Mechanism of APC/CCDC20 activation by mitotic phosphorylation.
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.
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Renping
R
Qiao
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Florian
F
Weissmann
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
Masaya
M
Yamaguchi
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
Nicholas G
NG
Brown
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105; Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105;
Ryan
R
VanderLinden
Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Richard
R
Imre
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Marc A
MA
Jarvis
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105;
Michael R
MR
Brunner
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Iain F
IF
Davidson
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Gabriele
G
Litos
Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, 37077 Göttingen, Germany;
David
D
Haselbach
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria; Institute of Molecular Biotechnology of the Austrian Academy of Sciences (IMBA), Vienna Biocenter (VBC), 1030 Vienna, Austria;
Karl
K
Mechtler
Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany; Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, 37077 Göttingen, Germany; Jan-Michael.Peters@imp.ac.at Brenda.Schulman@stjude.org hstark1@gwdg.de.
Holger
H
Stark
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105; Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, TN 38105; Department of Microbiology, Immunology and Biochemistry, University of Tennessee Health Sciences Center, Memphis, TN 38163 Jan-Michael.Peters@imp.ac.at Brenda.Schulman@stjude.org hstark1@gwdg.de.
Brenda A
BA
Schulman
Research Institute of Molecular Pathology (IMP), Vienna Biocenter (VBC), 1030 Vienna, Austria; Jan-Michael.Peters@imp.ac.at Brenda.Schulman@stjude.org hstark1@gwdg.de.
Jan-Michael
JM
Peters
D064173:Anaphase-Promoting Complex-Cyclosome / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D064199:Cdc20 Proteins / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D006367:HeLa Cells; D006801:Humans; D008938:Mitosis / Q000502:physiology*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D010766:Phosphorylation; D011485:Protein Binding; D014162:Transfection / Q000379:methods
D064199:Cdc20 Proteins; C577488:CDC20 protein, human; D064173:Anaphase-Promoting Complex-Cyclosome
APC/C; CDC20; cell cycle; mitosis; phosphorylation