#APC/C

Cell Cycle Regulation

Which regulatory factor is crucial for the metaphase-to-anaphase transition?

Metaphase to anaphase transition represents irreversible commitment executed by APC/C bound to Cdc20 coactivator. Throughout prometaphase chromosomes attach via kinetochores, generating unattached kinetochore signal converting Mad2 to active inhibitor forming mitotic checkpoint complex with BubR1, Bub3, and Cdc20 that binds APC/C core to block E2 recruitment. Upon achievement of biorientation of all chromosomes and establishment of tension, checkpoint protein recruitment stops, complex disassembles, and free Cdc20 associates with phosphorylated APC/C previously primed by CDK1 and Plk1. Active APC/C-Cdc20 polyubiquitinates securin and cyclin B with K11 chains for proteasomal destruction. Securin normally binds and locks separase protease in inactive conformation together with cyclin B-CDK1 phosphorylation. Degradation liberates separase that cleaves kleisin Rad21 subunit of cohesin ring embracing sister chromatids since S phase, opening ring, while cyclin B destruction drops CDK1 activity permitting phosphatase activation and mitotic exit. Blocking securin degradation prevents chromatid separation. This circuitry is highly conserved across eukaryotes, integrating growth factor signals, DNA damage surveillance, and developmental cues, and its disruption frequently underlies oncogenesis, providing targets for checkpoint inhibitors and cancer therapeutics.

Ref: Peters et al., Cell 1998, APC/C Regulation. Musacchio, Nat Rev Mol Cell Biol 2011, Anaphase.

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Cell Cycle Regulation

Which phase transition requires the Anaphase-Promoting Complex (APC/C)?

The anaphase-promoting complex/cyclosome, APC/C, is a 1.2 MDa cullin-RING E3 ubiquitin ligase comprising over a dozen core subunits including APC2, APC11 catalytic core, and scaffolding proteins APC1, APC3, APC6, plus transient coactivators Cdc20 and Cdh1 that confer substrate specificity through D-box and KEN motif recognition. During early mitosis its activity is tightly repressed by spindle assembly checkpoint that sequesters Cdc20 in mitotic checkpoint complex composed of Mad2, BubR1, and Bub3. Priming phosphorylation by cyclin B-CDK1 and Polo-like kinase Plk1 on APC3 and APC1 enhances Cdc20 binding affinity. Upon amphitelic attachment of all kinetochores and generation of inter-kinetochore tension, Mad2 production ceases, checkpoint complex disassembles, and free Cdc20 activates APC/C. Activated APC/C-Cdc20 polyubiquitinates securin and mitotic cyclin B with K11-linked chains targeting them for rapid 26S proteasomal degradation. Securin destruction releases separase protease that cleaves kleisin Rad21 subunit of cohesin, dissolving sister chromatid cohesion while cyclin B loss extinguishes CDK1 activity, permitting phosphatase-driven mitotic exit and faithful chromosome segregation.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 17: Cell Cycle Control, APC/C Regulation.

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Cell Cycle Regulation

APC/C-Cdh1 is required for:

After separase cleavage of cohesin and chromosome segregation, cells must eliminate remaining mitotic cyclins to establish low CDK environment permissive for origin licensing, transcription reactivation and cytokinesis in G1. Two sequential APC/C coactivators accomplish temporal ordering. APC/C-Cdc20 initiates anaphase by destroying securin and majority of cyclin B during metaphase to anaphase transition. Cdc20 itself then becomes substrate for APC/C-Cdh1. Cdh1, also known as Fzr1, associates with APC/C core from late anaphase through G1, recognizing extended degron repertoire including KEN-box, D-box and ABBA motifs. Targets include residual cyclin B, cyclin A, Plk1, Aurora kinases A and B, Cdc20, geminin, Skp2 and Ets transcription factors. Continued degradation prevents re-accumulation of CDK1-Cyclin B activity, maintaining stable G1 state that allows formation of pre-replicative complexes containing ORC, Cdc6, Cdt1 and MCM helicases. At G1/S border, Cdh1 phosphorylated by CDK2-Cyclin E and inhibited by Emi1 pseudosubstrate, allowing cyclin buildup for next S phase. Loss of Cdh1 causes persistent mitotic kinases, premature S entry and genomic instability. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Lindon, APC/C-Cdh1 Control of G1 Homeostasis, Biochem J 2008; Peters, Anaphase Promoting Complex Mechanism and Regulation.

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Cell Cycle Regulation

What happens if APC/C-Cdc20 is inhibited?

Anaphase-promoting complex bound to coactivator Cdc20 represents irreversible executor of metaphase exit, targeting securin and cyclin B. Inhibition of this ligase through multiple mechanisms arrests cells before chromosome separation. Endogenous inhibition occurs during spindle checkpoint activation where Mitotic Checkpoint Complex binds APC/C core blocking substrate recruitment. Exogenous inhibitors apcin occupying D-box binding pocket on Cdc20 and proTAME blocking APC/C activation, or depletion of Cdc20 protein itself, mimic checkpoint. Consequence is persistence of securin which continues to bind and suppress separase protease, and persistence of cyclin B maintaining high CDK1-phosphorylation of lamins, condensins and microtubule regulators. Cohesin rings at centromeres therefore remain intact, as separase cannot access Scc1 cleavage site, leaving sister chromatids interlinked despite bipolar spindle tension attempting to pull. Phenotypically cells display metaphase plate with fully aligned chromosomes but inability to initiate anaphase A movement. Prolonged arrest leads to mitotic slippage through slow cyclin B leakage, cohesion fatigue with partial separation, cell death or formation of tetraploid G1 cells after aberrant exit, illustrating essential role of APC/C-Cdc20 for timely anaphase.

Ref: Pines, Mitotic Exit Regulation by APC/C-Cdc20, Nat Rev Mol Cell Biol 2011; NCBI Bookshelf, Consequences of APC/C Inhibition.

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Cell Cycle Regulation

During mitosis, which event is triggered by APC/C activation?

Transition into anaphase is driven by activation of anaphase promoting complex cyclosome bound to Cdc20, a large 1.2 MDa cullin-RING E3 ubiquitin ligase. When SAC is satisfied, MCC disassembly through p31comet and TRIP13 releases Cdc20 permitting association with APC/C core. Active APC/C-Cdc20 recognizes destruction box degrons on two pivotal substrates: securin and Cyclin B. Polyubiquitination of securin triggers rapid proteolysis via 26S proteasome, discharging separase cysteine protease previously held inactive as pseudosubstrate. Free separase cleaves Scc1 or Rad21 kleisin subunit of cohesin rings at conserved E-x-x-R- site, opening tripartite ring and dissolving sister chromatid cohesion at centromeres remaining after Wapl-mediated arm removal. Simultaneously, cyclin B ubiquitination and degradation reduces CDK1 activity, permitting phosphatases PP1 and PP2A-B55 to dephosphorylate mitotic substrates, soften cortex and allow anaphase A chromatid-to-pole movement and anaphase B spindle elongation driven by Eg5 kinesin and cortical dynein. APC/C activation therefore couples cohesion destruction to mitotic exit preparation ensuring ordered segregation and genome stability. Additional feedback loops involving polo-like kinases, phosphatases and SCF-mediated degradation reinforce irreversibility and protect against premature progression that would compromise genome integrity and viability.

Ref: Pines, APC/C Regulation of Anaphase Onset, Nat Rev Mol Cell Biol 2011; NCBI Bookshelf, Anaphase Triggered by APC/C.

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Cell Cycle Regulation

Which component of the Mitotic Checkpoint Complex (MCC) directly inhibits APC/C?

Mitotic checkpoint complex inhibition of anaphase-promoting complex relies on multiple contacts that block substrate recognition and catalytic activity. MCC consists of BubR1, Bub3, Mad2, and Cdc20, assembled at unattached kinetochores. Closed conformer of Mad2, generated through Mad1-Mad2 template mechanism, wraps around Cdc20 as safety belt, sequestering it and presenting inhibitory surface. Although BubR1 contains KEN boxes that mimic APC/C substrates and directly blocks substrate binding via pseudo-substrate inhibition, Mad2 provides crucial initial locking of Cdc20 in MCC, preventing it from associating with and activating APC/C. Studies using purified components show Mad2 alone can partially inhibit APC/C, but full potency requires BubR1 which inserts into APC/C central cavity and obstructs E2 ubiquitin-conjugating enzyme UbcH10 recruitment. Bub3 serves as adaptor recognizing phosphorylated MELT motifs on Knl1. Mutations that prevent closed Mad2 formation bypass checkpoint, allowing premature anaphase. Therefore, within MCC, Mad2 is considered direct inhibitor that captures Cdc20 and nucleates complex assembly that culminates in APC/C suppression until bi-orientation achieved.

Ref: Alfieri et al., Nature 2016, APC/C-MCC Structure; De Antoni et al., Curr Biol 2005, Mad2 Conformers.

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Cell Cycle Regulation

Which protein complex is responsible for delaying mitosis in case of spindle defects?

Accurate chromosome segregation requires that all kinetochores achieve amphitelic attachment to spindle microtubules from opposite poles and generate tension. Unattached or improperly attached kinetochores generate wait signal that delays onset of anaphase to prevent mis-segregation. This delay is enforced by mitotic checkpoint complex, abbreviated MCC, composed of Mad2, Bub3, BubR1 (Mad3 in yeast), and Cdc20. At unattached kinetochores, scaffold protein Knl1 is phosphorylated by Mps1, recruiting Bub1-Bub3 and Mad1-Mad2 complexes that catalyze conformational conversion of open Mad2 to closed form that sequesters Cdc20, essential co-activator of anaphase-promoting complex/cyclosome (APC/C). MCC-bound Cdc20 cannot activate APC/C, preventing ubiquitination and degradation of securin and cyclin B, thereby keeping separase inactive and CDK1-cyclin B high. Once all kinetochores attach and tension is established, MCC production ceases, existing complexes are disassembled by p31comet and TRIP13, freeing Cdc20 to activate APC/C and drive anaphase onset and mitotic exit. The inhibitory signal is generated catalytically, with single unattached kinetochore producing multiple MCC complexes through Mad1-Mad2 template, allowing amplification and sustained APC/C inhibition throughout cell, illustrating how local attachment defect generates global cell cycle arrest via diffusible inhibitor and kinetochore-based signaling platform.

Ref: Musacchio & Salmon, Nature Rev Mol Cell Biol 2007, Spindle Assembly Checkpoint; Lara-Gonzalez et al., Curr Biol 2012, MCC Functions.

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