Budding yeast Cdc20: a target of the spindle checkpoint

The spindle checkpoint regulates the cell division cycle by keeping cells with defective spindles from leaving mitosis. In the two-hybrid system, three proteins that are components of the checkpoint, Mad1, Mad2 (see Drosophila Mad2), and Mad3, were shown to interact with Cdc20, a protein required for exit from mitosis. Mad2 and Mad3 coprecipitate with Cdc20 at all stages of the cell cycle. The binding of Mad2 depends on Mad1, and Mad3 depends on Mad1 and Mad2. Overexpression of Cdc20 allows cells with a depolymerized spindle or damaged DNA to leave mitosis but does not overcome the arrest caused by unreplicated DNA. Mutants in Cdc20 that are resistant to the spindle checkpoint no longer bind Mad proteins, suggesting that Cdc20 is the target of the spindle checkpoint (Huang, 1998).

The spindle assembly checkpoint mechanism delays anaphase initiation until all chromosomes are aligned at the metaphase plate. Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a target for the checkpoint intervention. The human checkpoint protein hMAD2 prevents activation of APC by forming a hMAD2-CDC20-APC complex. When injected into Xenopus embryos, hMAD2 arrests cells at mitosis with an inactive APC. The recombinant hMAD2 protein exists in two-folded states: a tetramer and a monomer. Both the tetramer and the monomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression. Thus, hMAD2 binding is not sufficient for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal. There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2-CDC20-APC ternary complex present at metaphase, a CDC20-APC binary complex active in degrading specific substrates at anaphase, and a CDH1-APC complex active later in mitosis and in G1. It is concluded that the checkpoint-mediated cell cycle arrest involves hMAD2 receiving an upstream signal to inhibit activation of APC (Fang, 1998).

The spindle checkpoint must detect the presence of unattached or improperly attached kinetochores and must then inhibit progression through the cell cycle until the offending condition is resolved. Detection probably involves attachment-sensitive kinetochore phosphorylation. A key player in the checkpoint's response is the Mad2 protein, which prevents activation of the anaphase-promoting complex (APC) by the Cdc20 protein. Microinjection of Mad2 antibodies results in premature anaphase onset, and excess Mad2 protein causes arrest in mitosis. Mad2 localizes to unattached kinetochores in vertebrate cells, and this localization ceases as kinetochores accumulate microtubules. But how is Mad2 binding limited to unattached kinetochores? This study used lysed PtK1 cells to study kinetochore phosphorylation and Mad2 binding. It was found that Mad2 binds to phosphorylated kinetochores, but not to unphosphorylated ones. These data suggest that it is kinetochore protein phosphorylation that promotes Mad2 binding to unattached kinetochores. Thus, a probable molecular link has been identified between attachment-sensitive kinetochore phosphorylation and the inhibition of anaphase. The complete pathway for error control in mitosis can now be outlined (Waters, 1999).

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 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. It is not known 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)]. 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 (Shirayama, 1999).

Exit from mitosis in budding yeast: biphasic inactivation of the Cdc28-Clb2 mitotic kinase and the role of Cdc20

Cdc20, an activator of the anaphase-promoting complex (APC), is also required for the exit from mitosis in Saccharomyces cerevisiae. During mitosis, both the inactivation of Cdc28-Clb2 kinase and the degradation of mitotic cyclin Clb2 occur in two steps. The first phase of Clb2 proteolysis, which commences at the metaphase-to-anaphase transition when Clb2 abundance is high, is dependent on Cdc20. The second wave of Clb2 destruction in telophase requires activation of the Cdc20 homolog, Hct1/Cdh1. The first phase of Clb2 destruction, which lowers the Cdc28-Clb2 kinase activity, is a prerequisite for the second. Thus, Clb2 proteolysis is not solely mediated by Hct1 as generally believed; instead, it requires a sequential action of both Cdc20 and Hct1 (Yeong, 2000).

Structure and degradation of Cdc20 in yeast

The precise order of molecular events during cell cycle progression depends upon ubiquitin-mediated proteolysis of cell cycle regulators. Hsl1p, a protein kinase that inhibits the Swe1p protein kinase in a bud morphogenesis checkpoint, is targeted for ubiquitin-mediated turnover by the anaphase-promoting complex (APC). Regions of Hsl1p that are critical both for binding to the APC machinery and for APC-mediated degradation have been investigated. Hsl1p contains both a destruction box (D box) and a KEN box motif that are necessary for Hsl1p turnover with either APC(Cdc20) or APC(Cdh1). In coimmunoprecipitation studies, the D box of full-length Hsl1p is critical for association with Cdc20p, whereas the KEN box is important for association with Cdh1p. Fusion of a 206-amino-acid fragment of Hsl1p containing these motifs to a heterologous protein results in APC-dependent degradation of the fusion protein that requires intact D box and KEN box motifs. Finally, this bacterially expressed Hsl1p fusion protein interacts with Cdc20p and Cdh1p either translated in vitro or expressed in and purified from insect cells. Binding to Cdc20p and Cdh1p is disrupted completely by a D box/KEN box double mutant. These results indicate that D box and KEN box motifs are important for direct binding to the APC machinery, leading to ubiquitination and subsequent protein degradation (Burton, 2001).

The anaphase inhibitor Pds1 binds to the APC/C-associated protein Cdc20 in a destruction box-dependent manner

An essential aspect of progression through mitosis is the sequential degradation of key mitotic regulators in a process that is mediated by the anaphase promoting complex/cyclosome (APC/C) ubiquitin ligase. In mitotic cells, two forms of the APC/C exist, APC/C(Cdc20) and APC/C(Cdh1), which differ in their associated WD-repeat proteins (Cdc20 and Cdh1, respectively), time of activation, and substrate specificity. How the WD-repeat proteins contribute to APC/C's activation and substrate specificity is not clear. Many APC/C substrates contain a destruction box element that is necessary for their ubiquitination. One such APC/C substrate, the budding yeast anaphase inhibitor Pds1 (securin), is degraded prior to anaphase initiation in a destruction box and APC/C(Cdc20)-dependent manner. Pds1 interacts directly with Cdc20 and that this interaction requires Pds1's destruction box. These results suggest that Cdc20 provides a link between the substrate and the core APC/C and that the destruction box is essential for efficient Cdc20-substrate interaction. Pds1 does not interact with Cdh1. Finally, the effect of spindle assembly checkpoint activation, known to inhibit APC/C function, on the Pds1-Cdc20 interaction was examined (Hilioti, 2001).

BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase

BubR1 is an essential mitotic checkpoint protein with multiple functional domains. It has been implicated in mitotic checkpoint control, as an active kinase at unattached kinetochores, and as a cytosolic inhibitor of APC/C(Cdc20) activity, as well as in mitotic timing and stable chromosome-spindle attachment. Using BubR1-conditional knockout cells and BubR1 domain mutants, it was demonstrated that the N-terminal Cdc20 binding domain of BubR1 is essential for all of these functions, whereas its C-terminal Cdc20-binding domain, Bub3-binding domain, and kinase domain are not. The BubR1 N terminus binds to Cdc20 in a KEN box-dependent manner to inhibit APC/C activity in interphase, thereby allowing accumulation of cyclin B in G(2) phase prior to mitosis onset. Together, these results suggest that kinetochore-bound BubR1 is nonessential and that soluble BubR1 functions as a pseudosubstrate inhibitor of APC/C(Cdc20) during interphase to prevent unscheduled degradation of specific APC/C substrates (Malureanu, 2009).

Structure of the APC and its interaction with the APC adaptors

The anaphase-promoting complex or cyclosome (APC) is an unusually complicated ubiquitin ligase, composed of 13 core subunits and either of two loosely associated regulatory subunits, Cdc20 and Cdh1. The architecture of the APC was analyzed using a recently constructed budding yeast strain that is viable in the absence of normally essential APC subunits. The largest subunit, Apc1, serves as a scaffold that associates independently with two separable subcomplexes, one that contains Apc2 (Cullin), Apc11 (RING), and Doc1/Apc10, and another that contains the three TPR subunits (Cdc27, Cdc16, and Cdc23). The three TPR subunits display a sequential binding dependency, with Cdc27 the most peripheral, Cdc23 the most internal, and Cdc16 between. Apc4, Apc5, Cdc23, and Apc1 associate interdependently, such that loss of any one subunit greatly reduces binding between the remaining three. Intriguingly, the cullin and TPR subunits both contribute to the binding of Cdh1 to the APC. Enzymatic assays performed with APC purified from strains lacking each of the essential subunits revealed that only cdc27Δ complexes retain detectable activity in the presence of Cdh1. This residual activity depends on the C-box domain of Cdh1, but not on the C-terminal IR domain, suggesting that the C-box mediates a productive interaction with an APC subunit other than Cdc27. The IR domain of Cdc20 is dispensable for viability, suggesting that Cdc20 can activate the APC through another domain. This study has provided an updated model for the subunit architecture of the APC (Thornton, 2006).

Inhibition of yeast CDC20-APC by checkpoint proteins

Anaphase-promoting complex (APC), a ubiquitin ligase, controls both sister chromatid separation and mitotic exit. The APC is activated in mitosis and G1 by CDC20 and CDH1, and inhibited by the checkpoint protein MAD2, a specific inhibitor of CDC20. A MAD2 homolog MAD2B also inhibits APC. In contrast to MAD2, MAD2B inhibits both CDH1-APC and CDC20-APC. This inhibition is targeted to CDH1 and CDC20, but not directly to APC. Unlike MAD2, whose interaction with MAD1 is required for mitotic checkpoint control, MAD2B does not interact with MAD1, suggesting that MAD2B may relay a different cellular signal to APC (Chen, 2001).

Accumulation of Mad2-Cdc20 complex during spindle checkpoint activation requires binding of open and closed conformers of Mad2 in Saccharomyces cerevisiae

The spindle assembly checkpoint (SAC) coordinates mitotic progression with sister chromatid alignment. In mitosis, the checkpoint machinery accumulates at kinetochores, which are scaffolds devoted to microtubule capture. The checkpoint protein Mad2 (mitotic arrest deficient 2) adopts two conformations: open (O-Mad2) and closed (C-Mad2). C-Mad2 forms when Mad2 binds its checkpoint target Cdc20 or its kinetochore receptor Mad1. When unbound to these ligands, Mad2 folds as O-Mad2. In HeLa cells, an essential interaction between C- and O-Mad2 conformers allows Mad1-bound C-Mad2 to recruit cytosolic O-Mad2 to kinetochores. This study shows that the interaction of the O and C conformers of Mad2 is conserved in Saccharomyces cerevisiae. MAD2 mutant alleles impaired in this interaction fail to restore the SAC in a mad2 deletion strain. The corresponding mutant proteins bind Mad1 normally, but their ability to bind Cdc20 is dramatically impaired in vivo. This biochemical and genetic evidence shows that the interaction of O- and C-Mad2 is essential for the SAC and is conserved in evolution (Nezi, 2006).

A mutual inhibition between APC/C and its substrate Mes1 required for meiotic progression in fission yeast

The anaphase-promoting complex/cyclosome (APC/C) is a cell-cycle-regulated essential E3 ubiquitin ligase; however, very little is known about its meiotic regulation. Fission yeast Mes1 is a substrate of the APC/C as well as an inhibitor, allowing autoregulation of the APC/C in meiosis. Mes1 blocks cyclin B/Cdc13 proteolysis in meiosis I to ensure that sufficient levels of cyclin B remain to initiate meiosis II. Both traits require a functional destruction box (D box) and KEN box. Mes1 directly binds the WD40 domain of the Fizzy family of APC/C activators. Intriguingly, expression of nonubiquitylatable Mes1 blocks cells in metaphase I with high levels of APC/C substrates, suggesting that ubiquitylation of Mes1 is required for partial degradation of cyclin B in meiosis I by alleviating Mes1 inhibitory function. Consistently, a ternary complex, APC/C-Fizzy/Cdc20-Mes1, is stabilized by inhibiting Mes1 ubiquitylation. These results demonstrate that the fine-tuning of the APC/C activity, by a substrate that is also an inhibitor, is required for the precise coordination and transition through meiosis (Kimata, 2008).

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, mice have been obtained which lack Cdc20 function. These mice show failed embryogenesis. The embryos arrest 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 (Li, 2007).

Persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression

Cell cycle progression is driven by waves of cyclin expression coupled with regulated protein degradation. An essential step for initiating mitosis is the inactivation of proteolysis mediated by the anaphase-promoting complex/cyclosome bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) has been proposed to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). In a normal cell cycle APC(Cdh1) is inactivated in a graded manner and is not extinguished until S phase. Complete inactivation of APC(Cdh1) requires S phase cyclins. Further, persistent APC(Cdh1) activity throughout G1 helps to ensure the proper timing of Cdc20p expression. This suggests that S phase cyclins have an important role in allowing the accumulation of mitotic cyclins and further suggests a regulatory loop among S phase cyclins, APC(Cdh1), and APC(Cdc20) (Huang, 2001).

The ordered activation of the ubiquitin protein ligase anaphase-promoting complex or cyclosome by CDC20 in metaphase and by CDH1 in telophase is essential for anaphase and for exit from mitosis, respectively. CDC20 can only bind to and activate the mitotically phosphorylated form of the Xenopus and the human APC in vitro. In contrast, the analysis of phosphorylated and nonphosphorylated forms of CDC20 suggests that CDC20 phosphorylation is neither sufficient nor required for APC activation. On the basis of these results and the observation that APC phosphorylation correlates with APC activation in vivo, it was proposed that mitotic APC phosphorylation is an important mechanism that controls the proper timing of APC(CDC20) activation. CDH1 is phosphorylated in vivo during S, G2, and M phase, and CDH1 levels fluctuate during the cell cycle. In vitro, phosphorylated CDH1 neither binds to nor activates the APC as efficiently as does nonphosphorylated CDH1. Nonphosphorylatable CDH1 mutants constitutively activate APC in vitro and in vivo, whereas mutants mimicking the phosphorylated form of CDH1 are constitutively inactive. These results suggest that mitotic kinases have antagonistic roles in regulating APC(CDC20) and APC(CDH1); the phosphorylation of APC subunits is required to allow APC activation by CDC20, whereas the phosphorylation of CDH1 prevents activation of the APC by CDH1. These mechanisms can explain the temporal order of APC activation by CDC20 and CDH1 and may help to ensure that exit from mitosis is not initiated before anaphase has occurred (Kramer, 2000).

Substrate recognition by the Cdc20 and Cdh1 components of the anaphase-promoting complex

The specificity of ubiquitin-mediated protein degradation with regard to the selection of substrates to be polyubiquitinated has only been determined rather recently. Substrate targeting by the N-end rule and HECT (homology to E6AP carboxyl terminus) domain ubiquitin ligases occurs through substrate-specific binding domains. In contrast, the SCF complex recruits substrates through a substrate adaptor protein, the F-box subunit. Despite evidence showing that Cdc20 and Cdh1 bind and activate the anaphase-promoting complex in a substrate-specific manner, there is no evidence that the activating protein and substrate interact directly; hence, no clear model exists for the mechanism of APC activation or recruitment of substrates. This study shows that the activators Cdc20 and Cdh1 can associate with substrates via their N termini. In the absence of APC, Cdc20 and Cdh1 bind substrates reflecting Cdc20-APC and Cdh1-APC specificity. The N termini of Cdc20 and Cdh1 provide specificity functionally, as demonstrated by the generation of active chimeras that display the specificity corresponding to their N termini. Thus, Cdc20 and Cdh1 act as both substrate recognition and activating modules for APC (Pfleger, 2001; full text of article).

Chromosome segregation and mitotic exit depend on activation of the anaphase-promoting complex (APC) by the substrate adaptor proteins CDC20 and CDH1. The APC is a ubiquitin ligase composed of at least 11 subunits. The interaction of APC2 and APC11 with E2 enzymes is sufficient for ubiquitination reactions, but the functions of most other subunits are unknown. Subcomplexes of the human APC have been biochemically characterized. One subcomplex, containing APC2/11, APC1, APC4, and APC5, can assemble multiubiquitin chains but is unable to bind CDH1 and to ubiquitinate substrates. The other subcomplex contains all known APC subunits except APC2/11. This subcomplex can recruit CDH1 but fails to support any ubiquitination reaction. In vitro, the C termini of CDC20 and CDH1 bind to the closely related TPR subunits APC3 and APC7. Homology modeling predicts that these proteins are similar in structure to the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. APC activation by CDH1 depends on a conserved C-terminal motif that is also found in CDC20 and APC10. It is concluded that APC1, APC4, and APC5 may connect APC2/11 with TPR subunits. TPR domains in APC3 and APC7 recruit CDH1 to the APC and may thereby bring substrates into close proximity of APC2/11 and E2 enzymes. By analogy to PEX5, the different TPR subunits of the APC might function as receptors that interact with the C termini of regulatory proteins such as CDH1, CDC20, and APC10 (Vodermaier, 2003).

The isolation and characterization of two subcomplexes of the human APC have provided first insight into the molecular interactions between APC's many subunits. The cullin subunit APC2 and its binding partner, the RING finger protein APC11, are found in a subcomplex with APC1, APC4, and APC5 and are essential for the assembly of multiubiquitin chains from ubiquitin residues donated by E2 enzymes. Substrate ubiquitination requires the activator proteins CDH1 and CDC20, which interact via their C termini with the TPR subunits APC3 and APC7. APC's TPR subunits are predicted to form structures that are similar to the one of the peroxisomal import receptor PEX5, which binds cargo proteins via their C termini. The APC may therefore contain multiple TPR subunits to allow modular interactions with different regulatory proteins. These results reveal a function for the TPR subunits of the APC, and they provide insight into how substrates are recruited to the ubiquitin ligase (Vodermaier, 2003).

Roles of the anaphase-promoting complex/cyclosome and of its activator Cdc20 in functional substrate binding

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit ubiquitin-protein ligase that targets for degradation cell-cycle regulatory proteins during exit from mitosis and in the G1 phase of the cell cycle. The activity of APC/C in mitosis and in G1 requires interaction with the activator proteins Cdc20 and Cdh1, respectively. Substrates of APC/C-Cdc20 contain a recognition motif called the 'destruction box' (D-box). The mode of the action of APC/C activators and their possible role in substrate binding remain poorly understood. Several investigators suggested that Cdc20 and Cdh1 mediate substrate recognition, whereas others proposed that substrates bind to APC/C or to APC/C-activator complexes. All these studies used binding assays, which do not necessarily indicate that substrate binding is functional and leads to product formation. In the present investigation this problem was examined by an 'isotope-trapping' approach that directly demonstrates productive substrate binding. With this method it was found that the simultaneous presence of both APC/C and Cdc20 is required for functional substrate binding. By contrast, with conventional binding assays it was found that either Cdc20 or APC/C can bind substrate by itself, but only at low affinity and relaxed selectivity for D-box. These results are consistent with models in which interaction of substrate with specific binding sites on both APC/C and Cdc20 is involved in selective and productive substrate binding (Eytan, 2006; full text of article).

