Phosphorylation of the cyclosome is required for its stimulation by Fizzy/cdc20.
Exit from mitosis in eukaryotic cells is regulated by the cyclosome (also called anaphase promoting complex or APC), a multisubunit ubiquitin ligase that acts on mitotic cyclins. Previous studies in a cell-free system from clam oocytes have shown that the activation of the cyclosome at the end of mitosis involves its phosphorylation by protein kinase Cdk1/cyclin B. Genetic and biochemical studies have furthermore indicated that cyclosome activity also requires a WD-40 repeat containing protein called Fizzy (FZY) or Cdc20. It has been suggested [Fang et al. (1998) Mol. Cell 2, 163-171] that in the presence of FZY, the phosphorylation of the cyclosome is not critical for its activation. By contrast, we find that the activity of the interphase, non-phosphorylated form of the cyclosome from clam embryos is not stimulated by FZY to a significant extent. However, when interphase cyclosome is first incubated with protein kinase Cdk1/cyclin B, the subsequent supplementation of FZY greatly stimulates its cyclin-ubiquitin ligase activity. Furthermore, phosphatase treatment of purified mitotic cyclosome prevents its stimulation by FZY, a process that can be reversed by the action of protein kinase Cdk1/cyclin B. We conclude that in the early embryonic cell cycles, the primary event in the activation of the cyclosome at the end of mitosis is its Cdk1-dependent phosphorylation and activation by FZY takes place in a subsequent process.
Shteinberg
M
M
Unit of Biochemistry, B. Rappaport Faculty of Medicine and the Rappaport Institute for Research in the Medical Sciences, Haifa, 31096, Israel.
Protopopov
Y
Y
Listovsky
T
T
Brandeis
M
M
Hershko
A
A
D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; D049872:Bivalvia / Q000196:embryology; D016203:CDC2 Protein Kinase / Q000494:pharmacology; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D019926:Cyclin B / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D008025:Ligases / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000494:pharmacology
D064191:Apc3 Subunit, Anaphase-Promoting Complex-Cyclosome; C577495:CDC20 protein, S cerevisiae; C577355:CDC27 protein, S cerevisiae; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; D019926:Cyclin B; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D016203:CDC2 Protein Kinase; D008025:Ligases