Requirement of ATM for rapid p53 phosphorylation at Ser46 without Ser/Thr-Gln sequences.
p53 phosphorylation at Ser46 following DNA damage is important for preferential transactivation of proapoptotic genes. Here, we report that ataxia-telangiectasia mutated (ATM) kinase is responsible for Ser46 phosphorylation of p53 during early-phase response to DNA damage. To elucidate the direct phosphorylation of p53 at Ser46 by ATM, an ATM mutant (ATM-AS) sensitive to ATP analogues was engineered. In vitro kinase assays revealed that p53 was phosphorylated at Ser46 by ATM-AS, even when ATP analogues were used as phosphate donors, although this phosphorylation site is not in an SQ motif, a consensus ATM site. Furthermore, Ser46 phosphorylation by ATM was dependent on the N- and C-terminal domains of p53, unlike Ser15 phosphorylation. Immunofluorescence analyses showed that Ser46-phosphorylated p53 was observed as foci in response to DNA damage and colocalized with gamma-H2AX or Ser1981-phosphorylated ATM. These results suggest that ATM phosphorylates a noncanonical serine residue on p53 by mechanisms different from those for the phosphorylation of Ser15.
Kodama
Masami
M
Radiobiology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Otsubo
Chihiro
C
Hirota
Toru
T
Yokota
Jun
J
Enari
Masato
M
Taya
Yoichi
Y
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005973:Glutamine / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013912:Threonine / Q000235:genetics* / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D011993:Recombinant Fusion Proteins; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D005973:Glutamine; D013912:Threonine; D012694:Serine; D000255:Adenosine Triphosphate; D004247:DNA; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases