title: ATR (AT mutated Rad3 related) activity stabilizes Cdc6 and delays G2/M-phase entry during hydroxyurea-induced S-phase arrest of HeLa cells.
abstract: The Cdc6 protein, a key DNA replication initiation factor, contributes to the long-term maintenance of the S-phase checkpoint by anchoring the Rad3-Rad26 complex to chromatin. Here, we demonstrate that ATR (AT mutated and Rad3 related) activity is essential for maintaining high chromatin levels of the Cdc6 protein, thereby delaying entry into mitosis during hydroxyurea (HU)-induced S-phase arrest of HeLa cells. Downregulation of ATR (AT mutated and Rad3 related) (i.e., using ATR-siRNA) reduced the protein levels of chromatin Cdc6 and significantly increased the cellular levels of phospho-histone H3 (Ser 10), an index of mitosis. Downregulation of Cdc6 was completely restored by pretreatment with MG132, a proteasome inhibitor. Moreover, mitotic entry of MG132-pretreated cells was significantly downregulated. Our results also show that ATR (AT mutated and Rad3 related) kinase phosphorylates Cdc6 at serine residue 6. Thus, this ATR (AT mutated and Rad3 related)-mediated phosphorylation of Cdc6 is likely associated with stabilization of Cdc6 protein, thereby maintaining high levels of chromatin Cdc6 and delaying premature mitotic entry. This novel mechanism likely contributes to the functional regulation of chromatin Cdc6, which delays the cell cycle of hydroxyurea-induced cells to enter mitosis at the S-phase checkpoint.
authors.affiliation: Division of Pharmaceutical Biosciences, Research Institute for Pharmaceutical Sciences, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea. liulinhua@hanmail.net
authors.forename: Linhua
authors.initials: L
authors.lastname: Liu
authors.forename: Jae Hyuk
authors.initials: JH
authors.lastname: Choi
authors.forename: Hyungshin
authors.initials: H
authors.lastname: Yim
authors.forename: Joon Seok
authors.initials: JS
authors.lastname: Choi
authors.forename: Byoung Duck
authors.initials: BD
authors.lastname: Park
authors.forename: Seung Ju
authors.initials: SJ
authors.lastname: Cho
authors.forename: Seung Ki
authors.initials: SK
authors.lastname: Lee
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D002453:Cell Cycle / Q000187:drug effects* / Q000235:genetics; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002455:Cell Division / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D006918:Hydroxyurea / Q000494:pharmacology*; D015151:Immunoblotting; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016196:S Phase / Q000187:drug effects*
chemical_list: C103706:CDC6 protein, human; D018797:Cell Cycle Proteins; D009687:Nuclear Proteins; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases; D006918:Hydroxyurea