title: In cellulo phosphorylation of XRCC4 Ser320 by DNA-PK induced by DNA damage.
abstract: XRCC4 is a protein associated with DNA Ligase IV, which is thought to join two DNA ends at the final step of DNA double-strand break repair through non-homologous end joining. In response to treatment with ionizing radiation or DNA damaging agents, XRCC4 undergoes DNA-PK-dependent phosphorylation. Furthermore, Ser260 and Ser320 (or Ser318 in alternatively spliced form) of XRCC4 were identified as the major phosphorylation sites by purified DNA-PK in vitro through mass spectrometry. However, it has not been clear whether these sites are phosphorylated in vivo in response to DNA damage. In the present study, we generated an antibody that reacts with XRCC4 phosphorylated at Ser320 and examined in cellulo phosphorylation status of XRCC4 Ser320. The phosphorylation of XRCC4 Ser320 was induced by γ-ray irradiation and treatment with Zeocin. The phosphorylation of XRCC4 Ser320 was detected even after 1 Gy irradiation and increased in a manner dependent on radiation dose. The phosphorylation was observed immediately after irradiation and remained mostly unchanged for up to 4 h. The phosphorylation was inhibited by DNA-PK inhibitor NU7441 and was undetectable in DNA-PKcs-deficient cells, indicating that the phosphorylation was mainly mediated by DNA-PK. These results suggested potential usefulness of the phosphorylation status of XRCC4 Ser320 as an indicator of DNA-PK functionality in living cells.
authors.affiliation: Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, N1-30 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8550, Japan Department of Zoology, R.R. Government College, Alwar 301001, India.
authors.forename: Mukesh Kumar
authors.initials: MK
authors.lastname: Sharma
authors.affiliation: Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, N1-30 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8550, Japan Division of Chemotherapy and Clinical Research, National Cancer Center Research Institute, Tokyo 104-0045, Japan.
authors.forename: Shoji
authors.initials: S
authors.lastname: Imamichi
authors.affiliation: Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, N1-30 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8550, Japan.
authors.forename: Mikoto
authors.initials: M
authors.lastname: Fukuchi
authors.affiliation: Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, N1-30 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8550, Japan Department of Research, Bhopal Memorial Hospital & Research Centre, Department of Health Research, Raisen Bypass Road, Bhopal 462038, India National Institute for Research in Environmental Health, Indian Council of Medical Research, Kamla Nehru Hospital Building, Gandhi Medical College Campus, Bhopal 462001, India.
authors.forename: Ravindra Mahadeo
authors.initials: RM
authors.lastname: Samarth
authors.affiliation: Radiation Safety Research Center, Central Research Institute of Electric Power Industry, Tokyo 201-8511, Japan.
authors.forename: Masanori
authors.initials: M
authors.lastname: Tomita
authors.affiliation: Research Laboratory for Nuclear Reactors, Tokyo Institute of Technology, N1-30 2-12-1, Ookayama, Meguro-ku, Tokyo 152-8550, Japan yoshim@nr.titech.ac.jp.
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Matsumoto
mesh_terms: D004249:DNA Damage; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004307:Dose-Response Relationship, Radiation; D005720:Gamma Rays; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation / Q000528:radiation effects; D010768:Phosphoserine / Q000378:metabolism*
chemical_list: D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C101467:XRCC4 protein, human; D010768:Phosphoserine; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human
keywords: DNA double-strand break repair; DNA-dependent protein kinase (DNA-PK); XRCC4; non-homologous end joining (NHEJ); phosphorylation