Involvement of novel autophosphorylation sites in ATM activation.
ATM kinase plays a central role in signaling DNA double-strand breaks to cell cycle checkpoints and to the DNA repair machinery. Although the exact mechanism of ATM activation remains unknown, efficient activation requires the Mre11 complex, autophosphorylation on S1981 and the involvement of protein phosphatases and acetylases. We report here the identification of several additional phosphorylation sites on ATM in response to DNA damage, including autophosphorylation on pS367 and pS1893. ATM autophosphorylates all these sites in vitro in response to DNA damage. Antibodies against phosphoserine 1893 revealed rapid and persistent phosphorylation at this site after in vivo activation of ATM kinase by ionizing radiation, paralleling that observed for S1981 phosphorylation. Phosphorylation was dependent on functional ATM and on the Mre11 complex. All three autophosphorylation sites are physiologically important parts of the DNA damage response, as phosphorylation site mutants (S367A, S1893A and S1981A) were each defective in ATM signaling in vivo and each failed to correct radiosensitivity, genome instability and cell cycle checkpoint defects in ataxia-telangiectasia cells. We conclude that there are at least three functionally important radiation-induced autophosphorylation events in ATM.
Kozlov
Sergei V
SV
The Queensland Institute of Medical Research, Post Office Royal Brisbane Hospital, Herston, Brisbane, Queensland, Australia.
Graham
Mark E
ME
Peng
Cheng
C
Chen
Philip
P
Robinson
Phillip J
PJ
Lavin
Martin F
MF
D000595:Amino Acid Sequence; D064007:Ataxia Telangiectasia Mutated Proteins; D001665:Binding Sites / Q000528:radiation effects; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D004249:DNA Damage / Q000502:physiology / Q000528:radiation effects; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation / Q000528:radiation effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D011836:Radiation Tolerance; D015398:Signal Transduction / Q000528:radiation effects; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000378:metabolism*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases