structure called the kinetochore to generate attachments between chromosomal DNA and spindle microtubules. It is essential for kinetochore components to be carefully regulated to guarantee successful cell division. Depletion, mutation or dysregulation of kinetochore proteins results in mitotic arrest and/or cell death. HEC1 (high expression in cancer) has been reported to be a kinetochore protein, depletion of which, by RNA interference, results in catastrophic mitotic exit. Materials and methods and results: To investigate how HEC1 protein is controlled post‐translation, we analysed the role of anaphase‐promoting complex/cyclosome (APC/C)‐Cdh1 in degradation of HEC1 protein. In this study, we show that HEC1 is an unstable protein and can be targeted by endogenous ubiquitin‐proteasome system in HEK293T cells. Results of RNA interference and in vivo ubiquitination assay indicated that HEC1 could be ubiquitinated and degraded by APC/C‐hCdh1 E3 ligase. The evolutionally conserved D‐box at the C‐terminus functioned as the degron of HEC1, destruction of which resulted in resistance to degradation mediated by APC/C‐Cdh1. Overexpression of non‐degradable HEC1 (D‐box destroyed) induced accumulation of cyclin B protein in vivo and triggered mitotic arrest. Conclusion: APC/C‐Cdh1 controls stability of HEC1, ensuring normal cell cycle progression.     

Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation

BACKGROUND: The kinetochore attachment (spindle assembly) checkpoint arrests cells in metaphase to prevent exit from mitosis until all the chromosomes are aligned properly at the metaphase plate. The checkpoint operates by preventing activation of the anaphase-promoting complex (APC), which triggers anaphase by degrading mitotic cyclins and other proteins. This checkpoint is active during normal mitosis and upon experimental disruption of the mitotic spindle. In yeast, the serine/threonine protein kinase Bub1 and the WD-repeat protein Bub3 are elements of a signal transduction cascade that regulates the kinetochore attachment checkpoint. In mammalian cells, activated MAPK is present on kinetochores during mitosis and activity is upregulated by the spindle assembly checkpoint. In vertebrate unfertilized eggs, a special form of meiotic metaphase arrest by cytostatic factor (CSF) is mediated by MAPK activation of the protein kinase p90(Rsk), which leads to inhibition of the APC. However, it is not known whether CSF-dependent metaphase arrest caused by p90(Rsk) involves components of the spindle assembly checkpoint. RESULTS: xBub1 is present in resting oocytes and its protein level increases slightly during oocyte maturation and early embryogenesis. In Xenopus oocytes, Bub1 is localized to kinetochores during both meiosis I and meiosis II, and the electrophoretic mobility of Bub1 upon SDS-PAGE decreases during meiosis I, reflecting phosphorylation and activation of the enzyme. The activation of Bub1 can be induced in interphase egg extracts by selective stimulation of the MAPK pathway by c-Mos, a MAPKKK. In oocytes treated with the MEK1 inhibitor U0126, the MAPK pathway does not become activated, and Bub1 remains in its low-activity, unshifted form. Injection of a constitutively active target of MAPK, the protein kinase p90(Rsk), restores the activation of Bub1 in the presence of U0126. Moreover, purified p90(Rsk) phosphorylates Bub1 in vitro and increases its protein kinase activity. CONCLUSIONS: Bub1, an upstream component of the kinetochore attachment checkpoint, is activated during meiosis in Xenopus in a MAPK-dependent manner. Moreover, a single substrate of MAPK, p90(Rsk), is sufficient to activate Bub1 in vitro and in vivo. These results indicate that in vertebrate eggs, kinetochore attachment/spindle assembly checkpoint proteins, including Bub1, are downstream of p90(Rsk) and may be effectors of APC inhibition and CSF-dependent metaphase arrest by p90(Rsk).     

The anaphase-promoting complex or cyclosome supports cell survival in response to endoplasmic reticulum stress

The anaphase-promoting complex or cyclosome (APC/C) is a multi-subunit ubiquitin ligase that regulates exit from mitosis and G1 phase of the cell cycle. Although the regulation and function of APC/C(Cdh1) in the unperturbed cell cycle is well studied, little is known of its role in non-genotoxic stress responses. Here, we demonstrate the role of APC/C(Cdh1) (APC/C activated by Cdh1 protein) in cellular protection from endoplasmic reticulum (ER) stress. Activation of APC/C(Cdh1) under ER stress conditions is evidenced by Cdh1-dependent degradation of its substrates. Importantly, the activity of APC/C(Cdh1) maintains the ER stress checkpoint, as depletion of Cdh1 by RNAi impairs cell cycle arrest and accelerates cell death following ER stress. Our findings identify APC/C(Cdh1) as a regulator of cell cycle checkpoint and cell survival in response to proteotoxic insults.     

APC/C-Mediated Degradation in Early Mitosis: How to Avoid Spindle Assembly Checkpoint Inhibition

The APC/C is an E3 ubiquitin ligase that, by targeting substrates for proteasomal degradation, plays a major role in cell cycle control. In complex with one of two WD40 activator proteins, Cdc20 or Cdh1, the APC/C is active from early mitosis through to late G1 and during this time targets many critical regulators of the cell cycle for degradation. However, this destruction is carefully ordered to ensure that cell cycle events are executed in a timely fashion. Recent studies have begun to shed light on how the APC/C selects different substrates at different times in the cell cycle. One particular problem is how the APC/C recognizes its first set of substrates, Nek2A and cyclin A, in early mitosis when, at this time, the spindle assembly checkpoint (SAC) inhibits most APC/C-dependent degradation. The answer may lie in how substrates are recruited to the APC/C. While checkpoint-dependent substrates appear to require Cdc20 for recruitment, experiments on the early mitotic substrate Nek2A demonstrate that it can bind the APC/C in the absence of Cdc20. The direct interaction of substrates with core subunits of the APC/C could allow their degradation to proceed unhindered even when the SAC is active.