APC/C-Cdh1: From cell cycle to cellular differentiation and genomic integrity

Anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin-protein ligase that targets various substrates for proteolysis inside and outside of the cell cycle. The activation of APC/C is dependent on two WD-40 domain proteins, Cdc20 and Cdh1. While APC/Cdc20 principally regulates mitotic progression, APC/Cdh1 shows a broad spectrum of substrates in and beyond cell cycle. In the past several years, numerous biochemical and mouse genetic studies have greatly attracted our attention to the emerging role of APC/Cdh1 in genomic integrity, cellular differentiation and human diseases. This review will aim to summarize the recently expanded understanding of APC/Cdh1 in regulating biological function and how its dysfunction may lead to diseases.Key words: APC/C, Cdh1, proteolysis, genomic integrity, signal transduction, differentiation, tumorigenesis     

APC/C-Cdh1

Anaphase-promoting complex/cyclosome (APC/C) is a multifunctional ubiquitin-protein ligase that targets various substrates for proteolysis inside and outside of cell cycle. The activation of APC/C is depended on two WD-40 domain proteins, Cdc20 and Cdh1. While APC/Cdc20 principally regulates mitotic progression, APC/Cdh1 shows a broad spectrum of substrates in and beyond cell cycle. In past several years, numerous biochemical and mouse genetic studies have greatly attracted our attention to the emerging role of APC/Cdh1 in genomic integrity, cellular differentiation and human diseases. This review will aim to summarize the recent expended understanding of APC/Cdh1 in regulating biological function and how its dysfunction may lead to diseases.     

Activity of the APC(Cdh1) form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p.

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 (APC/C) bound to its regulator Cdh1p/Hct1p. Yeast APC(Cdh1) was proposed previously to be inactivated at Start by G1 cyclin/cyclin-dependent kinase (CDK). Here, we demonstrate that 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).     

Nuclear translocation of Cyclin B1 marks the restriction point for terminal cell cycle exit in G2 phase

Upon DNA damage, cell cycle progression is temporally blocked to avoid propagation of mutations. While transformed cells largely maintain the competence to recover from a cell cycle arrest, untransformed cells past the G1/S transition lose mitotic inducers, and thus the ability to resume cell division. This permanent cell cycle exit depends on p21, p53, and APC/C^Cdh1. However, when and how permanent cell cycle exit occurs remains unclear. Here, we have investigated the cell cycle response to DNA damage in single cells that express Cyclin B1 fused to eYFP at the endogenous locus. We find that upon DNA damage Cyclin B1-eYFP continues to accumulate up to a threshold level, which is reached only in G2 phase. Above this threshold, a p21 and p53-dependent nuclear translocation required for APC/C^Cdh1-mediated Cyclin B1-eYFP degradation is initiated. Thus, cell cycle exit is decoupled from activation of the DNA damage response in a manner that correlates to Cyclin B1 levels, suggesting that G2 activities directly feed into the decision for cell cycle exit. Once Cyclin B1-eYFP nuclear translocation occurs, checkpoint inhibition can no longer promote mitotic entry or re-expression of mitotic inducers, suggesting that nuclear translocation of Cyclin B1 marks the restriction point for permanent cell cycle exit in G2 phase.     

Hsl1p, a Swe1p inhibitor, is degraded via the anaphase-promoting complex

Ubiquitination and subsequent degradation of critical cell cycle regulators is a key mechanism exploited by the cell to ensure an irreversible progression of cell cycle events. The anaphase-promoting complex (APC) is a ubiquitin ligase that targets proteins for degradation by the 26S proteasome. Here we identify the Hsl1p protein kinase as an APC substrate that interacts with Cdc20p and Cdh1p, proteins that mediate APC ubiquitination of protein substrates. Hsl1p is absent in G(1), accumulates as cells begin to bud, and disappears in late mitosis. Hsl1p is stabilized by mutations in CDH1 and CDC23, both of which result in compromised APC activity. Unlike Hsl1p, Gin4p and Kcc4p, protein kinases that have sequence homology to Hsl1p, were stable in G(1)-arrested cells containing active APC. Mutation of a destruction box motif within Hsl1p (Hsl1p(db-mut)) stabilized Hsl1p. Interestingly, this mutation also disrupted the Hsl1p-Cdc20p interaction and reduced the association between Hsl1p and Cdh1p in coimmunoprecipitation studies. These findings suggest that the destruction box motif is required for Cdc20p and, to a lesser extent, for Cdh1p to target Hsl1p to the APC for ubiquitination. Hsl1p has been previously shown to inhibit Swe1p, a protein kinase that negatively regulates the cyclin-dependent kinase Cdc28p, by promoting Swe1p degradation via SCF(Met30) in a bud morphogenesis checkpoint. Results of the present work indicate that Hsl1p is degraded in an APC-dependent manner and suggest a link between the SCF (Skp1-cullin-F box) and APC-proteolytic systems that may help to coordinate the proper progression of cell cycle events.     

The emerging role of APC/CCdh1 in development

The function of APC/C (anaphase-promoting complex/cyclosome) was initially implicated with the onset of anaphase during mitosis, where its association with Cdc20 targets securin for destruction, thereby allowing the separation of two duplicated daughter genomes. When combined with Cdh1, APC regulates G1/S transition and DNA replication during cell cycle. Beyond cell cycle control, results from recent biochemical and mouse genetic studies have attracted our attention to the unexpected impact of APC/C(Cdh1) in cellular differentiation, genomic integrity and pathogenesis of various diseases. This review will aim to summarize current understanding of APC/C(Cdh1) in regulating crucial events during development.     

Inhibition of APCCdh1 activity by Cdh1/Acm1/Bmh1 ternary complex formation

The anaphase-promoting complex (APC) is an essential E3 ubiquitin ligase responsible for catalyzing proteolysis of key regulatory proteins in the cell cycle. Cdh1 is a co-activator of the APC aiding in the onset and maintenance of G(1) phase, whereas phosphorylation of Cdh1 at the end of G(1) phase by cyclin-dependent kinases assists in the inactivation of APC(Cdh1). Here, we suggest additional components are involved in the inactivation of APC(Cdh1) independent of Cdh1 phosphorylation. We have identified proteins known as Acm1 and Bmh1, which bind and form a ternary complex with Cdh1. The presence of phosphorylated Acm1 is critical for the ternary complex formation, and Acm1 is predominantly expressed in S phase when APC(Cdh1) is inactive. The assembly of the ternary complex inhibits ubiquitination of Clb2 in vitro by blocking the interaction of Cdh1 with Clb2. In vivo, lethality caused by overexpression of constitutively active Cdh1 is rescued by overexpression of Acm1. Partially phosphorylated Cdh1 in the absence of ACM1 still binds to and activates the APC. However, the addition of Acm1 decreases Clb2 ubiquitination when using either phosphorylated or nonphosphorylated Cdh1. Taken together, our results suggest an additional inactivation mechanism exists for APC(Cdh1) that is independent of Cdh1 phosphorylation.     

A conserved cyclin-binding domain determines functional interplay between anaphase-promoting complex-Cdh1 and cyclin A-Cdk2 during cell cycle progression

Periodic activity of the anaphase-promoting complex (APC) ubiquitin ligase determines progression through multiple cell cycle transitions by targeting cell cycle regulators for destruction. At the G(1)/S transition, phosphorylation-dependent dissociation of the Cdh1-activating subunit inhibits the APC, allowing stabilization of proteins required for subsequent cell cycle progression. Cyclin-dependent kinases (CDKs) that initiate and maintain Cdh1 phosphorylation have been identified. However, the issue of which cyclin-CDK complexes are involved has been a matter of debate, and the mechanism of how cyclin-CDKs interact with APC subunits remains unresolved. Here we substantiate the evidence that mammalian cyclin A-Cdk2 prevents unscheduled APC reactivation during S phase by demonstrating its periodic interaction with Cdh1 at the level of endogenous proteins. Moreover, we identified a conserved cyclin-binding motif within the Cdh1 WD-40 domain and show that its disruption abolished the Cdh1-cyclin A-Cdk2 interaction, eliminated Cdh1-associated histone H1 kinase activity, and impaired Cdh1 phosphorylation by cyclin A-Cdk2 in vitro and in vivo. Overexpression of cyclin binding-deficient Cdh1 stabilized the APC-Cdh1 interaction and induced prolonged cell cycle arrest at the G(1)/S transition. Conversely, cyclin binding-deficient Cdh1 lost its capability to support APC-dependent proteolysis of cyclin A but not that of other APC substrates such as cyclin B and securin Pds1. Collectively, these data provide a mechanistic explanation for the mutual functional interplay between cyclin A-Cdk2 and APC-Cdh1 and the first evidence that Cdh1 may activate the APC by binding specific substrates.     

