title: Cdk-mediated phosphorylation of Chk1 is required for efficient activation and full checkpoint proficiency in response to DNA damage.
abstract: Here, we show that activation of the checkpoint effector kinase Chk1 in response to irradiation-induced DNA damage is minimal in G1, maximal during S-phase and diminishes as cells enter G2. In addition, formation of irradiation-induced replication protein A (RPA)-coated single-stranded DNA (RPA-ssDNA), a structure required for ATM and Rad3-related (ATR)-Chk1 activation, occurs in a broadly similar pattern. Cyclin-dependent kinase (Cdk) activity is thought to promote RPA-ssDNA formation by stimulating DNA strand resection at double-strand breaks (DSBs), providing one possible mechanism of imposing cell cycle dependence on DNA damage signaling. However, it has recently been shown that Chk1 itself is also subject to Cdk-mediated phosphorylation at serines 286 and 301 (S286 and 301). We show that Chk1 S301 phosphorylation increases as cells progress through S and G2 and that both Cdk1 and Cdk2 are likely to contribute to this modification in vivo. We also find that substitution of S286 and S301 with non-phosphorylatable alanine residues strongly attenuates DNA damage-induced Chk1 activation and G2 checkpoint proficiency, but does not eliminate the underlying cell cycle dependence of Chk1 regulation. Taken together, these data indicate that Cdk activity regulates multiple steps in the DNA damage response pathway including full activation of Chk1 and checkpoint proficiency.
authors.affiliation: Beatson Institute for Cancer Research, Garscube Estate, Glasgow, UK. xu.naihan@sz.tsinghua.edu.cn
authors.forename: N
authors.initials: N
authors.lastname: Xu
authors.forename: S
authors.initials: S
authors.lastname: Libertini
authors.forename: E J
authors.initials: EJ
authors.lastname: Black
authors.forename: Y
authors.initials: Y
authors.lastname: Lao
authors.forename: N
authors.initials: N
authors.lastname: Hegarat
authors.forename: M
authors.initials: M
authors.lastname: Walker
authors.forename: D A
authors.initials: DA
authors.lastname: Gillespie
mesh_terms: D016203:CDC2 Protein Kinase / Q000378:metabolism; D059447:Cell Cycle Checkpoints / Q000235:genetics / Q000528:radiation effects; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004249:DNA Damage / Q000528:radiation effects*; D004789:Enzyme Activation; D006801:Humans; D008954:Models, Biological; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*
chemical_list: D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases