Molecular mechanism of APC/C activation by mitotic phosphorylation

Abstract

In eukaryotes, the anaphase-promoting complex/cyclosome (APC/C) regulates the ubiquitin-dependent proteolysis of specific cell cycle proteins to coordinate chromosome segregation in mitosis and entry into G1 (refs 1,2). The APC/C’s catalytic activity and ability to specify the destruction of particular proteins at different phases of the cell cycle are controlled by its interaction with two structurally related coactivator subunits (Cdc20 and Cdh1). Coactivators recognize substrate degrons3, and enhance the APC/C’s affinity for its cognate E2 (refs 4–6). During mitosis, cyclin-dependent kinase and polo kinase control Cdc20 and Cdh1-mediated activation of the APC/C. Hyper-phosphorylation of APC/C subunits, notably Apc1 and Apc3, is required for Cdc20 to activate the APC/C7–12, whereas phosphorylation of Cdh1 prevents its association with the APC/C9,13,14. Since both coactivators associate with the APC/C through their common C box15 and IR (Ile-Arg) tail motifs16,17, the mechanism underlying this differential regulation is unclear, as is the role of specific APC/C phosphorylation sites. Here, using cryo-electron microscopy (cryo-EM) and biochemical analysis, we define the molecular basis of how APC/C phosphorylation allows for its control by Cdc20. An auto-inhibitory (AI) segment of Apc1 acts as a molecular switch that in apo unphosphorylated APC/C interacts with the C-box binding site and obstructs engagement of Cdc20. Phosphorylation of the AI segment displaces it from the C-box binding site. Efficient phosphorylation of the AI segment, and thus relief of auto-inhibition, requires the recruitment of Cdk-cyclin-Cks to a hyper-phosphorylated loop of Apc3. We also find that the small molecule inhibitor, tosyl-L-arginine methyl ester (TAME), preferentially suppresses APC/CCdc20 rather than APC/CCdh1, and interacts with both the C-box and IR-tail binding sites. Our results reveal the mechanism for the regulation of mitotic APC/C by phosphorylation and provide a rationale for the development of selective inhibitors of this state.

WD40 domain of Apc1 is critical for the coactivator-induced allosteric transition that stimulates APC/C catalytic activity

“…Thus, deletion of loop 2 of Apc1 WD40 (also termed the "300s loop") enables APC/C Cdc20 activation, mimicking the effects of mitotic APC/C phosphorylation. This finding is in agreement with recent studies (39,40). The loss of APC/C catalytic activity in the absence of Apc1 WD40 could result from the loss of either UbcH10 or substrate/coactivator interactions with the mutant APC/C.…”

“…2E). This result, indicating that loop 2 (residues 307-402) of Apc1 WD40 inhibits Cdc20 activity, is in agreement with the identification of an autoinhibitory segment within this loop that blocks the binding site of the coactivator C-box on Apc8B (39,40). Phosphorylation of loop 2 (referred to as the "300s loop" in ref.…”

“…Interestingly, the conformation of APC/C ΔApc1−WD40 -Cdh1-Hsl1 resembles the hybrid class observed in the EM datasets of wild-type ternary APC/C-coactivator-substrate complexes (∼10% of APC/C particles) (12,19,39). In the hybrid state, the catalytic module adopts the inactive conformation, and EM densities for Apc1 WD40 and Apc15 are absent.…”

“…Numerous mitotic phosphorylation sites are located within these loops, implicating a potential role in regulating Cdc20 interactions with the APC/C. This idea has been confirmed recently by structural and biochemical studies (39,40). We therefore addressed the requirement of these Apc1 WD40 loops for APC/C activity.…”

“…Phosphorylation of loop 2 (referred to as the "300s loop" in ref. 39) by mitotic kinases displaces the autoinhibitory segment, relieving the steric blockade of the C-box binding site, thereby permitting APC/C-Cdc20 interactions (39). Thus, deletion of loop 2 of Apc1 WD40 (also termed the "300s loop") enables APC/C Cdc20 activation, mimicking the effects of mitotic APC/C phosphorylation.…”