Structured DNA promotes phosphorylation of p53 by DNA-dependent protein kinase at serine 9 and threonine 18.
Phosphorylation at multiple sites within the N-terminus of p53 promotes its dissociation from hdm2/mdm2 and stimulates its transcriptional regulatory potential. The large phosphoinositide 3-kinase-like kinases ataxia telangiectasia mutated gene product and the ataxia telangectasia and RAD-3-related kinase promote phosphorylation of human p53 at Ser15 and Ser20, and are required for the activation of p53 following DNA damage. DNA-dependent protein kinase (DNA-PK) is another large phosphoinositide 3-kinase-like kinase with the potential to phosphorylate p53 at Ser15, and has been proposed to enhance phosphorylation of these sites in vivo. Moreover, recent studies support a role for DNA-PK in the regulation of p53-mediated apoptosis. We have shown previously that colocalization of p53 and DNA-PK to structured single-stranded DNA dramatically enhances the potential for p53 phosphorylation by DNA-PK. We report here the identification of p53 phosphorylation at two novel sites for DNA-PK, Thr18 and Ser9. Colocalization of p53 and DNA-PK on structured DNA was required for efficient phosphorylation of p53 at multiple sites, while specific recognition of Ser9 and Thr18 appeared to be dependent upon additional determinants of p53 beyond the N-terminal 65 amino acids. Our results suggest a role for DNA-PK in the modulation of p53 activity resultant from the convergence of p53 and DNA-PK on structured DNA.
Soubeyrand
Sébastien
S
Department of Medicine, University of Ottawa, The Ottawa Health Research Institute, Ottawa, Ontario, Canada. ssoubeyrand@ohri.ca
Schild-Poulter
Caroline
C
Haché
Robert J G
RJ
D000595:Amino Acid Sequence; D004247:DNA / Q000378:metabolism*; D004277:DNA, Single-Stranded / Q000378:metabolism; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D009687:Nuclear Proteins; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012694:Serine / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013912:Threonine / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D016159:Tumor Suppressor Protein p53; D013912:Threonine; D012694:Serine; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases