APC(Cdc20) promotes exit from mitosis by destroying the anaphase inhibitor Pds1 and cyclin Clb5

Ubiquitin-mediated proteolysis due to the anaphase-promoting complex/cyclosome (APC/C) is essential for separation of sister chromatids, requiring degradation of the anaphase inhibitor Pds1, and for exit from mitosis, requiring inactivation of cyclin B Cdk1 kinases. Exit from mitosis in yeast involves accumulation of the cyclin kinase inhibitor Sic1 as well as cyclin proteolysis mediated by APC/C bound by the activating subunit Cdh1/Hct1 (APC(Cdh1)). Both processes require the Cdc14 phosphatase, whose release from the nucleolus during anaphase causes dephosphorylation and thereby activation of Cdh1 and accumulation of another protein, Sic1 (refs 4-7). We do not know what determines the release of Cdc14 and enables it to promote Cdk1 inactivation, but it is known to be dependent on APC/C bound by Cdc20 (APC(Cdc20)) (ref. 4). Here we show that APC(Cdc20) allows activation of Cdc14 and promotes exit from mitosis by mediating proteolysis of Pds1 and the S phase cyclin Clb5 in the yeast Saccharomyces cerevisiae. Degradation of Pds1 is necessary for release of Cdc14 from the nucleolus, whereas degradation of Clb5 is crucial if Cdc14 is to overwhelm Cdk1 and activate its foes (Cdh1 and Sic1). Remarkably, cells lacking both Pds1 and Clb5 can proliferate in the complete absence of Cdc20.     

Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex

Cell-cycle transitions are driven by waves of ubiquitin-dependent degradation of key cell-cycle regulators. SCF (Skp1/Cullin/F-box protein) complexes and anaphase-promoting complexes (APC) represent two major classes of ubiquitin ligases whose activities are thought to regulate primarily the G1/S and metaphase/anaphase cell-cycle transitions, respectively. The major target of the Skp1/Cul1/Skp2 (SCF(SKP2)) complex is thought to be the Cdk inhibitor p27 during S phase, whereas the principal targets for the APC are thought to be involved in chromatid separation (securin) and exit from mitosis (cyclin B). Although the role of the APC in mitosis is relatively clear, there is mounting evidence that APCs containing Cdh1 (APC(CDH1)) also have a function in the G1 phase of the cell cycle. Here, we show that the F-box protein Skp2 is polyubiquitinated, and hence earmarked for destruction, by APC(CDH1). As a result, accumulation of SCF(SKP2) requires prior inactivation of APC(CDH1). These findings provide an insight into the orchestration of SCF and APC activities during cell-cycle progression, and into the involvement of the APC in G1.     

APC-dependent proteolysis of the mitotic cyclin Clb2 is essential for mitotic exit

Cyclin degradation is central to regulation of the cell cycle. Mitotic exit was proposed to require degradation of the S phase cyclin Clb5 by the anaphase-promoting complex activated by Cdc20 (APC(Cdc20)). Furthermore, Clb5 degradation was thought to be necessary for effective dephosphorylation and activation of the APC regulatory subunit Cdh1 (also known as Hct1) and the cyclin-dependent kinase inhibitor Sic1 by the phosphatase Cdc14, allowing mitotic kinase inactivation and mitotic exit. Here we show, however, that spindle disassembly and cell division occur without significant APC(Cdc20)-mediated Clb5 degradation, as well as in the absence of both Cdh1 and Sic1. We find instead that destruction-box-dependent degradation of the mitotic cyclin Clb2 is essential for mitotic exit. APC(Cdc20) may be required for an essential early phase of Clb2 degradation, and this phase may be sufficient for most aspects of mitotic exit. Cdh1 and Sic1 may be required for further inactivation of Clb2-Cdk1, regulating cell size and the length of G1.     

Positive feedback sharpens the anaphase switch

At the onset of anaphase, sister-chromatid cohesion is dissolved abruptly and irreversibly, ensuring that all chromosome pairs disjoin almost simultaneously. The regulatory mechanisms that generate this switch-like behaviour are unclear. Anaphase is initiated when a ubiquitin ligase, the anaphase-promoting complex (APC), triggers the destruction of securin, thereby allowing separase, a protease, to disrupt sister-chromatid cohesion. Here we demonstrate that the cyclin-dependent kinase 1 (Cdk1)-dependent phosphorylation of securin near its destruction-box motif inhibits securin ubiquitination by the APC. The phosphatase Cdc14 reverses securin phosphorylation, thereby increasing the rate of securin ubiquitination. Because separase is known to activate Cdc14 (refs 5 and 6), our results support the existence of a positive feedback loop that increases the abruptness of anaphase. Consistent with this model, we show that mutations that disrupt securin phosphoregulation decrease the synchrony of chromosome segregation. Our results also suggest that coupling securin degradation with changes in Cdk1 and Cdc14 activities helps coordinate the initiation of sister-chromatid separation with changes in spindle dynamics.     

Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor

The ubiquitylation of cell-cycle regulatory proteins by the large multimeric anaphase-promoting complex (APC/C) controls sister chromatid segregation and the exit from mitosis. Selection of APC/C targets is achieved through recognition of destruction motifs, predominantly the destruction (D)-box and KEN (Lys-Glu-Asn)-box. Although this process is known to involve a co-activator protein (either Cdc20 or Cdh1) together with core APC/C subunits, the structural basis for substrate recognition and ubiquitylation is not understood. Here we investigate budding yeast APC/C using single-particle electron microscopy and determine a cryo-electron microscopy map of APC/C in complex with the Cdh1 co-activator protein (APC/C(Cdh1)) bound to a D-box peptide at ～10 ? resolution. We find that a combined catalytic and substrate-recognition module is located within the central cavity of the APC/C assembled from Cdh1, Apc10--a core APC/C subunit previously implicated in substrate recognition--and the cullin domain of Apc2. Cdh1 and Apc10, identified from difference maps, create a co-receptor for the D-box following repositioning of Cdh1 towards Apc10. Using NMR spectroscopy we demonstrate specific D-box-Apc10 interactions, consistent with a role for Apc10 in directly contributing towards D-box recognition by the APC/C(Cdh1) complex. Our results rationalize the contribution of both co-activator and core APC/C subunits to D-box recognition and provide a structural framework for understanding mechanisms of substrate recognition and catalysis by the APC/C.     

Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities

The spindle checkpoint prevents chromosome mis-segregation by delaying sister chromatid separation until all chromosomes have achieved bipolar attachment to the mitotic spindle. Its operation is essential for accurate chromosome segregation, whereas its dysregulation can contribute to birth defects and tumorigenesis. The target of the spindle checkpoint is the anaphase-promoting complex (APC), a ubiquitin ligase that promotes sister chromatid separation and progression to anaphase. Using a short hairpin RNA screen targeting components of the ubiquitin-proteasome pathway in human cells, we identified the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) as a critical regulator of the spindle checkpoint. USP44 is not required for the initial recognition of unattached kinetochores and the subsequent recruitment of checkpoint components. Instead, it prevents the premature activation of the APC by stabilizing the APC-inhibitory Mad2-Cdc20 complex. USP44 deubiquitinates the APC coactivator Cdc20 both in vitro and in vivo, and thereby directly counteracts the APC-driven disassembly of Mad2-Cdc20 complexes (discussed in an accompanying paper). Our findings suggest that a dynamic balance of ubiquitination by the APC and deubiquitination by USP44 contributes to the generation of the switch-like transition controlling anaphase entry, analogous to the way that phosphorylation and dephosphorylation of Cdk1 by Wee1 and Cdc25 controls entry into mitosis.     

Structure of the mitotic checkpoint complex

In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31(comet) (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31(comet) disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.     

Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry

Oscillations in cyclin-dependent kinase (CDK) activity drive the somatic cell cycle. After entry into mitosis, CDKs activate the anaphase-promoting complex (APC), which then promotes cyclin degradation and mitotic exit. The re-accumulation of cyclin A causes the inactivation of APC and entry into S phase, but how cyclin A can accumulate in the presence of active APC has remained unclear. Here we show that, during G1, APC autonomously switches to a state permissive for cyclin A accumulation. Crucial to this transition is the APC(Cdh1)-dependent autoubiquitination and proteasomal degradation of the ubiquitin-conjugating enzyme (E2) UbcH10. Because APC substrates inhibit the autoubiquitination of UbcH10, but not its E2 function, APC activity is maintained as long as G1 substrates are present. Thus, through UbcH10 degradation and cyclin A stabilization, APC autonomously downregulates its activity. This indicates that the core of the metazoan cell cycle could be described as a self-perpetuating but highly regulated oscillator composed of alternating CDK and APC activities.     

Calcium triggers exit from meiosis II by targeting the APC/C inhibitor XErp1 for degradation

Vertebrate eggs awaiting fertilization are arrested at metaphase of meiosis II by a biochemical activity termed cytostatic factor (CSF). This activity inhibits the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that triggers anaphase onset and mitotic/meiotic exit by targeting securin and M-phase cyclins for destruction. On fertilization a transient rise in free intracellular calcium causes release from CSF arrest and thus APC/C activation. Although it has previously been shown that calcium induces the release of APC/C from CSF inhibition through calmodulin-dependent protein kinase II (CaMKII), the relevant substrates of this kinase have not been identified. Recently, we characterized XErp1 (Emi2), an inhibitor of the APC/C and key component of CSF activity in Xenopus egg extract. Here we show that calcium-activated CaMKII triggers exit from meiosis II by sensitizing the APC/C inhibitor XErp1 for polo-like kinase 1 (Plx1)-dependent degradation. Phosphorylation of XErp1 by CaMKII leads to the recruitment of Plx1 that in turn triggers the destruction of XErp1 by phosphorylating a site known to serve as a phosphorylation-dependent degradation signal. These results provide a molecular explanation for how the fertilization-induced calcium increase triggers exit from meiosis II.     

