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 cel ls with defective spindles from leaving mitosis interacts with Cdc20 and pr events its stimulatory action on the cyclosome. The activity of Cdh1 is neg atively 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 remai ned unknown. Here we show that phosphorylation of Cdc20 by Cdk1/cyclin B ab rogates its ability to activate cyclosome/APC from mitotic HeLa cells. A no nphosphorylatable derivative of Cdc20 stimulates cyclin-ubiquitin ligation in extracts from nocodazole-arrested cells to a much greater extent than do es wildtype Cdc20. It is suggested that inhibitory phosphorylation of Cdc20 /Fizzy may have a role in keeping the cyclosome inactive in early mitosis a nd under conditions of mitotic checkpoint arrest. (C) 2000 Academic Press.