Mechanism of APC/CCDC20 Activation by Mitotic Phosphorylation

Proc Natl Acad Sci U S A. 2016 May 10;113(19):E2570-8. doi: 10.1073/pnas.1604929113. Epub 2016 Apr 25.

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 phospho-sites 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.

Keywords: APC/C; CDC20; cell cycle; mitosis; phosphorylation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaphase-Promoting Complex-Cyclosome / chemistry
  • Anaphase-Promoting Complex-Cyclosome / metabolism*
  • Binding Sites
  • Cdc20 Proteins / chemistry
  • Cdc20 Proteins / metabolism*
  • Enzyme Activation
  • HeLa Cells
  • Humans
  • Mitosis / physiology*
  • Mutagenesis, Site-Directed / methods
  • Phosphorylation
  • Protein Binding
  • Transfection / methods

Substances

  • Cdc20 Proteins
  • CDC20 protein, human
  • Anaphase-Promoting Complex-Cyclosome