Cell cycle- and cell growth-regulated proteolysis of mammalian CDC6 is dependent on APC-CDH1.
CDC6 is conserved during evolution and is essential and limiting for the initiation of eukaryotic DNA replication. Human CDC6 activity is regulated by periodic transcription and CDK-regulated subcellular localization. Here, we show that, in addition to being absent from nonproliferating cells, CDC6 is targeted for ubiquitin-mediated proteolysis by the anaphase promoting complex (APC)/cyclosome in G(1). A combination of point mutations in the destruction box and KEN-box motifs in CDC6 stabilizes the protein in G(1) and in quiescent cells. Furthermore, APC, in association with CDH1, ubiquitinates CDC6 in vitro, and both APC and CDH1 are required and limiting for CDC6 proteolysis in vivo. Although a stable mutant of CDC6 is biologically active, overexpression of this mutant or wild-type CDC6 is not sufficient to induce multiple rounds of DNA replication in the same cell cycle. The APC-CDH1-dependent proteolysis of CDC6 in early G(1) and in quiescent cells suggests that this process is part of a mechanism that ensures the timely licensing of replication origins during G(1).
Petersen
B O
BO
Department of Experimental Oncology, European Institute of Oncology, 20141 Milan, Italy.
Wagener
C
C
Marinoni
F
F
Kramer
E R
ER
Melixetian
M
M
Lazzerini Denchi
E
E
Gieffers
C
C
Matteucci
C
C
Peters
J M
JM
Helin
K
K
D000595:Amino Acid Sequence; D064173:Anaphase-Promoting Complex-Cyclosome; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002455:Cell Division; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D004261:DNA Replication; D005455:Fluorescent Antibody Technique; D016193:G1 Phase; D006801:Humans; D008025:Ligases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D046988:Proteasome Endopeptidase Complex; D012333:RNA, Messenger / Q000378:metabolism; D018741:Replication Origin; D029701:Saccharomyces cerevisiae Proteins; D043743:Ubiquitin-Protein Ligase Complexes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D014452:Ubiquitins; D043743:Ubiquitin-Protein Ligase Complexes; D064173:Anaphase-Promoting Complex-Cyclosome; D044767:Ubiquitin-Protein Ligases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases