The spindle checkpoint,APC/CCdc20, and APC/CCdh1 play distinct roles in connecting mitosis to S phase

DNA replication depends on a preceding licensing event by Cdt1 and Cdc6. In animal cells, relicensing after S phase but before mitosis is prevented by the Cdt1 inhibitor geminin and mitotic cyclin activity. Here, we show that geminin, like cyclin B1 and securin, is a bona fide target of the spindle checkpoint and APC/C^Cdc20. Cyclin B1 and geminin are degraded simultaneously during metaphase, which directs Cdt1 accumulation on segregating sister chromatids. Subsequent activation of APC/C^Cdh1 leads to degradation of Cdc6 well before Cdt1 becomes unstable in a replication-coupled manner. In mitosis, the spindle checkpoint supports Cdt1 accumulation, which promotes S phase onset. We conclude that the spindle checkpoint, APC/C^Cdc20, and APC/C^Cdh1 act successively to ensure that the disappearance of licensing inhibitors coincides exactly with a peak of Cdt1 and Cdc6. Whereas cell cycle entry from quiescence requires Cdc6 resynthesis, our results indicate that proliferating cells use a window of time in mitosis, before Cdc6 is degraded, as an earlier opportunity to direct S phase.     

Cdh1 is an antagonist of the spindle assembly checkpoint

The spindle assembly checkpoint (SAC) monitors unsatisfied connections of microtubules to kinetochores and prevents anaphase onset by inhibition of the ubiquitin ligase E3 anaphase-promoting complex or cyclosome (APC/C) in association with the activator Cdc20. Another APC/C activator, Cdh1, exists permanently throughout the cell cycle but it becomes active from telophase to G1. Here, we show that Cdh1 is partially active and mediates securin degradation even in SAC-active metaphase cells. Additionally, Cdh1 mediates Cdc20 degradation in metaphase, promoting formation of the APC/C-Cdh1. These results indicate that Cdh1 opposes the SAC and promotes anaphase transition.     

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.     

How APC/C-Cdc20 changes its substrate specificity in mitosis

Progress through mitosis requires that the right protein be degraded at the right time. One ubiquitin ligase, the anaphase-promoting complex or cyclosome (APC/C) targets most of the crucial mitotic regulators by changing its substrate specificity throughout mitosis. The spindle assembly checkpoint (SAC) acts on the APC/C co-activator, Cdc20 (cell division cycle 20), to block the degradation of metaphase substrates (for example, cyclin B1 and securin), but not others (for example, cyclin A). How this is achieved is unclear. Here we show that Cdc20 binds to different sites on the APC/C depending on the SAC. Cdc20 requires APC3 and APC8 to bind and activate the APC/C when the SAC is satisfied, but requires only APC8 to bind the APC/C when the SAC is active. Moreover, APC10 is crucial for the destruction of cyclin B1 and securin, but not cyclin A. We conclude that the SAC causes Cdc20 to bind to different sites on the APC/C and this alters APC/C substrate specificity.     

Loss of Cdc20 causes a securin-dependent metaphase arrest in two-cell mouse embryos

The anaphase-promoting complex/cyclosome (APC/C) is an E3 ubiquitin ligase mediating targeted proteolysis through ubiquitination of protein substrates to control the progression of mitosis. The APC/C recognizes its substrates through two adapter proteins, Cdc20 and Cdh1, which contain similar C-terminal domains composed of seven WD-40 repeats believed to be involved in interacting with their substrates. During the transition from metaphase to anaphase, APC/C-Cdc20 mediates the ubiquitination of securin and cyclin B1, allowing the activation of separase and the onset of anaphase and mitotic exit. APC/C-Cdc20 and APC/C-Cdh1 have overlapping substrates. It is unclear whether they are redundant for mitosis. Using a gene-trapping approach, we have obtained mice which lack Cdc20 function. These mice show failed embryogenesis. The embryos were arrested in metaphase at the two-cell stage with high levels of cyclin B1, indicating an essential role of Cdc20 in mitosis that is not redundant with that of Cdh1. Interestingly, Cdc20 and securin double mutant embryos could not maintain the metaphase arrest, suggesting a role of securin in preventing mitotic exit.     

Bub1, a gatekeeper for Cdc20-dependent mitotic exit

The mitotic spindle assembly checkpoint arrests cells at metaphase by suppressing Cdc20, a protein required to trigger ubiquitination and consequent degradation of cyclin B. New evidence from Tang et al. appearing in the November 5th issue of Molecular Cell finds that one of the checkpoint proteins, Bub1, specifically phosphorylates Cdc20 to suppress APC/C activation.     

The spindle and kinetochore–associated (Ska) complex enhances binding of the anaphase-promoting complex/cyclosome (APC/C) to chromosomes and promotes mitotic exit

The spindle and kinetochore–associated (Ska) protein complex is a heterotrimeric complex required for timely anaphase onset. The major phenotypes seen after small interfering RNA–mediated depletion of Ska are transient alignment defects followed by metaphase arrest that ultimately results in cohesion fatigue. We find that cells depleted of Ska3 arrest at metaphase with only partial degradation of cyclin B1 and securin. In cells arrested with microtubule drugs, Ska3-depleted cells exhibit slower mitotic exit when the spindle checkpoint is silenced by inhibition of the checkpoint kinase, Mps1, or when cells are forced to exit mitosis downstream of checkpoint silencing by inactivation of Cdk1. These results suggest that in addition to a role in fostering kinetochore–microtubule attachment and chromosome alignment, the Ska complex has functions in promoting anaphase onset. We find that both Ska3 and microtubules promote chromosome association of the anaphase-promoting complex/cyclosome (APC/C). Chromosome-bound APC/C shows significantly stronger ubiquitylation activity than cytoplasmic APC/C. Forced localization of Ska complex to kinetochores, independent of microtubules, results in enhanced accumulation of APC/C on chromosomes and accelerated cyclin B1 degradation during induced mitotic exit. We propose that a Ska-microtubule-kinetochore association promotes APC/C localization to chromosomes, thereby enhancing anaphase onset and mitotic exit.     

The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.     

Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes.

Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis [1--3]. In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation [4--7]. We investigated the role of xFizzy during meiosis using an antisense depletion approach. xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II. However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I [8]. Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I. Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk). Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity [7], but also to stimulate cyclin B synthesis in Meiosis II.     

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.     

How cyclin A destruction escapes the spindle assembly checkpoint

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C.     

Evidence that the spindle assembly checkpoint does not regulate APC(Fzy) activity in Drosophila female meiosis

The spindle assembly checkpoint (SAC) plays an important role in mitotic cells to sense improper chromosome attachment to spindle microtubules and to inhibit APC(Fzy)-dependent destruction of cyclin B and Securin; consequent initiation of anaphase until correct attachments are made. In Drosophila , SAC genes have been found to play a role in ensuring proper chromosome segregation in meiosis, possibly reflecting a similar role for the SAC in APC(Fzy) inhibition during meiosis. We found that loss of function mutations in SAC genes, Mad2, zwilch, and mps1, do not lead to the predicted rise in APC(Fzy)-dependent degradation of cyclin B either globally throughout the egg or locally on the meiotic spindle. Further, the SAC is not responsible for the inability of APC(Fzy) to target cyclin B and promote anaphase in metaphase II arrested eggs from cort mutant females. Our findings support the argument that SAC proteins play checkpoint independent roles in Drosophila female meiosis and that other mechanisms must function to control APC activity.     

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.     

Evidence That the Yeast Spindle Assembly Checkpoint Has a Target Other Than the Anaphase Promoting Complex

The spindle assembly checkpoint monitors biorientation of chromosomes on the metaphase spindle and inhibits the Anaphase Promoting Complex (APC) specificity factor Cdc20. If APC-Cdc20 is the sole target of the spindle checkpoint, then cells lacking APC and its targets, B-type cyclin and securin, would lack spindle checkpoint function. We tested this hypothesis in yeast cells that are APC-null. Surprisingly, we find that such yeast cells are able to activate the spindle assembly checkpoint, delaying cell cycle progression in G2/M phase. These data suggest that the spindle checkpoint has a non-APC target that can restrain anaphase onset.     

Inefficient degradation of cyclin B1 re-activates the spindle checkpoint right after sister chromatid disjunction

Sister chromatid separation creates a sudden loss of tension on kinetochores, which could, in principle, re-activate the spindle checkpoint in anaphase. This so-called “anaphase problem” is probably avoided by timely inactivation of cyclin B1-Cdk1, which may prevent the spindle tension sensing Aurora B kinase from destabilizing kinetochore–microtubule interactions as they lose tension in anaphase. However, exactly how spindle checkpoint re-activation is prevented remains unclear.

