title: HCLK2 is required for activity of the DNA damage response kinase ATR.
abstract: ATR is a protein kinase that orchestrates the cellular response to replication problems and DNA damage. HCLK2 has previously been reported to stabilize ATR and Chk1. Here we provide evidence that human HCLK2 acts at an early step in the ATR signaling pathway and contributes to full-scale activation of ATR kinase activity. We show that HCLK2 forms a complex with ATR-ATRIP and the ATR activator TopBP1. We demonstrate that HCLK2-induced ATR kinase activity toward substrates requires TopBP1 and vice versa and provides evidence that HCLK2 facilitates efficient ATR-TopBP1 association. Consistent with its role in ATR activation, HCLK2 depletion severely impaired phosphorylation of multiple ATR targets including Chk1, Nbs1, and Smc1 after DNA damage. We show that HCLK2 is required for and stimulates ATR autophosphorylation and activity toward different substrates in vitro. Furthermore, HCLK2 depletion abrogated the G(2) checkpoint and decreased survival of cells after exposure to DNA damaging agents and replicative stress. Overall, our data suggest that HCLK2 facilitates ATR activation and, therefore, contributes to ATR-mediated checkpoint signaling. Importantly, our results suggest that HCLK2 functions in the same pathway as TopBP1 but that the two proteins regulate different steps in ATR activation.
authors.affiliation: Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark.
authors.forename: Jannie M
authors.initials: JM
authors.lastname: Rendtlew Danielsen
authors.forename: Dorthe Helena
authors.initials: DH
authors.lastname: Larsen
authors.forename: Kenneth Bødtker
authors.initials: KB
authors.lastname: Schou
authors.forename: Raimundo
authors.initials: R
authors.lastname: Freire
authors.forename: Jacob
authors.initials: J
authors.lastname: Falck
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.forename: Jiri
authors.initials: J
authors.lastname: Lukas
mesh_terms: D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004790:Enzyme Induction / Q000502:physiology; D004795:Enzyme Stability / Q000502:physiology; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D000098550:Structural Maintenance of Chromosome Protein 1
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; C112156:NBN protein, human; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D000098550:Structural Maintenance of Chromosome Protein 1; D011494:Protein Kinases; C066780:Clk dual-specificity kinases; D011505:Protein-Tyrosine Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases