title: Essential role for Cdk2 inhibitory phosphorylation during replication stress revealed by a human Cdk2 knockin mutation.
abstract: Cyclin-dependent kinases (Cdks) coordinate cell division, and their activities are tightly controlled. Phosphorylation of threonine 14 (T14) and tyrosine 15 (Y15) inhibits Cdks and regulates their activities in numerous physiologic contexts. Although the roles of Cdk1 inhibitory phosphorylation during mitosis are well described, studies of Cdk2 inhibitory phosphorylation during S phrase have largely been indirect. To specifically study the functions of Cdk2 inhibitory phosphorylation, we used gene targeting to make an endogenous Cdk2 knockin allele in human cells, termed Cdk2AF, which prevents Cdk2 T14 and Y15 phosphorylation. Cdk2AF caused premature S-phase entry, rapid cyclin E degradation, abnormal DNA replication, and genome instability. Cdk2AF cells also exhibited strikingly abnormal responses to replication stress, accumulated irreparable DNA damage, and permanently exited the cell cycle after transient exposure to S-phase inhibitors. Our results reveal the specific and essential roles of Cdk2 inhibitory phosphorylation in the successful execution of the replication stress checkpoint response and in maintaining genome integrity.
authors.affiliation: Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA.
authors.forename: Bridget T
authors.initials: BT
authors.lastname: Hughes
authors.forename: Julia
authors.initials: J
authors.lastname: Sidorova
authors.forename: Jherek
authors.initials: J
authors.lastname: Swanger
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Monnat
authors.forename: Bruce E
authors.initials: BE
authors.lastname: Clurman
mesh_terms: D018797:Cell Cycle Proteins / Q000378:metabolism; D002478:Cells, Cultured; D019927:Cyclin E / Q000378:metabolism; D051357:Cyclin-Dependent Kinase 2 / Q000235:genetics / Q000378:metabolism*; D004249:DNA Damage / Q000502:physiology; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000378:metabolism; D016521:Electrophoresis, Gel, Pulsed-Field; D005434:Flow Cytometry; D055879:Gene Knock-In Techniques; D042822:Genomic Instability / Q000502:physiology; D006801:Humans; D044085:Microfluidics; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D016196:S Phase / Q000502:physiology*; D059807:S Phase Cell Cycle Checkpoints / Q000502:physiology; D015398:Signal Transduction / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D019927:Cyclin E; D004268:DNA-Binding Proteins; C078341:MYT1 protein, human; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases; C499349:WEE1 protein, human; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2
keywords: Wee1; cyclin A