Cdt1 phosphorylation by cyclin A-dependent kinases negatively regulates its function without affecting geminin binding.
The current concept regarding cell cycle regulation of DNA replication is that Cdt1, together with origin recognition complex and CDC6 proteins, constitutes the machinery that loads the minichromosome maintenance complex, a candidate replicative helicase, onto chromatin during the G(1) phase. The actions of origin recognition complex and CDC6 are suppressed through phosphorylation by cyclin-dependent kinases (Cdks) after S phase to prohibit rereplication. It has been suggested in metazoan cells that the function of Cdt1 is blocked through binding to an inhibitor protein, geminin. However, the functional relationship between the Cdt1-geminin system and Cdks remains to be clarified. In this report, we demonstrate that human Cdt1 is phosphorylated by cyclin A-dependent kinases dependent on its cyclin-binding motif. Cdk phosphorylation resulted in the binding of Cdt1 to the F-box protein Skp2 and subsequent degradation. In contrast, in vitro DNA binding activity of Cdt1 was inhibited by the phosphorylation. However, geminin binding to Cdt1 was not affected by the phosphorylation. Finally we provide evidence that inactivation of Cdk1 results in Cdt1 dephosphorylation and rebinding to chromatin in murine FT210 cells synchronized around the G(2)/M phase. Taken together, these findings suggest that Cdt1 function is also negatively regulated by the Cdk phosphorylation independent of geminin binding.
Virology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuohku, Tokyo 104-0045.
Nozomi
N
Sugimoto
Yasutoshi
Y
Tatsumi
Tatsuya
T
Tsurumi
Akio
A
Matsukage
Tohru
T
Kiyono
Hideo
H
Nishitani
Masatoshi
M
Fujita
D020816:Amino Acid Motifs; D000818:Animals; D016203:CDC2 Protein Kinase / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D016193:G1 Phase; D064248:Geminin; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D051379:Mice; D009687:Nuclear Proteins; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D013696:Temperature; D013997:Time Factors; D014162:Transfection
C066503:CDC6 protein, S cerevisiae; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C577995:GMNN protein, human; D064248:Geminin; C577997:Gmnn protein, mouse; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D044786:S-Phase Kinase-Associated Proteins; D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; D005982:Glutathione Transferase; D016203:CDC2 Protein Kinase