Here, we investigated how different degrees of cyclin B1 stabilization affected the spindle checkpoint in metaphase and anaphase. Cells expressing a strongly stabilized (R42A) mutant of cyclin B1 degraded APC/C^Cdc20 substrates normally, showing that checkpoint release was not inhibited by high cyclin B1-Cdk1 activity. However, after this initial wave of APC/C^Cdc20 activity, the spindle checkpoint returned in cells with uncohesed sister chromatids. Expression of a lysine mutant of cyclin B1 that is degraded only slightly inefficiently allowed a normal metaphase-to-anaphase transition. Strikingly, however, the spindle checkpoint returned in cells that had not degraded the cyclin B1 mutant 10–15 min after anaphase onset. When cyclin B1 remained in late anaphase, cytokinesis stalled, and translocation of INCENP from separated sister chromatids to the spindle midzone was blocked. This late anaphase arrest required the activity of Aurora B and Mps1. In conclusion, our results reveal that complete removal of cyclin B1 is essential to prevent the return of the spindle checkpoint following sister chromatid disjunction. Speculatively, increasing activity of APC/C^Cdc20 in late anaphase helps to keep cyclin B1 levels low.     

The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction

The spindle assembly checkpoint (SAC) is required to block sister chromatid separation until all chromosomes are properly attached to the mitotic apparatus. The SAC prevents cells from entering anaphase by inhibiting the ubiquitylation of cyclin B1 and securin by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. The target of the SAC is the essential APC/C activator Cdc20. It is unclear how the SAC inactivates Cdc20 but most current models suggest that Cdc20 forms a stable complex with the Mad2 checkpoint protein. Here we show that most Cdc20 is not in a complex with Mad2; instead Mad2 is required for Cdc20 to form a complex with another checkpoint protein, BubR1. We further show that during the SAC, the APC/C ubiquitylates Cdc20 to target it for degradation. Thus, ubiquitylation of human Cdc20 is not required to release it from the checkpoint complex, but to degrade it to maintain mitotic arrest.     

To cell cycle, swing the APC/C

For successful mitosis, Cyclin B1 and Securin must be degraded efficiently before anaphase. Destruction of these mitotic regulators by the 26S proteasome is the result of their poly-ubiquitination by a multi-subunit E3 ligase: the Anaphase-Promoting Complex or Cyclosome (APC/C). Clearly, the APC/C is not just important for mitosis. Destruction of APC/C substrates such as Cdc20, Plk1, Aurora A and Skp2 directs events in G1. Strikingly, the APC/C needs to stay active even in quiescent cells to keep them out of the cell cycle and forms an intriguing link with pRb. An inactive APC/C stabilizes Geminin, Cyclin A and Cyclin B1, thereby securing completion of DNA synthesis and progression through G2-phase. In prometaphase the APC/C becomes active again, but is controlled by the spindle assembly checkpoint. Here we discuss how the APC/C is either held in check or released. We argue that shedding more light on the APC/C is also important to understand cancer and could help the design of treatment.     

Cdc20 and Cks direct the spindle checkpoint-independent destruction of cyclin A

Successful mitosis requires the right protein be degraded at the right time. Central to this is the spindle checkpoint that prevents the destruction of securin and cyclin B1 when there are improperly attached chromosomes. The principal target of the checkpoint is Cdc20, which activates the anaphase-promoting complex/cyclosome (APC/C). A Drosophila Cdc20/fizzy mutant arrests in mitosis with high levels of cyclins A and B, but paradoxically the spindle checkpoint does not stabilize cyclin A. Here, we investigated this paradox and found that Cdc20 is rate limiting for cyclin A destruction. Indeed, Cdc20 binds efficiently to cyclin A before and in mitosis, and this complex has little associated Mad2. Furthermore, the cyclin A complex must bind to a Cks protein to be degraded independently of the checkpoint. Thus, we identify a crucial role for the Cks proteins in mitosis and one mechanism by which the APC/C can target substrates independently of the spindle checkpoint.     

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.     

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.