Spatiotemporal regulation of the anaphase-promoting complex in mitosis

Abstract

The appropriate timing of events that lead to chromosome segregation during mitosis and cytokinesis is essential to prevent aneuploidy, and defects in these processes can contribute to tumorigenesis. Key mitotic regulators are controlled through ubiquitylation and proteasome-mediated degradation. The APC/C (anaphase-promoting complex; also known as the cyclosome) is an E3 ubiquitin ligase that has a crucial function in the regulation of the mitotic cell cycle, particularly at the onset of anaphase and during mitotic exit. Co-activator proteins, inhibitor proteins, protein kinases and phosphatases interact with the APC/C to temporally and spatially control its activity and thus ensure accurate timing of mitotic events.

Figure 1. Ordered degradation of APC/C substrates

The APC/C ubiquitylates proteins, marking their degradation at specific times and driving forward progression of the cell cycle. APC/C-Cdc20 ubiquitylates substrates during early and mid mitosis while APC/C-Cdh1 ubiquitylates substrates after anaphase onset, during mitotic exit and in G1. APC/C-Cdc20 ubiquitylates Cyclin A and Nek2A in prometaphase. During prometaphase APC/C-Cdc20 activity toward late substrates, Securin and Cyclin B1 is suppressed by the spindle checkpoint. At metaphase, the spindle checkpoint is silenced and ubiquitylation of Securin and Cyclin B1 is maximized. At mitotic exit, APC/C-Cdh1 ubiquitylates Cdc20, Aurora kinases and Plk1. At the G1-S transition, APC/C-Cdh1 is inactivated by a combination of binding to the APC/C inhibitor Emi1, degradation of its E2 UbcH10, Cdh1 phosphorylation, and ubiquitylation and degradation of Cdh1.

Figure 2. Structural organization of the APC/C

A) The subunits of the APC/C can be largely organized into three sub-complexes: the platform (APC1, APC4, APC5, APC15), the catalytic core (APC2, APC11, APC10/Doc1) and the TPR lobe or Arc lamp (APC8, APC6, APC3, APC7) sub-complex . (For APC/C subunit nomenclature used in yeast see Table 1. The APC1 subunit in the platform is the largest APC/C subunit and acts to bridge the other sub-complexes: the catalytic core and TPR lobe ^, . APC2 acts as a scaffold for the catalytic core. APC11 potentiates interaction with E2 enzymes and APC10 forms part of the substrate-binding pocket ^, . The TPR lobe has multiple subunits that form homodimers and provide important scaffolding functions to the APC/C. Accessory proteins stabilize subunits in the TPR lobe: APC12 stabilizes APC6; APC13 interacts with the TPR repeats of APC3, APC6, APC8; APC16 interacts with TPR repeats of APC3 and APC7 subunits . While most subunits exist as monomers, APC3, APC6, APC7, APC8 and APC12 are present as dimers. B) Cryo-electron microscopy reconstruction of the human APC/C-Cdh1 complex depicting the location of the individual subunits along with their underlying secondary structures. Figure 1B is reproduced with permission from reference .

Figure 3. Conformational changes during APC/C activation and inactivation

The APC/C undergoes conformational changes upon coactivator-substrate binding to bring the E2-Ub close to the substrate and this conformational activation is inhibited by the Mitotic checkpoint complex (MCC). A) Diagram of the conformational activation of the APC/C upon coactivator and substrate binding. Coactivator binding disrupts interaction between APC8 and APC1 causing a downward shift of the platform that is accompanied by an upward shift of the catalytic module (APC2-APC11). This might bring the E2-Ub close to the substrate and potentiate attachment of the initiating ubiquitin ^, . Cdc20 is also required for the activity of the chain-elongating E2 (Ube2S) . A distinct region on APC2, near the APC2-APC4 junction is required to bind Ube2S . The APC/C also tethers the distal molecule of an emerging ubiquitin conjugate close to Ube2S thereby potentiating efficient ubiquitin chain elongation. B) Diagram of APC/C bound to MCC. MCC components inhibit recruitment of late mitotic substrates that rely upon recognition though D box and KEN box motif and hence inhibit APC/C activity toward these substrates. Mad2 and BubR1 bind Cdc20 and prevent its ability to recruit substrates. Cdc20 as part of the MCC is also pushed downwards towards platform subunits and prevented from forming the D box co-receptor with APC10 . This position of Cdc20 might also facilitate its own ubiquitylation and subsequent degradation during active spindle checkpoint signaling.

Figure 4. MCC turnover during mitosis

A) In the presence of unattached kinetochores, Mad2-BubR1-Bub3-Cdc20 interact to form a diffusible mitotic checkpoint complex (MCC) that binds and inhibits the APC/C. APC/C ubiquitylates and promotes degradation of its co-activator Cdc20. Cdc20 is continually synthesized during mitosis. In the continued presence of unattached kinetochores, spindle checkpoint proteins Mad2 and BubR1-Bub3 can be recycled to bind newly synthesized Cdc20, form MCC and inhibit APC/C. B) Once all sister kinetochores achieve bipolar attachment to spindle and are under mechanical tension, MCC formation is inhibited and MCC disassembly dominates. Cdc20 is released from MCC and/or freshly synthesized Cdc20 binds and generates the APC/C-Cdc20 with high activity toward late mitotic substrates ^{, ,} . Several mechanisms contribute to loss of MCC activity. MCC catalysis at kinetochores is inhibited by transport of several checkpoint components, including Mad2 and BuBR1 from kinetochores by the minus-end directed motor protein Dynein ^{, –}. P31comet competes with BuBR1 for binding Mad2 and prevents conformational activation of Mad2 . MCC disassembly allows APC/C activation leading to ubiquitylation and degradation of Securin and Cyclin B1 for anaphase onset and mitotic exit.

Figure 5. Positive and negative modulators control rapid changes in APC/C activity

Mitotic progression is primarily regulated through two main activators, Cdk1 kinase and the APC/C. These work through a feedback mechanism whereby Cdk1 activation of APC/C ultimately induces degradation of Cyclin B1 and Cdk1 inactivation (red arrows). APC/C activity is further modulated by a host of other components that are themselves regulated by posttranslational modification and by subcellular localization, particularly at kinetochores. The resulting regulatory networks control APC/C activity and allow the APC/C to respond to rapid changes in kinetochore attachment/detachment. The spindle checkpoint proteins Mad1, Mad2, BuBR1, Bub3 inhibit APC/C activity. These spindle checkpoint proteins are themselves activated by mitotic protein kinases Mps1, Bub1, Aurora B, Cyclin B1/Cdk1 and inhibited by p31comet, protein phosphatases (PP1, PP2A) and Dynein. These regulators affect localization or activity of the spindle checkpoint proteins. While the spindle checkpoint inhibits APC/C, regulators of the spindle checkpoint also directly modulate APC/C activity. This results in complex regulatory networks that fine-tune APC/C activity during mitosis. In addition, some proteins have roles in both inhibiting and promoting APC/C activity. For example Cdk1 has inhibitory roles in phosphorylating Cdc20, Cdh1, and spindle checkpoint proteins. At the same time Cdk1 phosphorylation enhances APC/C-Cdc20 activity. The interplay of these regulators and the existence of subcellular pools of APC/C that differ in post-translational modification and inhibitor or activator binding is likely to play important roles in the dynamic regulation of APC/C activity during progressive stages of the cell cycle.

Figure 6. Hypothesis for the spatiotemporal regulation of the APC/C in mitosis

In prometaphase, APC/C activity is inhibited toward late mitotic substrates to prevent anaphase onset and mitotic exit until all kinetochores are bi-oriented on mitotic spindle and attached to microtubules properly. During mitosis subcellular localization of APC/C and its substrates might play important roles in mitotic progression. Some APC/C is concentrated at centrosomes where it is bound and potentially inhibited by binding to a protein complex containing Emi1, Numa and Dynein-Dynactin . Spindle assembly factors are localized to microtubules and thereby protected from APC/C-mediated degradation until completion of spindle formation . The spindle checkpoint generates the diffusible mitotic checkpoint complex (MCC) catalyzed at unattached kinetochores to inhibit the soluble cytosolic APC/C (intensity of red color denotes degree of APC/C inhibition, green indicates APC/C activation). A small pool of active APC/C-Cdc20 might remain associated with chromosomes in prometaphase, potentially escaping checkpoint inhibition and contributing to the basal Cyclin B1 degradation seen in cells arrested in mitosis with microtubule drugs. Upon proper microtubule attachment at metaphase, active APC/C-Cdc20 further accumulates on chromosomes dependent on the SKA complex . Loss of inhibition by spindle checkpoint proteins generates globally strong APC/C activity throughout the cytoplasm. Final activation of APC/C might occur on chromosomes to allow rapid Cohesin cleavage and synchronous anaphase chromatid separation . Thus APC/C activity is regulated spatially and temporally to control proper mitotic progression.