Coactivator functions in a stoichiometric complex with anaphase-promoting complex/cyclosome to mediate substrate recognition

The anaphase-promoting complex/cyclosome (APC/C) is a multisubunit E3 ligase required for ubiquitin-dependent proteolysis of cell-cycle-regulatory proteins, including mitotic cyclins and securin/Pds1. Regulation of APC/C activity and substrate recognition, mediated by the coactivators Cdc20 and Cdh1, is fundamental to cell-cycle control. However, the precise mechanism by which coactivators stimulate APC/C ubiquitylation activity and the nature of the substrate-binding sites on the activated APC/C are not understood. This study shows that the optimal interaction of substrate with APC/C is dependent specifically on the simultaneous association of coactivator. This is consistent with a model whereby both core APC/C subunits and coactivators contribute recognition sites for substrates, accounting for the bipartite nature (D and KEN boxes) of most APC/C degradation signals. A direct and stoichiometric function for the coactivators could explain how specific substrates are recognized by APC/C in a cell-cycle-specific manner, and how coactivator stimulates APC/C ubiquitylation activity (Passmore, 2006; full text of article).

A new Mad2-interacting domain of Cdc20 is critical for the function of Mad2-Cdc20 complex in the spindle assembly checkpoint

Interaction between Mad2 and Cdc20 (cell division cycle 20) is a key event during spindle assembly checkpoint activation. In the past, an N-terminal peptide containing amino acid residues 111-150 of Cdc20 was shown to bind Mad2 much better than the full-length Cdc20 protein. Using co-localization, co-immunoprecipitation and peptide inhibition analysis with different deletion mutants of Cdc20, another Mad2-binding domain has been identified on Cdc20 from amino acids 342-355 within the WD repeat region. An intervening region between these two domains interferes with its Mad2 binding when present individually with any of these two Mad2-binding sites. It is suggested that these three domains together determine the overall strength of Mad2 binding with Cdc20. Functional analysis suggests that an optimum Mad2 binding efficiency of Cdc20 is required during checkpoint arrest and release. Further, a unique polyhistidine motif with metal binding property has been identified adjacent to this second binding domain that may be important for maintaining the overall conformation of Cdc20 for its binding to Mad2 (Mondal, 2006).

Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1

The mitotic spindle assembly checkpoint delays anaphase until all chromosomes achieve bipolar attachment to the spindle microtubules. The spindle assembly checkpoint protein BubR1 is thought to act by forming an inhibitory complex with Cdc20. Two Cdc20 binding sites have been identified on BubR1. A strong Cdc20 binding site is located between residues 490 and 560, but mutations that disrupt Cdc20 binding to this region have no effect upon checkpoint function. A second Cdc20 binding site present between residues 1 and 477 is highly specific for Cdc20 already bound to Mad2. Mutation of a conserved lysine in this region weakened Cdc20 binding and correspondingly reduced checkpoint function. These results indicate that there may be more than one checkpoint complex containing BubR1, Mad2, and Cdc20. They also lead to the propose that in vivo checkpoint inhibition of Cdc20 is a two-step process in which prior binding of Mad2 to Cdc20 is required to make Cdc20 sensitive to inhibition by BubR1. Thus, Mad2 and BubR1 must cooperate to inhibit Cdc20 activity (Davenport, 2006).

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. This study shows 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, these results suggest a mechanistic basis for how cancer cells can have a compromised spindle checkpoint without corresponding mutations in checkpoint genes (Reddy, 2007).

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, the deubiquitinating enzyme USP44 (ubiquitin-specific protease 44) was identified 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. These 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 (Stegmeier, 2007).

Inhibitory factors associated with anaphase-promoting complex/cylosome in mitotic checkpoint

The mitotic (or spindle assembly) checkpoint system ensures accurate chromosome segregation by preventing anaphase initiation until all chromosomes are correctly attached to the mitotic spindle. It affects the activity of the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets inhibitors of anaphase initiation for degradation. The mechanisms by which this system regulates APC/C remain obscure. Some models propose that the system promotes sequestration of the APC/C activator Cdc20 by binding to the checkpoint proteins Mad2 and BubR1. A different model suggests that a mitotic checkpoint complex (MCC) composed of BubR1, Bub3, Cdc20, and Mad2 inhibits APC/C in mitotic checkpoint. This problem was examined by using extracts from nocodazole-arrested cells that reproduce some downstream events of the mitotic checkpoint system, such as lag kinetics of the degradation of APC/C substrate. Incubation of extracts with adenosine-5'-(gamma-thio)triphosphate (ATP[gammaS]) stabilized the checkpoint-arrested state, apparently by stable thiophosphorylation of some proteins. By immunoprecipitation of APC/C from stably checkpoint-arrested extracts, followed by elution with increased salt concentration, inhibitory factors were isolated associated with APC/C. A part of the inhibitory material consists of Cdc20 associated with BubR1 and Mad2, and is thus similar to MCC. Contrary to the original MCC hypothesis, it was found that MCC disassembles upon exit from the mitotic checkpoint. Thus, the requirement of the mitotic checkpoint system for the binding of Mad2 and BubR1 to Cdc20 may be for the assembly of the inhibitory complex rather than for Cdc20 sequestration (Braunstein, 2007).

Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint

Inappropriate attachment/tension between chromosomal kinetochores and the kinetochore microtubules activates the spindle assembly checkpoint, which delays anaphase by blocking the ubiquitin-mediated degradation of securin/Pds1p by APCCdc20. The checkpoint proteins Mad2 and Mad3/BubR1 bind to Cdc20, although how they inhibit APCCdc20 is unclear. The roles of two evolutionarily conserved KEN boxes and a D box within Mad3/BubR1 were investigated. Although such motifs usually mediate APC-substrate recognition and ubiquitination, they have no apparent role in Mad3p turnover in Saccharomyces cerevisiae. Instead, these motifs are important for Mad3p function in the checkpoint and for binding to Cdc20p. The Mad3p D box and KEN boxes function together to mediate Cdc20p-Mad3p interaction and Mad3p and an anaphase-promoting complex (APC) substrate, Hsl1p, compete for Cdc20p binding in a D-box- and KEN-box-dependent manner. In vivo, an increased binding of Cdc20p to Mad3p and decreased binding to Hsl1p is observed upon checkpoint activation. Furthermore, Mad2p stimulates the association between Mad3p and Cdc20p and this stimulated binding requires KEN box 1 within Mad3p. These findings implicate Mad3p as a pseudosubstrate inhibitor of APCCdc20, competing with APC substrates for Cdc20p binding. A model is presented aimed at unifying previous analyses of checkpoint function by focusing on the Mad3-Cdc20 interaction (Burton, 2007).

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 focus was placed on Mad3p and how it acts, in concert with Mad2p, to efficiently inhibit Cdc20p. The function of two motifs in Mad3p, KEN30 and KEN296, were identified and analysed; These motifs 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. 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, the consequences of MAD3 overexpression was analyzed and it was 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 (King, 2007).

The CRY box: a second APCcdh1-dependent degron in mammalian cdc20

Cdc20 and cdh1 are coactivators of the anaphase-promoting complex (APC). APC(cdc20) is necessary for the metaphase-anaphase transition and, at the end of mitosis, vertebrate cdc20 itself becomes a target for degradation through KEN-box-dependent APC(cdh1) activity. By studying the degradation of fluorescent protein chimaeras in mammalian oocytes and early embryos, it was found that cdc20 was degraded through two independent degradation signals (degrons), the KEN box and a newly described CRY box. In both oocytes and G1-stage embryos, the rate of degradation through the CRY box was greater than through the KEN box, although both were mediated by APC(cdh1). Thus, mammalian oocytes and embryos have the capacity to recognize two degrons in cdc20 (Reis, 2006).

Mammalian Emi2 mediates cytostatic arrest and transduces the signal for meiotic exit via Cdc20

Fertilizable mammalian oocytes are arrested at the second meiotic metaphase (mII) by the cyclinB-Cdc2 heterodimer, maturation promoting factor (MPF). MPF is stabilized via the activity of an unidentified cytostatic factor (CSF), thereby suspending meiotic progression until fertilization. Evidence that a conserved 71 kDa mammalian orthologue of Xenopus XErp1/Emi2, which is termed endogenous meiotic inhibitor 2 (Emi2) is an essential CSF component. Depletion in situ of Emi2 by RNA interference elicited precocious meiotic exit in maturing mouse oocytes. Reduction of Emi2 released mature mII oocytes from cytostatic arrest, frequently inducing cytodegeneration. Mos levels autonomously declined to undetectable levels in mII oocytes. Recombinant Emi2 reduced the propensity of mII oocytes to exit meiosis in response to activating stimuli. Emi2 and Cdc20 proteins mutually interact and Cdc20 ablation negated the ability of Emi2 removal to induce metaphase release. Consistent with this, Cdc20 removal prevented parthenogenetic or sperm-induced meiotic exit. These studies show in intact oocytes that the interaction of Emi2 with Cdc20 links activating stimuli to meiotic resumption at fertilization and during parthenogenesis in mammals (Shoji, 2006).

Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding

Premature anaphase onset is prevented by the mitotic checkpoint through production of a 'wait anaphase' inhibitor(s) that blocks recognition of cyclin B and securin by Cdc20-activated APC/C, an E3 ubiquitin ligase that targets them for destruction. Using physiologically relevant levels of Mad2, Bub3, BubR1, and Cdc20, this study demonstrates that unattached kinetochores on purified chromosomes catalytically generate a diffusible Cdc20 inhibitor or inhibit Cdc20 already bound to APC/C. Furthermore, the chromosome-produced inhibitor requires both recruitment of Mad2 by Mad1 that is stably bound at unattached kinetochores and dimerization-competent Mad2. Purified chromosomes promote BubR1 binding to APC/C-Cdc20 by acting directly on Mad2, but not BubR1. These results support a model in which immobilized Mad1/Mad2 at kinetochores provides a template for initial assembly of Mad2 bound to Cdc20 that is then converted to a final mitotic checkpoint inhibitor with Cdc20 bound to BubR1 (Kulukian, 2009).

