title: Checkpoint kinase ATR phosphorylates Cdt2, a substrate receptor of CRL4 ubiquitin ligase, and promotes the degradation of Cdt1 following UV irradiation.
abstract: The DNA replication-licensing factor Cdt1 is present during the G1 phase of the cell cycle. When cells initiate S phase or are UV-irradiated, Cdt1 is recruited to chromatin-bound PCNA and ubiquitinated by CRL4(Cdt2) for degradation. In both situations, the substrate-recognizing subunit Cdt2 is detected as a highly phosphorylated form. Here, we show that both caffeine-sensitive kinase and MAP kinases are responsible for Cdt2 phosphorylation following UV irradiation. We found that Cdt1 degradation was attenuated in the presence of caffeine. This attenuation was also observed in cells depleted of ATR, but not ATM. Following UV irradiation, Cdt2 was phosphorylated at the S/TQ sites. ATR phosphorylated Cdt2 in vitro, mostly in the C-terminal region. Cdt1 degradation was also induced by DNA damaging chemicals such as methyl methanesulfonate (MMS) or zeocin, depending on PCNA and CRL4-Cdt2, though it was less caffeine-sensitive. These findings suggest that ATR, activated after DNA damage, phosphorylates Cdt2 and promotes the rapid degradation of Cdt1 after UV irradiation in the G1 phase of the cell cycle.
authors.affiliation: Graduate School of Life Science, University of Hyogo, Kamigori, Ako-gun, Hyogo, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Sakaguchi
authors.forename: Toshihiro
authors.initials: T
authors.lastname: Takami
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Yasutani
authors.forename: Takeshi
authors.initials: T
authors.lastname: Maeda
authors.forename: Masayuki
authors.initials: M
authors.lastname: Morino
authors.forename: Takashi
authors.initials: T
authors.lastname: Ishii
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Shiomi
authors.forename: Hideo
authors.initials: H
authors.lastname: Nishitani
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D044842:Cullin Proteins / Q000378:metabolism; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D055785:Gene Knockdown Techniques; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational / Q000528:radiation effects*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D059748:Proteolysis / Q000528:radiation effects*; D034622:RNA Interference; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism*; D014466:Ultraviolet Rays
chemical_list: C467199:CDT1 protein, human; C114234:CUL4A protein, human; D018797:Cell Cycle Proteins; D044842:Cullin Proteins; D004268:DNA-Binding Proteins; C429530:DTL protein, human; D009687:Nuclear Proteins; D025521:Tumor Suppressor Proteins; D002110:Caffeine; D044767:Ubiquitin-Protein Ligases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D020928:Mitogen-Activated Protein Kinases