Phosphorylation of Cdc20/fizzy negatively regulates the mammalian cyclosome/APC in the mitotic checkpoint.
The cyclosome/anaphase promoting complex (APC) is a multisubunit ubiquitin ligase that targets mitotic regulators for degradation in exit from mitosis. It is activated at the end of mitosis by phosphorylation and association with the WD-40 protein Cdc20/Fizzy and is then kept active in the G1 phase by association with Cdh1/Hct1. The mitotic checkpoint system that keeps cells with defective spindles from leaving mitosis interacts with Cdc20 and prevents its stimulatory action on the cyclosome. The activity of Cdh1 is negatively regulated by phosphorylation, while the abundance of Cdc20 is cell cycle regulated, with a peak in M-phase. Cdc20 is also phosphorylated in G2/M and in mitotically arrested cells, but the role of phosphorylation remained unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B abrogates its ability to activate cyclosome/APC from mitotic HeLa cells. A nonphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than does wild-type Cdc20. It is suggested that inhibitory phosphorylation of Cdc20/Fizzy may have a role in keeping the cyclosome inactive in early mitosis and under conditions of mitotic checkpoint arrest.
Yudkovsky
Y
Y
Unit of Biochemistry, B. Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, 31096, Israel.
Shteinberg
M
M
Listovsky
T
T
Brandeis
M
M
Hershko
A
A
D064173:Anaphase-Promoting Complex-Cyclosome; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D019926:Cyclin B / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D006367:HeLa Cells; D006801:Humans; D008025:Ligases / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis; D010766:Phosphorylation; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases
C577495:CDC20 protein, S cerevisiae; D064199:Cdc20 Proteins; D064200:Cdh1 Proteins; D018797:Cell Cycle Proteins; D019926:Cyclin B; C577584:FZR1 protein, human; D029701:Saccharomyces cerevisiae Proteins; C577488:CDC20 protein, human; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D008025:Ligases