Mechanisms for the temporal regulation of substrate ubiquitination by the anaphase-promoting complex/cyclosome

Abstract

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit, multifunctional ubiquitin ligase that controls the temporal degradation of numerous cell cycle regulatory proteins to direct the unidirectional cell cycle phases. Several different mechanisms contribute to ensure the correct order of substrate modification by the APC/C complex. Recent advances in biochemical, biophysical and structural studies of APC/C have provided a deep mechanistic insight into the working of this complex ubiquitin ligase. This complex displays remarkable conformational flexibility in response to various binding partners and post-translational modifications, which together regulate substrate selection and catalysis of APC/C. Apart from this, various features and modifications of the substrates also influence their recognition and affinity to APC/C complex. Ultimately, temporal degradation of substrates depends on the kind of ubiquitin modification received, the processivity of APC/C, and other extrinsic mechanisms. This review discusses our current understanding of various intrinsic and extrinsic mechanisms responsible for 'substrate ordering' by the APC/C complex.

Keywords: APC/C; Anaphase-promoting complex; Cell cycle; Substrate ordering; Ubiquitination.

Fig. 1

Structural organization of the APC/C. a, b EM construction of the APC/C^CDH1 in complex with the inhibitory protein Emi1. Reproduced with permission from [9]. c Schematic of the Apo-APC/C. d active APC/C. Positions of IR tail binding sites and C-box binding sites are shown. Change in the mobility of the platform and repositioning of the catalytic core upon substrate binding is indicated. e Domain organization and modification sites of CDC20 (upper) and CDH1 (lower) with known CDK1 phosphorylation sites (dark green)

Fig. 2

Activation of APC/C^CDC20 by phosphorylation. In the unphosphorylated APC/C, the C-box binding site on APC8 is occupied by an auto-inhibitory loop of APC1, thus preventing the C-box of CDC20 to bind to APC/C. Phosphorylation of a loop in APC3 subunits results in recruitment of CDK1-CyclinB-CKS complex to this loop, leading to phosphorylation of the APC1 loop and displacing it from the C-box binding site, and allowing the CDC20 C-box to associate with APC8. The IR tail of CDC20 interacts with APC3A while the phosphorylated loop of APC3B interacts with the IR tail of APC10. Coactivator binding induces the movement of the catalytic core in the ‘up’ position

Fig. 3

Post-translational modifications and the order of substrate degradation. Temporal pattern of APC/C activity in context with CDC20 (in blue) and with CDH1 (in red) in different cell cycle phases is shown on the left. Substrates are shown on top in the order of their degradation. The blue box denotes the CDC20-mediated, and orange box shows the CDH1 mediated degradation of the substrates. The substrates undergo various post-translational modifications that also contribute to the order in which they are degraded. These PTMs are color coded, with darker to lighter shading, where darker shade represents higher amount of protein and vice versa

Fig. 4

Different modes of binding of UBE2C and UBE2S to APC/C and effects on ubiquitination of the substrates. Upon engagement of the coactivator (purple) bound to the substrate (shown as a solid red line, with D- and KEN-boxes), UBE2C interacts with APC11 and the WHB domain of APC2 interacts with the backside of UBE2C. This arrangement restricts the sample space that can be explored by UBE2C and allows only a few ubiquitin molecules to be attached to the substrates. UBE2S interacts with a different region of APC11 that is away from the RING domain, while the C-terminal peptide of UBE2S binds to a site between APC2 and APC4 via a flexible linker. The RING domain of APC11 interacts with the acceptor ubiquitin (yellow) on the substrate and presents its K11 residue for accepting a ubiquitin (orange) from UBE2S. Flexible linkers of APC11 and UBE2S are shown by dashed lines

Fig. 5

Spindle assembly checkpoint (SAC) independent ubiquitination of Cyclin A2. a Phosphorylation of inactive, apo-APC/C leads to binding of CDK1-Cyclin A2-CKS1 complex to CDC20. The degrons of Cyclin A2 can bind in two different modes to APC/C^CDC20, only one mode is shown here that engages KEN- (K) and non-canonical D-box (D2), and activates ubiquitination of Cyclin A2. b Cyclin A can also bind to APC/C^CDC20-MCC complex. In the closed APC/C–MCC conformation, BUBR1 forms a lariat like structure between APC/C^CDC20 and CDC20^MCC via its multiple degrons. ABBA-box of Cyclin A2 (A) competes with the ABBA-box 2 of BUBR1 (A2) and can bridge both CDC20 molecules in APC/C by interaction of its D2- and KEN- boxes. This is proposed to induce the open conformation of APC/C–MCC and facilitates Cyclin A2 ubiquitination. For clarity, only relevant subunits are shown here. Yellow circle shows ubiquitination, degrons bounds with dashed lines indicate no interaction, those with solid lines indicate binding. Flexible linkers are shown by dashed lines. P denotes phosphorylation