title: Werner protein is a target of DNA-dependent protein kinase in vivo and in vitro, and its catalytic activities are regulated by phosphorylation.
abstract: Human Werner Syndrome is characterized by early onset of aging, elevated chromosomal instability, and a high incidence of cancer. Werner protein (WRN) is a member of the recQ gene family, but unlike other members of the recQ family, it contains a unique 3'-->5' exonuclease activity. We have reported previously that human Ku heterodimer interacts physically with WRN and functionally stimulates WRN exonuclease activity. Because Ku and DNA-PKcs, the catalytic subunit of DNA-dependent protein kinase (DNA-PK), form a complex at DNA ends, we have now explored the possibility of functional modulation of WRN exonuclease activity by DNA-PK. We find that although DNA-PKcs alone does not affect the WRN exonuclease activity, the additional presence of Ku mediates a marked inhibition of it. The inhibition of WRN exonuclease by DNA-PKcs requires the kinase activity of DNA-PKcs. WRN is a target for DNA-PKcs phosphorylation, and this phosphorylation requires the presence of Ku. We also find that treatment of recombinant WRN with a Ser/Thr phosphatase enhances WRN exonuclease and helicase activities and that WRN catalytic activity can be inhibited by rephosphorylation of WRN with DNA-PK. Thus, the level of phosphorylation of WRN appears to regulate its catalytic activities. WRN forms a complex, both in vitro and in vivo, with DNA-PKC. WRN is phosphorylated in vivo after treatment of cells with DNA-damaging agents in a pathway that requires DNA-PKcs. Thus, WRN protein is a target for DNA-PK phosphorylation in vitro and in vivo, and this phosphorylation may be a way of regulating its different catalytic activities, possibly in the repair of DNA dsb.
authors.affiliation: Laboratory of Molecular Gerontology, NIA, National Institutes of Health, Baltimore, Maryland 21224, USA.
authors.forename: Parimal
authors.initials: P
authors.lastname: Karmakar
authors.forename: Jason
authors.initials: J
authors.lastname: Piotrowski
authors.forename: Robert M
authors.initials: RM
authors.lastname: Brosh
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Sommers
authors.forename: Susan P Lees
authors.initials: SP
authors.lastname: Miller
authors.forename: Wen-Hsing
authors.initials: WH
authors.lastname: Cheng
authors.forename: Carey M
authors.initials: CM
authors.lastname: Snowden
authors.forename: Dale A
authors.initials: DA
authors.lastname: Ramsden
authors.forename: Vilhelm A
authors.initials: VA
authors.lastname: Bohr
mesh_terms: D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D020134:Catalytic Domain; D004249:DNA Damage; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D005090:Exodeoxyribonucleases; D006367:HeLa Cells; D006801:Humans; D007313:Insecta; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D053484:RecQ Helicases; D011994:Recombinant Proteins / Q000378:metabolism; D014898:Werner Syndrome / Q000378:metabolism*; D000071657:Werner Syndrome Helicase
chemical_list: D017931:DNA Primers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D005090:Exodeoxyribonucleases; D004265:DNA Helicases; D053484:RecQ Helicases; C098785:WRN protein, human; D000071657:Werner Syndrome Helicase