While unattached kinetochores have been widely inferred to be the source of a 'wait anaphase' mitotic checkpoint inhibitor, this study has now demonstrated that kinetochores can, in fact, catalyze production of an initial Mad2-Cdc20 inhibitor, significantly accelerating the initial rate of its production. Unattached kinetochores did not affect inhibition by Bub3/BubR1 in the absence of Mad2. Production of at least two inhibitors can be enhanced by unattached kinetochores: one containing diffusible Cdc20 and another in which Cdc20 is already bound in a megadalton complex to APC/C, consistent with reports that Cdc20 and checkpoint proteins are present in two complexes with differing sizes during mitosis. Both inhibitors prevent recognition by APC/C of cyclin B as an ubiquitination substrate. Disruption of cyclin B ubiquitination by a kinetochore-derived inhibitor even while Cdc20 remains bound to APC/C provides a potential explanation for the differential timing of destruction of cyclins A and B. Instead of simple sequestration of Cdc20, a kinetochore-derived mitotic checkpoint inhibitor bound to APC/CCdc20 may block recognition of cyclin B as an ubiquitination substrate, while permitting APC/CCdc20-mediated ubiquitination and destruction of cyclin A, an event that is known to initiate immediately after mitotic entry (Kulukian, 2009).

Despite amplification of Cdc20 inhibition when equal molar levels of BubR1, Mad2, and Cdc20 were added, no evidence was found for assembly of a quaternary mitotic checkpoint- (MCC-) like complex as a bona fide inhibitor produced by unattached kinetochores. Rather, almost all Cdc20 shifted to a complex comigrating with the majority of BubR1 but containing very little Mad2. Also arguing against a contribution in kinetochore-derived checkpoint signaling, it is noted that MCC-like complexes in animal cells are present outside of mitosis, and their formation in yeast continues in the absence of a functional centromere/kinetochore. All of this supports an MCC-like, premade Cdc20 inhibitor produced in a kinetochore-independent manner in interphase that restrains APC/C ubiquitination activity for cyclin B just after mitotic entry, which has been referred to as a 'timer' (Kulukian, 2009).

More importantly, at physiologically relevant concentrations of unattached kinetochores and Mad2, chromosomes catalyzed production of Cdc20 inhibition of cyclin B recognition by APC/C by at least 8-fold relative to inhibitors formed spontaneously in the absence of chromosomes. The actual in vivo effect is likely to be much greater than observed in vitro, since chromosome purification resulted in partial loss of signaling molecules from kinetochores, including a proportion of Mad1 and kinases that include Bub1, BubR1, and Aurora B (Kulukian, 2009).

Chromosome amplification of Cdc20 inhibition required Mad1 recruitment of Mad2 to kinetochores and dimerization-competent Mad2, thereby providing a direct demonstration that a Mad1:Mad2 core complex recruits and converts soluble 'inactive' Mad2 into a more potent inhibitor of Cdc20. At least part of this is from action of kinetochores on Mad2. Although it has previously been argued that the kinetochore may sensitize the APC/C for checkpoint-mediated inhibition, direct contact of chromosomes with APC/C was not required to amplify inhibition. While a kinetochore-dependent function of BubR1 can by no means be excluded from roles in microtubule attachment and chromosome alignment or from further amplification of a kinetochore derived signal, kinetochore-mediated enhancement of Cdc20 inhibition did not require BubR1 localization to or contact with kinetochores. It is concluded that immobilized, kinetochore-bound Mad1/Mad2, but not BubR1, catalyzes conversion at the kinetochore of soluble, open Mad2 into a form with its seatbelt domain poised for Cdc20 capture. Further support for this conclusion includes evidence that kinetochore-bound BubR1 is nonessential (Kulukian, 2009).

Moreover, incubation of physiologically relevant concentrations of each component ultimately produced most Cdc20 bound to BubR1, not Mad2, whether or not chromosomes were present. In fact, amplification of Cdc20 inhibition by unattached kinetochores was accompanied by a shift to a more rapidly eluting Bub3/BubR1-Cdc20 complex, without a stable pool of Mad2-Cdc20. Evidence also demonstrated that most Cdc20 is complexed with BubR1 in vivo, rather than Mad2. A model from all of this is proposed in which Mad1/Mad2 immobilized at kinetochores templates conversion of an inactive, open Mad2 to one capable of transient capture of Cdc20 followed by relay to BubR1 as sequentially produced mitotic checkpoint inhibitors that may be soluble or APC/C bound. This evidence supports Mad2-Cdc20, and perhaps an MCC-like complex, as a transient intermediate in kinetochore-mediated checkpoint signaling and one that is a precursor to BubR1-Cdc20. Further, Bub3/BubR1 binds to APC/C, but only in a Mad2-dependent manner that is stimulated by unattached kinetochores, demonstrating that kinetochores facilitate loading of Bub3/BubR1 onto APC/C. That BubR1-APC/CCdc20 is produced indirectly by unattached kinetochores as the final Cdc20 inhibitor would also support suggestions that BubR1 acts as a nonproductive pseudosubstrate of the APC/C or mediates Cdc20 proteolytic turnover (Kulukian, 2009).

