Cdc20 proteolysis requires p38 MAPK signaling and Cdh1‐independent APC/C ubiquitination during spindle assembly checkpoint activation by cadmium

Cdc20, an activator of the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase, initiates the destruction of key mitotic regulators to facilitate mitosis, while it is negatively regulated by the spindle assembly checkpoint (SAC) to prevent premature anaphase entry. Activation of the p38 mitogen‐activated protein kinase could contribute to mitotic arrest, but the underlying mechanism is unknown. Here we report a novel pathway in which the p38 signaling triggers Cdc20 destruction under SAC elicited by cadmium, a human carcinogen. We found that the cadmium‐induced prometaphase arrest was linked to decreased Cdc20 and accumulated cyclin A protein levels in human cells, whereas the activity of cyclin B1–Cdk1 was unaffected. The Cdc20 half‐life was markedly shortened along with its ubiquitination and degradation via 26S proteasome in cadmium‐treated asynchronous or G₂‐enriched cells. Depletion of APC3 markedly suppressed the cadmium‐induced Cdc20 ubiquitination and proteolysis, while depletion of Cdh1, another activator of APC/C, did not. Intriguingly, blockage of p38 activity restored the Cdc20 levels for continuing mitosis under cadmium, while inhibition of JNK activity had no effect. The cadmium‐induced Cdc20 proteolysis was also suppressed during transient depletion of p38α or stable expression a dominant negative form of p38. Inhibition of p38 abolished the induction of Mad2–Cdc20–APC3 complex by cadmium. Moreover, forced expression of MKK6–p38 signaling could promote Cdc20 degradation in a Cdh1‐independent APC/C pathway. In summary, accelerated ubiquitination and proteolysis of Cdc20 is essential for prometaphase arrest that is mediated via the p38 signaling during SAC activation. J. Cell. Physiol. 223: 327–334, 2010. © 2010 Wiley‐Liss, Inc.     

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.     

Cdc20: a WD40 activator for a cell cycle degradation machine

Cdc20 is an essential cell-cycle regulator required for the completion of mitosis in organisms from yeast to man and contains at its C terminus a WD40 repeat domain that mediates protein-protein interactions. In mitosis, Cdc20 binds to and activates the ubiquitin ligase activity of a large molecular machine called the anaphase-promoting complex/cyclosome (APC/C) and enables the ubiquitination and degradation of securin and cyclin B, thus promoting the onset of anaphase and mitotic exit. APC/C(Cdc20) is temporally and spatially regulated during the somatic and embryonic cell cycle by numerous mechanisms, including the spindle checkpoint and the cytostatic factor (CSF). Therefore, Cdc20 serves as an integrator of multiple intracellular signaling cascades that regulate progression through mitosis. This review summarizes recent progress toward the understanding of the functions of Cdc20, the mechanisms by which it activates APC/C, and its regulation by phosphorylation and by association with its binding proteins.     

Mitotic regulation of the human anaphase-promoting complex by phosphorylation

The anaphase-promoting complex (APC) or cyclosome is a ubiquitin ligase that initiates anaphase and mitotic exit. APC activation is thought to depend on APC phosphorylation and Cdc20 binding. We have identified 43 phospho-sites on APC of which at least 34 are mitosis specific. Of these, 32 sites are clustered in parts of Apc1 and the tetratricopeptide repeat (TPR) subunits Cdc27, Cdc16, Cdc23 and Apc7. In vitro, at least 15 of the mitotic phospho-sites can be generated by cyclin-dependent kinase 1 (Cdk1), and 3 by Polo-like kinase 1 (Plk1). APC phosphorylation by Cdk1, but not by Plk1, is sufficient for increased Cdc20 binding and APC activation. Immunofluorescence microscopy using phospho-antibodies indicates that APC phosphorylation is initiated in prophase during nuclear uptake of cyclin B1. In prometaphase phospho-APC accumulates on centrosomes where cyclin B ubiquitination is initiated, appears throughout the cytosol and disappears during mitotic exit. Plk1 depletion neither prevents APC phosphorylation nor cyclin A destruction in vivo. These observations imply that APC activation is initiated by Cdk1 already in the nuclei of late prophase cells.     

