title: Serines 440 and 467 in the Werner syndrome protein are phosphorylated by DNA-PK and affects its dynamics in response to DNA double strand breaks.
abstract: WRN protein, defective in Werner syndrome (WS), a human segmental progeria, is a target of serine/threonine kinases involved in sensing DNA damage. DNA-PK phosphorylates WRN in response to DNA double strand breaks (DSBs). However, the main phosphorylation sites and functional importance of the phosphorylation of WRN has remained unclear. Here, we identify Ser-440 and -467 in WRN as major phosphorylation sites mediated by DNA-PK.In vitro, DNA-PK fails to phosphorylate a GST-WRN fragment with S440A and/or S467A substitution. In addition, full length WRN with the mutation expressed in 293T cells was not phosphorylated in response to DSBs produced by bleomycin. Accumulation of the mutant WRN at the site of laser-induced DSBs occurred with the same kinetics as wild type WRN in live HeLa cells. While the wild type WRN relocalized to the nucleoli after 24 hours recovery from etoposide-induced DSBs, the mutant WRN remained mostly in the nucleoplasm. Consistent with this, WS cells expressing the mutants exhibited less DNA repair efficiency and more sensitivity to etoposide, compared to those expressing wild type. Our findings indicate that phosphorylation of Ser-440 and -467 in WRN are important for relocalization of WRN to nucleoli, and that it is required for efficient DSB repair.
authors.affiliation: Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD 21224, USA.
authors.forename: Rika
authors.initials: R
authors.lastname: Kusumoto-Matsuo
authors.forename: Deblina
authors.initials: D
authors.lastname: Ghosh
authors.forename: Parimal
authors.initials: P
authors.lastname: Karmakar
authors.forename: Alfred
authors.initials: A
authors.lastname: May
authors.forename: Dale
authors.initials: D
authors.lastname: Ramsden
authors.forename: Vilhelm A
authors.initials: VA
authors.lastname: Bohr
mesh_terms: D001761:Bleomycin / Q000494:pharmacology; D002466:Cell Nucleolus / Q000187:drug effects / Q000201:enzymology*; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000235:genetics / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D005047:Etoposide / Q000494:pharmacology; D005090:Exodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D053484:RecQ Helicases / Q000235:genetics / Q000378:metabolism*; D012694:Serine; D014162:Transfection; D000071657:Werner Syndrome Helicase
chemical_list: D009687:Nuclear Proteins; D001761:Bleomycin; D012694:Serine; D005047:Etoposide; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D005090:Exodeoxyribonucleases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase