The APC/C targets tens of substrates for degradation. However, despite the fact that the large majority of these targets are present in the cell when the APC/C is activated in mitosis, these targets are not destroyed simultaneously. The APC/C destroys their substrates in a strict order that is highly conserved, the degradation of these targets in the wrong order or the presence of non-degradable substrates can have profound effects on the cell cycle.
|S-phase entry cyclin-5 (CLB5)||metaphase arrest|
|Securin (PDS1)||loss of sister chromatid separation|
|G2/mitotic-specific cyclin-2 (CLB2)||telophase arrest|
|DDK kinase regulatory subunit DBF4 (DBF4)||minor|
|Cell cycle serine/threonine-protein kinase CDC5/MSD2 (CDC5)||anaphase arrest|
|APC/C activator protein CDC20 (CDC20)||SAC defect|
|APC/C-CDH1 modulator 1 (ACM1)||-|
|Ras GTPase-activating-like protein IQG1 (IQG1)||minor septum formation defects|
|Filament protein FIN1 (FIN1)||minor |
|Serine/threonine-protein kinase Nek2 (NEK2)||-|
|Cyclin-A2 (CCNA2)||metaphase arrest|
|Shugoshin-like 1 (SGOL1)||minor|
|Securin (PTTG1)||loss of sister chromatid separation|
|Kinesin-like protein KIF22 (KIF22)||spindle movement defects|
|G2/mitotic-specific cyclin-B1 (CCNB1)||metaphase arrest |
|Serine/threonine-protein kinase PLK1 (PLK1)||cytokinesis defects, delayed mitotic exit|
|G2/mitotic-specific cyclin-B3 (CCNB3)||late anaphase arrest|
|Cell division cycle protein 20 homolog (CDC20)||SAC defect|
|Kinesin-like protein KIFC1 (KIFC1)||increased number of monopolar spindles |
|Aurora kinase B (AURKB)||minor|
|Actin-binding protein anillin (ANLN)||minor |
|Ubiquitin-conjugating enzyme E2 C (UBE2C)||G1 delay|
|S-phase kinase-associated protein 2 (SKP2)||premature S phase entry|
|Cell division control protein 6 homolog (CDC6)||minor|
Table 1. Degradation timing of selected APC/C substrates
A selection of the key substrates destroyed by APC/C in the human and yeast proteomes for which the degradation timing has been characterised. The phenotype of a non-degradable mutant of the protein is given if such an experiment has been carried out.
In vertebrate cells, degradation of the first APC/CCdc20 targets begins immediately after nuclear envelope breakdown at the onset of prometaphase. These early APC/CCdc20 substrates include cyclin A and the protein kinase Nek2A, the destruction of which presumably allows dephosphorylation of cyclin A-Cdk and Nek2A substrates. Among its many functions, cyclin A promotes kinetochore-microtubule detachment to facilitate error correction and proper chromosome segregation; thus, cyclin A destruction helps stabilize these attachments in metaphase . Nek2A is a regulator of centrosome organization, centrosome separation and chromatin condensation ; little is known about the reasons for the early dephosphorylation of its targets. When APC/CCdc20 forms in early mitosis, its activity is initially suppressed in many cell types by the spindle assembly checkpoint (SAC), a regulatory system that is activated when chromosomes are not yet aligned correctly on the mitotic spindle. Activation of the SAC leads to formation of the mitotic checkpoint complex (MCC), which interacts tightly with APC/CCdc20 and blocks its interaction with degrons on specific targets, such as securin, that are required for anaphase onset. In mammals, the early targets of the APC/C, cyclin A and Nek2A, are partially resistant to the SAC, primarily because these substrates employ SAC-resistant degrons and other mechanisms to interact with the MCC-bound APC/C. Similar to cyclin A, the yeast S-phase cyclin Clb5, is also degraded early in mitosis in the presence of the SAC , but not to the same degree as the SAC-resistant degradation seen with cyclin A; consequently, most Clb5 is destroyed after the SAC has been satisfied.
Following chromosome biorientation at the metaphase plate, the SAC is satisfied and APC/CCdc20 becomes fully active. The major metaphase targets of APC/CCdc20 are securin and the M cyclins. Securin destruction liberates separase from the inactive securin-separase complex, and separase then cleaves cohesin to trigger sister chromatid separation and the initiation of anaphase. The metaphase degradation of M cyclins, including cyclin B in vertebrates, reduces Cdk activity and thereby allows phosphatases to dephosphorylate numerous Cdk targets. These targets include the APC/C activator Cdh1, dephosphorylation of which contributes to its activation in early anaphase. Cyclin B destruction also promotes the activation of separase. The budding yeast M-cyclin, Clb2, exhibits biphasic degradation: a fraction of Clb2 is degraded during metaphase, while the remainder is degraded during anaphase .
A small number of additional APC/CCdc20 substrates are degraded in parallel with securin and M cyclins. These include the shugoshin proteins, which recruit protein phosphatase 2A to pericentromeric regions to govern cohesin function and bi-orientation in yeast . In budding yeast (but not in vertebrates), APC/CCdc20 also targets the kinase regulatory subunit Dbf4, which binds and thereby activates the protein kinase Cdc7. Dbf4 degradation is thought to prevent the premature Cdc7-dependent phosphorylation of the Mcm helicase, a key regulatory modification controlling the initiation of DNA replication. Finally, another key APC/CCdc20 target in budding yeast is the Cdh1 inhibitor Acm1, which interacts tightly with Cdh1 to inhibit its interaction with substrates. APC/CCdc20-mediated Acm1 destruction, together with Cdh1 dephosphorylation, triggers activation of APC/CCdh1 in early anaphase.
Anaphase and mitotic exit
APC/CCdh1 has a wide range of targets in early anaphase and beyond. These include the cell cycle kinases of the Polo and Aurora kinase families. PLK1 is degraded first, thereby promoting dephosphorylation of substrates involved in spindle elongation, cleavage furrow formation and cytokinesis. Aurora kinase A is degraded after PLK1, thereby promoting the reorganisation of the mitotic spindle . In yeast, degradation of the PLK homolog Cdc5 in late mitosis helps promote inactivation of the mitotic phosphatase Cdc14 . Numerous other regulators of the kinetochore, mitotic spindle, and cytokinetic furrow are also destroyed via APC/CCdh1 to promote disassembly of these structures before the next cell cycle commences . Several spindle motor kinesins are also targeted to modulate spindle dynamics and facilitate sister chromatid separation . In late mitosis, the APC/C targets several proteins involved in cytokinesis, including the actin-binding protein anillin , Cytoskeleton-associated protein 2 (CKAP2) , and Aurora kinase B .
In G1, APC/CCdh1 continues to degrade the cyclins, thereby creating a window of low Cdk activity that blocks premature entry into S phase and allows assembly of pre-replicative complexes (pre-RCs) at replication origins . APC/CCdh1 also regulates Cdk activity by targeting SKP2, a subunit of the ubiquitin ligase SCF, thereby blocking the degradation of G1 phase Cdk inhibitors such as p27Kip1 . APC/CCdh1 also controls DNA replication through degradation of geminin, an inhibitor of pre-RC formation. In vertebrates, the origin licensing factor CDC6 is an APC/CCdh1 target , providing a mechanism by which pre-RC formation is restrained until Cdc6 is stabilized in late G1 by cyclin E-Cdk2. The inactivation of APC/CCdh1 in late G1 leads to stabilization of geminin and the cyclins, ensuring that origins cannot be relicensed until the APC/C is activated again in late mitosis.