Mechanism of APC/C ^CDC20 activation by mitotic phosphorylation

Abstract

Chromosome segregation and mitotic exit are initiated by the 1.2-MDa ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome) and its coactivator CDC20 (cell division cycle 20). To avoid chromosome missegregation, APC/C CDC20 activation is tightly controlled. CDC20 only associates with APC/C in mitosis when APC/C has become phosphorylated and is further inhibited by a mitotic checkpoint complex until all chromosomes are bioriented on the spindle. APC/C contains 14 different types of subunits, most of which are phosphorylated in mitosis on multiple sites. However, it is unknown which of these phospho-sites enable APC/C CDC20 activation and by which mechanism. Here we have identified 68 evolutionarily conserved mitotic phosphosites on human APC/C bound to CDC20 and have used the biGBac technique to generate 47 APC/C mutants in which either all 68 sites or subsets of them were replaced by nonphosphorylatable or phospho-mimicking residues. The characterization of these complexes in substrate ubiquitination and degradation assays indicates that phosphorylation of an N-terminal loop region in APC1 is sufficient for binding and activation of APC/C by CDC20. Deletion of the N-terminal APC1 loop enables APC/C CDC20 activation in the absence of mitotic phosphorylation or phospho-mimicking mutations. These results indicate that binding of CDC20 to APC/C is normally prevented by an autoinhibitory loop in APC1 and that its mitotic phosphorylation relieves this inhibition. The predicted location of the N-terminal APC1 loop implies that this loop controls interactions between the N-terminal domain of CDC20 and APC1 and APC8. These results reveal how APC/C phosphorylation enables CDC20 to bind and activate the APC/C in mitosis.cell cycle | mitosis | APC/C | CDC20 | phosphorylation

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.…”

“…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.…”

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes

“…These results indicate that biGBac vectors can be used to express fully assembled enzymatically active recombinant human APC/C, which is sensitive to inhibition by EMI1-SKP1 and whose structural homogeneity allowed us to study its structure by cryo-EM. This conclusion is further supported by the accompanying manuscript by Qiao et al (22), which describes the generation and functional characterization of 47 different APC/C variants mutated in up to 68 mitotic phosphorylation sites. Furthermore, the recombinant forms of APC/C described in Brown et al (8,23) were also expressed from biGBac constructs.…”

“…Finally, biGBac vectors are designed in a way that enables a flexible mix and match utilization of genes, cDNAs, or cassettes containing multiples of these. As is illustrated by our generation of APC/C expression constructs in this and the accompanying manuscript by Qiao et al (22), this flexibility is particularly useful for the generation of constructs that encode subcomplexes, mutants, or differently tagged versions of a given protein complex. Based on these advantageous features of biGBac (summarized in Table S5), we expect that this method will further advance structural and functional studies of large protein complexes.…”