Activation of Cdh1-dependent APC is required for G1 cell cycle arrest and DNA damage-induced G2 checkpoint in vertebrate cells.

Anaphase-promoting complex (APC) is activated by two regulatory proteins, Cdc20 and Cdh1. In yeast and Drosophila, Cdh1-dependent APC (Cdh1-APC) activity targets mitotic cyclins from the end of mitosis to the G1 phase. To investigate the function of Cdh1 in vertebrate cells, we generated clones of chicken DT40 cells disrupted in their Cdh1 loci. Cdh1 was dispensable for viability and cell cycle progression. However, similarly to yeast and Drosophila, loss of Cdh1 induced unscheduled accumulation of mitotic cyclins in G1, resulting in abrogation of G1 arrest caused by treatment with rapamycin, an inducer of p27(Kip1). Further more, we found that Cdh1(-/-) cells fail to maintain DNA damage-induced G2 arrest and that Cdh1-APC is activated by X-irradiation-induced DNA damage. Thus, activation of Cdh1-APC plays a crucial role in both cdk inhibitor-dependent G1 arrest and DNA damage-induced G2 arrest.     

New origin firing is inhibited by APC/CCdh1 activation in S-phase after severe replication stress

Defects in DNA replication and repair are known to promote genomic instability, a hallmark of cancer cells. Thus, eukaryotic cells have developed complex mechanisms to ensure accurate duplication of their genomes. While DNA damage response has been extensively studied in tumour cells, the pathways implicated in the response to replication stress are less well understood especially in non-transformed cells. Here we show that in non-transformed cells, APC/C^Cdh1 is activated upon severe replication stress. Activation of APC/C^Cdh1 prevents new origin firing and induces permanent arrest in S-phase. Moreover, Rad51-mediated homologous recombination is also impaired under these conditions. APC/C^Cdh1 activation in S-phase occurs after replication forks have been processed into double strand breaks. Remarkably, this activation, which correlates with decreased Emi1 levels, is not prevented by ATR/ATM inhibition, but it is abrogated in cells depleted of p53 or p21. Importantly, we found that the lack of APC/C^Cdh1 activity correlated with an increase in genomic instability. Taken together, our results define a new APC/C^Cdh1 function that prevents cell cycle resumption after prolonged replication stress by inhibiting origin firing, which may act as an additional mechanism in safeguarding genome integrity.     

Proteolysis of Rad17 by Cdh1/APC regulates checkpoint termination and recovery from genotoxic stress

Recent studies have shown a critical function for the ubiquitin‐proteasome system (UPS) in regulating the signalling network for DNA damage responses and DNA repair. To search for new UPS targets in the DNA damage signalling pathway, we have carried out a non‐biased assay to identify fast‐turnover proteins induced by various types of genotoxic stress. This endeavour led to the identification of Rad17 as a protein exhibiting a distinctive pattern of upregulation followed by subsequent degradation after exposure to UV radiation in human primary cells. Our characterization showed that UV‐induced Rad17 oscillation is mediated by Cdh1/APC, a ubiquitin‐protein ligase. Studies using a degradation‐resistant Rad17 mutant demonstrated that Rad17 stabilization prevents the termination of checkpoint signalling, which in turn attenuates the cellular re‐entry into cell‐cycle progression. The findings provide an insight into how the proteolysis of Rad17 by Cdh1/APC regulates the termination of checkpoint signalling and the recovery from genotoxic stress.     

Mammalian Cdh1/Fzr mediates its own degradation

The Anaphase-Promoting Complex/Cyclosome (APC/C) ubiquitin ligase mediates degradation of cell cycle proteins during mitosis and G1. Cdc20/Fzy and Cdh1/Fzr are substrate-specific APC/C activators. The level of mammalian Cdh1 is high in mitosis, but it is inactive and does not bind the APC/C. We show that when Cdh1 is active in G1 and G0, its levels are considerably lower and almost all of it is APC/C associated. We demonstrate that Cdh1 is subject to APC/C-specific degradation in G1 and G0, and that this degradation depends upon two RXXL-type destruction boxes. We further demonstrate that addition of Cdh1 to Xenopus interphase extracts, which have an inactive APC/C, activates it to degrade Cdh1. These observations indicate that Cdh1 mediates its own degradation by activating the APC/C to degrade itself. Elevated levels of Cdh1 are deleterious for cell cycle progression in various organisms. This auto-regulation of Cdh1 could thus play a role in ensuring that the level of Cdh1 is reduced during G1 and G0, allowing it to be switched off at the correct time.