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

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

The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 and Cdh1 leads to ubiquitin-dependent degradation of securin and cyclin B and thereby promotes the initiation of anaphase and exit from mitosis. Cyclin B and securin ubiquitination depend on a destruction box (D box) sequence in these proteins, but how APC/C bound to Cdc20 or Cdh1 recognizes the D box is poorly understood. By using site-specific photocrosslinking in combination with mutational analyses, we show that the D box directly interacts with an evolutionarily conserved surface on the predicted WD40 propeller structure of Cdh1 and that this interaction is essential for processive substrate ubiquitination. We further show that Cdh1 specifically crosslinks to the APC/C subunit Cdc27 and that Cdh1 binding to APC/C depends on the presence of Cdc27. Our data imply that APC/C is activated by the association of Cdh1 with Cdc27, which enables APC/C to recognize the D box of substrates via Cdh1's propeller domain.     

Emi1 is a mitotic regulator that interacts with Cdc20 and inhibits the anaphase promoting complex

We have discovered an early mitotic inhibitor, Emi1, which regulates mitosis by inhibiting the anaphase promoting complex/cyclosome (APC). Emi1 is a conserved F box protein containing a zinc binding region essential for APC inhibition. Emi1 accumulates before mitosis and is ubiquitylated and destroyed in mitosis, independent of the APC. Emi1 immunodepletion from cycling Xenopus extracts strongly delays cyclin B accumulation and mitotic entry, whereas nondestructible Emi1 stabilizes APC substrates and causes a mitotic block. Emi1 binds the APC activator Cdc20, and Cdc20 can rescue an Emi1-induced block to cyclin B destruction. Our results suggest that Emi1 regulates progression through early mitosis by preventing premature APC activation, and may help explain the well-known delay between cyclin B/Cdc2 activation and cyclin B destruction.     

Defining pathways of spindle checkpoint silencing: functional redundancy between Cdc20 ubiquitination and p31(comet)

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.     

Cdc20 protein contains a destruction-box but, unlike Clb2, its proteolysisis not acutely dependent on the activity of anaphase-promoting complex.

Both chromosome segregation and the final exit from mitosis require a ubiquitin-protein ligase called anaphase-promoting complex (APC) or cyclosome. This multiprotein complex ubiquitinates various substrates, such as the anaphase inhibitor Pds1 and mitotic cyclins, and thus targets them for proteolysis by the 26S proteasome. The ubiquitination by APC is dependent on the presence of a destruction-box sequence in the N-terminus of target proteins. Recent reports have strongly suggested that Cdc20, a WD40 repeat-containing protein required for nuclear division in the budding yeast Saccharomyces cerevisiae, is essential for the APC-mediated proteolysis. To understand the function of CDC20, we have studied its regulation in some detail. The expression of the CDC20 gene is cell-cycle regulated such that it is transcribed only during late S phase and mitosis. Although the protein is unstable to some extent through out the cell cycle, its degradation is particularly enhanced in G1. Cdc20 contains a destruction box sequence which, when mutated or deleted, stabilizes it considerably in G1. Surprisingly, we find that while the inactivation of APC subunits Cdc16, Cdc23 or Cdc27 results in stabilization of the mitotic cyclin Clb2 in G1, the proteolytic destruction of Cdc20 remains largely unaffected. This suggests the existence of proteolytic mechanisms in G1 that can degrade destruction-box containing proteins, such as Cdc20, in an APC-independent manner.     

The destruction box of the cyclin Clb2 binds the anaphase-promoting complex/cyclosome subunit Cdc23

Properly regulated cyclin proteolysis is critical for normal cell cycle progression. A nine-amino acid peptide motif called the destruction box (D box) is present at the N terminus of the yeast mitotic cyclins. This short sequence is required for cyclin ubiquitination and subsequent proteolysis. The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 required for cyclin ubiquitination. We have tested the D box of five mitotic cyclins for interaction with six APC/C subunits. The APC/C subunit Cdc23, but not five other subunits tested, interacted by two-hybrid analysis with the N terminus of wild-type Clb2. None of these subunits interacted with the N termini of the cyclins Clb1, Clb3, or Clb5. Mutations in the D box sequences of Clb2 inhibited interaction with Cdc23 both in vivo and in vitro. Our results provide the first evidence for a direct interaction between an APC/C substrate (Clb2) and an APC/C subunit (Cdc23).     