Combining kinetochore-derived Bub3/BubR1-Cdc20 with evidence for two Cdc20 binding sites on BubR1 further suggests that the spontaneous and kinetochore-derived Bub3/BubR1-Cdc20 complexes may represent generation of Cdc20 bound at the two different sites, respectively, a point now testable with the appropriate BubR1 mutants (Kulukian, 2009).

A centrosomal Cdc20-APC pathway controls dendrite morphogenesis in postmitotic neurons

The ubiquitin ligase anaphase-promoting complex (APC) recruits the coactivator Cdc20 to drive mitosis in cycling cells. However, the nonmitotic functions of Cdc20-APC have remained unexplored. This study report that Cdc20-APC plays an essential role in dendrite morphogenesis in postmitotic neurons. Knockdown of Cdc20 in cerebellar slices and in postnatal rats in vivo profoundly impairs the formation of granule neuron dendrite arbors in the cerebellar cortex. Remarkably, Cdc20 is enriched at the centrosome in neurons, and the centrosomal localization is critical for Cdc20-dependent dendrite development. The centrosome-associated protein histone deacetylase 6 (HDAC6) promotes the polyubiquitination of Cdc20, stimulates the activity of centrosomal Cdc20-APC, and drives the differentiation of dendrites. These findings define a postmitotic function for Cdc20-APC in the morphogenesis of dendrites in the mammalian brain. The identification of a centrosomal Cdc20-APC ubiquitin signaling pathway holds important implications for diverse biological processes, including neuronal connectivity and plasticity (Kim, 2009).

APC(Cdc20) suppresses apoptosis through targeting Bim for ubiquitination and destruction

Anaphase-promoting complex Cdc20 (APCCdc20; Fizzy in Drosophila) plays pivotal roles in governing mitotic progression. By suppressing APCCdc20, antimitotic agents activate the spindle-assembly checkpoint and induce apoptosis after prolonged treatment, whereas depleting endogenous Cdc20 suppresses tumorigenesis in part by triggering mitotic arrest and subsequent apoptosis. However, the molecular mechanism(s) underlying apoptosis induced by Cdc20 abrogation remains poorly understood. This study reports the BH3-only proapoptotic protein BCL2-like 11 (Bim) as an APCCdc20 target, such that depletion of Cdc20 sensitizes cells to apoptotic stimuli. Strikingly, Cdc20 and multiple APC-core components were identified in a small interfering RNA screen that, upon knockdown, sensitizes otherwise resistant cancer cells to chemoradiation in a Bim-dependent manner. Consistently, human adult T cell leukemia cells that acquire elevated APCCdc20 activity via expressing the Tax viral oncoprotein exhibit reduced Bim levels and resistance to anticancer agents. These results reveal an important role for APCCdc20 in governing apoptosis, strengthening the rationale for developing specific Cdc20 inhibitors as effective anticancer agents (Wan, 2014).

APC/CCdh1 enables removal of Shugoshin-2 from the arms of bivalent chromosomes by moderating Cyclin-dependent kinase activity

In mammalian females, germ cells remain arrested as primordial follicles. Resumption of meiosis is heralded by germinal vesicle breakdown, condensation of chromosomes, and their eventual alignment on metaphase plates. At the first meiotic division, anaphase-promoting complex/cyclosome associated with Cdc20 (APC/CCdc20; see Drosophila Cdc20) activates separase (see Drosophila Separase) and thereby destroys cohesion along chromosome arms. Because cohesion around centromeres is protected by shugoshin-2 (see Drosophila mei-S332), sister chromatids remain attached through centromeric/pericentromeric cohesin. This study shows that, by promoting proteolysis of cyclins and Cdc25B (see Drosophila String) at the germinal vesicle (GV) stage, APC/C associated with the Cdh1 protein (APC/CCdh1; see Drosophila Fizzy-related) delays the increase in Cdk1 (see Drosophila Cdk2) activity, leading to germinal vesicle breakdown (GVBD). More surprisingly, by moderating the rate at which Cdk1 is activated following GVBD, APC/CCdh1 creates conditions necessary for the removal of shugoshin-2 from chromosome arms by the Aurora B/C kinase (see Drosophila Aurora B), an event crucial for the efficient resolution of chiasmata (Rattani, 2017).

Kinetochores accelerate or delay APC/C activation by directing Cdc20 to opposing fates

Mitotic duration is determined by activation of the anaphase-promoting complex/cyclosome (APC/C) bound to its coactivator, Cdc20 (see Drosophila Fizzy. Kinetochores, the microtubule-interacting machines on chromosomes, restrain mitotic exit when not attached to spindle microtubules by generating a Cdc20-containing complex that inhibits the APC/C. This study, shows that flux of Cdc20 through kinetochores also accelerates mitotic exit by promoting its dephosphorylation by kinetochore-localized protein phosphatase 1 (see Drosophila Flapwing), which allows Cdc20 to activate the APC/C. Both APC/C activation and inhibition depend on Cdc20 fluxing through the same binding site at kinetochores. The microtubule attachment status of kinetochores therefore optimizes mitotic duration by controlling the balance between opposing Cdc20 fates (Kim, 2017).

fizzy: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | References

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