Emi1 is required for cytostatic factor arrest in vertebrate eggs

Vertebrate eggs are arrested at metaphase of meiosis II with stable cyclin B and high cyclin B/Cdc2 kinase activity. The ability of the anaphase-promoting complex/cyclosome (APC), an E3 ubiquitin ligase, to trigger cyclin B destruction and metaphase exit is blocked in eggs by the activity of cytostatic factor (CSF) (reviewed in ref. 1). CSF was defined as an activity in mature oocytes that caused mitotic arrest when injected into dividing embryos. Fertilization causes a transient increase in cytoplasmic calcium concentration leading to CSF inactivation, APC activation, cyclin B destruction and mitotic exit. The APC activator Cdc20 is required for APC activation after fertilization. We show here that the APC(cdc20) inhibitor Emi1 (ref. 6) is necessary and sufficient to inhibit the APC and to prevent mitotic exit in CSF-arrested eggs. CSF extracts immunodepleted of Emi1 degrade cyclin B, and exit from mitosis prematurely in the absence of calcium. Addition of Emi1 to these Emi1-depleted extracts blocks premature inactivation of the CSF-arrested state. Emi1 is required to arrest unfertilized eggs at metaphase of meiosis II and seems to be the long-sought mediator of CSF activity.     

Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation

Eukaryotic cells rely on a surveillance mechanism known as the spindle checkpoint to ensure accurate chromosome segregation. The spindle checkpoint prevents sister chromatids from separating until all kinetochores achieve bipolar attachments to the mitotic spindle. Checkpoint proteins tightly inhibit the anaphase-promoting complex (APC), a ubiquitin ligase required for chromosome segregation and progression to anaphase. Unattached kinetochores promote the binding of checkpoint proteins Mad2 and BubR1 to the APC-activator Cdc20, rendering it unable to activate APC. Once all kinetochores are properly attached, however, cells inactivate the checkpoint within minutes, allowing for the rapid and synchronous segregation of chromosomes. How cells switch from strong APC inhibition before kinetochore attachment to rapid APC activation once attachment is complete remains a mystery. Here we show that checkpoint inactivation is an energy-consuming process involving APC-dependent multi-ubiquitination. Multi-ubiquitination by APC leads to the dissociation of Mad2 and BubR1 from Cdc20, a process that is reversed by a Cdc20-directed de-ubiquitinating enzyme. The mutual regulation between checkpoint proteins and APC leaves the cell poised for rapid checkpoint inactivation and ensures that chromosome segregation promptly follows the completion of kinetochore attachment. In addition, our results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes.     

Control of the SCF(Skp2-Cks1) ubiquitin ligase by the APC/C(Cdh1) ubiquitin ligase

Skp2 and its cofactor Cks1 are the substrate-targeting subunits of the SCF(Skp2-Cks1) (Skp1/Cul1/F-box protein) ubiquitin ligase complex that regulates entry into S phase by inducing the degradation of the cyclin-dependent kinase inhibitors p21 and p27 (ref. 1). Skp2 is an oncoprotein that often shows increased expression in human cancers; however, the mechanism that regulates its cellular abundance is not well understood. Here we show that both Skp2 and Cks1 proteins are unstable in G1 and that their degradation is mediated by the ubiquitin ligase APC/C(Cdh1) (anaphase-promoting complex/cyclosome and its activator Cdh1). Silencing of Cdh1 by RNA interference in G1 cells stabilizes Skp2 and Cks1, with a consequent increase in p21 and p27 proteolysis. Depletion of Cdh1 also increases the percentage of cells in S phase, whereas concomitant downregulation of Skp2 reverses this effect, showing that Skp2 is an essential target of APC/C(Cdh1). Expression of a stable Skp2 mutant that cannot bind APC/C(Cdh1) induces premature entry into S phase. Thus, the induction of Skp2 and Cks1 degradation in G1 represents a principal mechanism by which APC/C(Cdh1) prevents the unscheduled degradation of SCF(Skp2-Cks1) substrates and maintains the G1 state.     

Stabilization of microtubule dynamics at anaphase onset promotes chromosome segregation

Microtubules of the mitotic spindle form the structural basis for chromosome segregation. In metaphase, microtubules show high dynamic instability, which is thought to aid the 'search and capture' of chromosomes for bipolar alignment on the spindle. Microtubules suddenly become more stable at the onset of anaphase, but how this change in microtubule behaviour is regulated and how important it is for the ensuing chromosome segregation are unknown. Here we show that in the budding yeast Saccharomyces cerevisiae, activation of the phosphatase Cdc14 at anaphase onset is both necessary and sufficient for silencing microtubule dynamics. Cdc14 is activated by separase, the protease that triggers sister chromatid separation, linking the onset of anaphase to microtubule stabilization. If sister chromatids separate in the absence of Cdc14 activity, microtubules maintain high dynamic instability; this correlates with defects in both the movement of chromosomes to the spindle poles (anaphase A) and the elongation of the anaphase spindle (anaphase B). Cdc14 promotes localization of microtubule-stabilizing proteins to the anaphase spindle, and dephosphorylation of the kinetochore component Ask1 contributes to both the silencing of microtubule turnover and successful anaphase A.