Degradation of Cdt1 during S phase is Skp2-independent and is required for efficient progression of mammalian cells through S phase.
Previous reports have shown that the N terminus of Cdt1 is required for its degradation during S phase (Li, X., Zhao, Q., Liao, R., Sun, P., and Wu, X. (2003) J. Biol. Chem. 278, 30854-30858; Nishitani, H., Lygerou, Z., and Nishimoto, T. (2004) J. Biol. Chem. 279, 30807-30816). The stabilization was attributed to deletion of the cyclin binding motif (Cy motif), which is required for its phosphorylation by cyclin-dependent kinases. Phosphorylated Cdt1 is subsequently recognized by the F-box protein Skp2 and targeted for proteasomal mediated degradation. Using phosphopeptide mapping and mutagenesis studies, we found that threonine 29 within the N terminus of Cdt1 is phosphorylated by Cdk2 and required for interaction with Skp2. However, threonine 29 and the Cy motif are not necessary for proteolysis of Cdt1 during S phase. Mutants of Cdt1 that do not stably associate with Skp2 or cyclins are still degraded in S phase to the same extent as wild type Cdt1, indicating that other determinants within the N terminus of Cdt1 are required for degrading Cdt1. We localized the region necessary for Cdt1 degradation to the first 32 residues. Overexpression of stable forms of Cdt1 significantly delayed entry into and completion of S phase, suggesting that failure to degrade Cdt1 prevents normal progression through S phase. In contrast, Cdt1 mutants that fail to interact with Skp2 and cyclins progress through S phase with similar kinetics as wild type Cdt1 but stimulate the re-replication caused by overexpressing Cdt1. Therefore, a Skp2-independent pathway that requires the N-terminal 32 residues of Cdt1 is critical for the degradation of Cdt1 in S phase, and this degradation is necessary for the optimum progression of cells through S phase.
Takeda
David Y
DY
Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Parvin
Jeffrey D
JD
Dutta
Anindya
A
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D002460:Cell Line; D002469:Cell Separation; D004268:DNA-Binding Proteins / Q000378:metabolism; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D007700:Kinetics; D013058:Mass Spectrometry; D064168:Minichromosome Maintenance Complex Component 7; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010449:Peptide Mapping; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D016196:S Phase; D044786:S-Phase Kinase-Associated Proteins / Q000378:metabolism / Q000502:physiology*; D013912:Threonine / Q000737:chemistry; D014162:Transfection
C113571:CDC45 protein, human; C467199:CDT1 protein, human; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010455:Peptides; D044786:S-Phase Kinase-Associated Proteins; D013912:Threonine; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7