Cell cycle dependence of DNA-dependent protein kinase phosphorylation in response to DNA double strand breaks.
DNA-dependent protein kinase (DNA-PK), consisting of Ku and DNA-PKcs subunits, is the key component of the non-homologous end-joining (NHEJ) pathway of DNA double strand break (DSB) repair. Although the kinase activity of DNA-PKcs is essential for NHEJ, thus far, no in vivo substrate has been conclusively identified except for an autophosphorylation site on DNA-PKcs itself (threonine 2609). Here we report the ionizing radiation (IR)-induced autophosphorylation of DNA-PKcs at a novel site, serine 2056, the phosphorylation of which is required for the repair of DSBs by NHEJ. Interestingly, IR-induced DNA-PKcs autophosphorylation is regulated in a cell cycle-dependent manner with attenuated phosphorylation in the S phase. In contrast, DNA replication-associated DSBs resulted in DNA-PKcs autophosphorylation and localization to DNA damage sites. These results indicate that although IR-induced DNA-PKcs phosphorylation is attenuated in the S phase, DNA-PKcs is preferentially activated by the physiologically relevant DNA replication-associated DSBs at the sites of DNA synthesis.
Chen
Benjamin P C
BP
Department of Radiation Oncology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9187, USA. benjamin.chen@utsouthwestern.edu <benjamin.chen@utsouthwestern.edu>
Chan
Doug W
DW
Kobayashi
Junya
J
Burma
Sandeep
S
Asaithamby
Aroumougame
A
Morotomi-Yano
Keiko
K
Botvinick
Elliot
E
Qin
Jun
J
Chen
David J
DJ
D000595:Amino Acid Sequence; D002453:Cell Cycle; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry* / Q000378:metabolism*; D011839:Radiation, Ionizing; D016196:S Phase; D012694:Serine / Q000737:chemistry; D013912:Threonine / Q000737:chemistry
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D013912:Threonine; D012694:Serine; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases