title: Chemical genetics reveals a specific requirement for Cdk2 activity in the DNA damage response and identifies Nbs1 as a Cdk2 substrate in human cells.
abstract: The cyclin-dependent kinases (CDKs) that promote cell-cycle progression are targets for negative regulation by signals from damaged or unreplicated DNA, but also play active roles in response to DNA lesions. The requirement for activity in the face of DNA damage implies that there are mechanisms to insulate certain CDKs from checkpoint inhibition. It remains difficult, however, to assign precise functions to specific CDKs in protecting genomic integrity. In mammals, Cdk2 is active throughout S and G2 phases, but Cdk2 protein is dispensable for survival, owing to compensation by other CDKs. That plasticity obscured a requirement for Cdk2 activity in proliferation of human cells, which we uncovered by replacement of wild-type Cdk2 with a mutant version sensitized to inhibition by bulky adenine analogs. Here we show that transient, selective inhibition of analog-sensitive (AS) Cdk2 after exposure to ionizing radiation (IR) enhances cell-killing. In extracts supplemented with an ATP analog used preferentially by AS kinases, Cdk2(as) phosphorylated the Nijmegen Breakage Syndrome gene product Nbs1-a component of the conserved Mre11-Rad50-Nbs1 complex required for normal DNA damage repair and checkpoint signaling-dependent on a consensus CDK recognition site at Ser432. In vivo, selective inhibition of Cdk2 delayed and diminished Nbs1-Ser432 phosphorylation during S phase, and mutation of Ser432 to Ala or Asp increased IR-sensitivity. Therefore, by chemical genetics, we uncovered both a non-redundant requirement for Cdk2 activity in response to DNA damage and a specific target of Cdk2 within the DNA repair machinery.
authors.affiliation: Department of Structural and Chemical Biology, Mount Sinai School of Medicine, New York, New York, USA.
authors.forename: Lara
authors.initials: L
authors.lastname: Wohlbold
authors.forename: Karl A
authors.initials: KA
authors.lastname: Merrick
authors.forename: Saurav
authors.initials: S
authors.lastname: De
authors.forename: Ramon
authors.initials: R
authors.lastname: Amat
authors.forename: Jun Hyun
authors.initials: JH
authors.lastname: Kim
authors.forename: Stéphane
authors.initials: S
authors.lastname: Larochelle
authors.forename: Jasmina J
authors.initials: JJ
authors.lastname: Allen
authors.forename: Chao
authors.initials: C
authors.lastname: Zhang
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
authors.forename: John H J
authors.initials: JH
authors.lastname: Petrini
authors.forename: Robert P
authors.initials: RP
authors.lastname: Fisher
mesh_terms: D017766:Acid Anhydride Hydrolases; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism*; D004249:DNA Damage / Q000528:radiation effects*; D004260:DNA Repair; D045643:DNA Repair Enzymes / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D000076228:MRE11 Homologue Protein; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011839:Radiation, Ionizing
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; C112156:NBN protein, human; D009687:Nuclear Proteins; D051357:Cyclin-Dependent Kinase 2; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes