DNA-PK-dependent phosphorylation of Ku70/80 is not required for non-homologous end joining.
The Ku70/80 heterodimer is a major player in non-homologous end joining and the repair of DNA double-strand breaks. Studies suggest that once bound to a DNA double-strand break, Ku recruits the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) to form the DNA-dependent protein kinase holoenzyme complex (DNA-PK). We previously identified four DNA-PK phosphorylation sites on the Ku70/80 heterodimer: serine 6 of Ku70, serine 577 and 580 and threonine 715 of Ku80. This raised the interesting possibility that DNA-PK-dependent phosphorylation of Ku could provide a mechanism for the regulation of non-homologous end joining. Here, using mass spectrometry and phosphospecific antibodies we confirm that these sites are phosphorylated in vitro by purified DNA-PK. However, we show that neither DNA-PK nor the related protein kinase ataxia-telangiectasia mutated (ATM) is required for phosphorylation of Ku at these sites in vivo. Furthermore, Ku containing serine/threonine to alanine mutations at these sites was fully able to complement the radiation sensitivity of Ku negative mammalian cells indicating that phosphorylation at these sites is not required for non-homologous end joining. Interestingly, both Ku70 and Ku80 were phosphorylated in cells treated with the protein phosphatase inhibitor okadaic acid under conditions known to inactivate protein phosphatase 2A-like protein phosphatases. Moreover, okadaic acid-induced phosphorylation of Ku80 was inhibited by nanomolar concentrations of the protein kinase inhibitor staurosporine. These results suggest that the phosphorylation of Ku70 and Ku80 is regulated by a protein phosphatase 2A-like protein phosphatase and a staurosporine sensitive protein kinase in vivo, but that DNA-PK-mediated phosphorylation of Ku is not required for DNA double-strand break repair.
Douglas
Pauline
P
Department of Biochemistry & Molecular Biology and Southern Alberta Cancer Research Institute, University of Calgary, 3330 Hospital Drive N.W., Calgary, Alta., Canada T2N 4N1.
Gupta
Shikha
S
Morrice
Nick
N
Meek
Katheryn
K
Lees-Miller
Susan P
SP
D000818:Animals; D034961:Antigens, Nuclear / Q000276:immunology / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D011088:DNA Ligases; D004260:DNA Repair / Q000502:physiology*; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000276:immunology / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D000072200:Ku Autoantigen; D009687:Nuclear Proteins; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051381:Rats; D011995:Recombination, Genetic; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D034961:Antigens, Nuclear; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D025521:Tumor Suppressor Proteins; D011494:Protein Kinases; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; C000606509:Xrcc6 protein, human; C000606511:Xrcc6 protein, rat; D000072200:Ku Autoantigen; D011088:DNA Ligases