Methods to measure ubiquitin-dependent proteolysis mediated by the anaphase-promoting complex

The anaphase-promoting complex (APC) or cyclosome is a multi-subunit ubiquitin ligase that controls progression through mitosis and the G1-phase of the cell cycle. The APC ubiquitinates regulatory proteins such as securin and cyclin B and thereby targets them for destruction by the 26S proteasome. Activation of the APC depends on the activator proteins Cdc20 and Cdh1, which are thought to recruit substrates to the APC. In vitro, APC's RING finger subunit Apc11 alone can also function as a ubiquitin ligase. Here, we review different methods that have been used to measure the ubiquitination activity of the APC in vitro and to analyze APC-mediated degradation reactions either in vitro or in vivo. We describe procedures to isolate the APC from human cells or from Xenopus eggs, to activate purified APC with recombinant Cdc20 or Cdh1 and to measure the ubiquitination activity of the resulting APC(Cdc20) and APC(Cdh1) complexes. We also describe procedures to analyze the ubiquitination activity associated with recombinant Apc11.     

TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast.

The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.     

CDKs promote DNA replication origin licensing in human cells by protecting Cdc6 from APC/C-dependent proteolysis

Cyclin-dependent kinases (CDKs) restrict DNA replication origin firing to once per cell cycle by preventing the assembly of prereplicative complexes (pre-RCs; licensing) outside of G1 phase. Paradoxically, under certain circumstances, CDKs such as cyclin E-cdk2 are also required to promote licensing. Here, we show that CDK phosphorylation of the essential licensing factor Cdc6 stabilizes it by preventing its association with the anaphase promoting complex/cyclosome (APC/C). APC/C-dependent Cdc6 proteolysis prevents pre-RC assembly in quiescent cells and, when cells reenter the cell cycle from quiescence, CDK-dependent Cdc6 stabilization allows Cdc6 to accumulate before the licensing inhibitors geminin and cyclin A which are also APC/C substrates. This novel mechanism for regulating protein stability establishes a window of time prior to S phase when pre-RCs can assemble which we propose represents a critical function of cyclin E.     

Anaphase-promoting complex/cyclosome-dependent proteolysis of human cyclin A starts at the beginning of mitosis and is not subject to the spindle assembly checkpoint

Cyclin A is a stable protein in S and G2 phases, but is destabilized when cells enter mitosis and is almost completely degraded before the metaphase to anaphase transition. Microinjection of antibodies against subunits of the anaphase-promoting complex/cyclosome (APC/C) or against human Cdc20 (fizzy) arrested cells at metaphase and stabilized both cyclins A and B1. Cyclin A was efficiently polyubiquitylated by Cdc20 or Cdh1-activated APC/C in vitro, but in contrast to cyclin B1, the proteolysis of cyclin A was not delayed by the spindle assembly checkpoint. The degradation of cyclin B1 was accelerated by inhibition of the spindle assembly checkpoint. These data suggest that the APC/C is activated as cells enter mitosis and immediately targets cyclin A for degradation, whereas the spindle assembly checkpoint delays the degradation of cyclin B1 until the metaphase to anaphase transition. The "destruction box" (D-box) of cyclin A is 10-20 residues longer than that of cyclin B. Overexpression of wild-type cyclin A delayed the metaphase to anaphase transition, whereas expression of cyclin A mutants lacking a D-box arrested cells in anaphase.     

The Polo-like kinase Cdc5p and the WD-repeat protein Cdc20p/fizzy are regulators and substrates of the anaphase promoting complex in Saccharomyces cerevisiae.

Proteolysis mediated by the anaphase promoting complex (APC) has a crucial role in regulating the passage of cells through anaphase. Destruction of the anaphase inhibitor Pds1p is necessary for separation of sister chromatids, whereas destruction of the mitotic cyclin Clb2p is important for disassembly of the mitotic spindle, cytokinesis and re-replication of the genome. Pds1p proteolysis precedes that of Clb2p by at least 15 min, which helps to ensure that cells never re-replicate their genome before they have separated sister chromatids at the previous mitosis. What triggers Pds1p proteolysis and why does it not also trigger that of Clb2p? Apart from sharing a dependence on the APC, these two proteolytic events differ in their dependence on other cofactors. Pds1p proteolysis depends on a WD-repeat protein called Cdc20p, whereas Clb2p proteolysis depends on another, related WD protein called Hct1/Cdh1p. On the other hand, destruction of Clb2p, but not that of Pds1p, depends on the Polo-like kinase, Cdc5p. Cdc20p is essential for separation of sister chromatids, whereas Cdc5p is not. We show that both Cdc5p and Cdc20p are unstable proteins whose proteolysis is regulated by the APC. Both proteins accumulate during late G2/M phase and disappear at a late stage of anaphase. Accumulation of Cdc20p contributes to activation of Pds1p proteolysis in metaphase, whereas accumulation of Cdc5p facilitates the activation of Clb2p proteolysis.     

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.     

Overexpression of ubiquitin specific protease 44 (USP44) induces chromosomal instability and is frequently observed in human T-cell leukemia

Cdc20-anaphase promoting complex/cyclosome (Cdc20-APC/C) E3 ubiquitin ligase activity is essential for orderly mitotic progression. The deubiqituinase USP44 was identified as a key regulator of APC/C and has been proposed to suppress Cdc20-APC/C activity by maintaining its association with the inhibitory protein Mad2 until all chromosomes are properly attached to the mitotic spindle. However, this notion has been challenged by data in which a lysine-less mutant of Cdc20 leads to premature anaphase, suggesting that it's ubiquitination is not required for APC/C activation. To further evaluate its role in checkpoint function and chromosome instability, we studied the consequences of over-expression of mouse Usp44 in non-transformed murine embryonic fibroblasts. Here we show that cells with high Usp44 are prone to chromosome segregation errors and aneuploidization. We find that high Usp44 promotes association of Mad2 with Cdc20 and reinforces the mitotic checkpoint. Surprisingly, the APC/C-Cdc20 substrate cyclin B1 is stabilized in G2 when Usp44 is over-expressed, but is degraded with normal kinetics once cells enter mitosis. Furthermore, we show that USP44 expression is elevated in subset of T-cell leukemias. These data are consistent with an important role for USP44 in regulating Cdc20-APC/C activity and suggest that high levels of this enzyme may contribute to the pathogenesis of T-ALL.     

Cdc28 and Cdc14 control stability of the anaphase-promoting complex inhibitor Acm1

The anaphase-promoting complex (APC) regulates the eukaryotic cell cycle by targeting specific proteins for proteasomal degradation. Its activity must be strictly controlled to ensure proper cell cycle progression. The co-activator proteins Cdc20 and Cdh1 are required for APC activity and are important regulatory targets. Recently, budding yeast Acm1 was identified as a Cdh1 binding partner and APC(Cdh1) inhibitor. Acm1 disappears in late mitosis when APC(Cdh1) becomes active and contains conserved degron-like sequences common to APC substrates, suggesting it could be both an inhibitor and substrate. Surprisingly, we found that Acm1 proteolysis is independent of APC. A major determinant of Acm1 stability is phosphorylation at consensus cyclin-dependent kinase sites. Acm1 is a substrate of Cdc28 cyclin-dependent kinase and Cdc14 phosphatase both in vivo and in vitro. Mutation of Cdc28 phosphorylation sites or conditional inactivation of Cdc28 destabilizes Acm1. In contrast, inactivation of Cdc14 prevents Acm1 dephosphorylation and proteolysis. Cdc28 stabilizes Acm1 in part by promoting binding of the 14-3-3 proteins Bmh1 and Bmh2. We conclude that the opposing actions of Cdc28 and Cdc14 are primary factors limiting Acm1 to the interval from G(1)/S to late mitosis and are capable of establishing APC-independent expression patterns similar to APC substrates.     

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.     

Prophase destruction of Emi1 by the SCF(betaTrCP/Slimb) ubiquitin ligase activates the anaphase promoting complex to allow progression beyond prometaphase

Progression through mitosis occurs because cyclin B/Cdc2 activation induces the anaphase promoting complex (APC) to cause cyclin B destruction and mitotic exit. To ensure that cyclin B/Cdc2 does not prematurely activate the APC in early mitosis, there must be a mechanism delaying APC activation. Emi1 is a protein capable of inhibiting the APC in S and G2. We show here that Emi1 is phosphorylated by Cdc2, and on a DSGxxS consensus site, is subsequently recognized by the SCF(betaTrCP/Slimb) ubiquitin ligase and destroyed, thus providing a delay for APC activation. Failure of betaTrCP-dependent Emi1 destruction stabilizes APC substrates and results in mitotic catastrophe including centrosome overduplication, potentially explaining mitotic deficiencies in Drosophila Slimb/betaTrCP mutants. We hypothesize that Emi1 destruction relieves a late prophase checkpoint for APC activation.