The p53 tumor suppressor protein is activated and phosphorylated on serine-15 in response to various DNA damaging agents. The gene product mutated in ataxia telangiectasia, ATM, acts upstream of p53 in a signal transduction pathway initiated by ionizing radiation. Immunoprecipitated ATM had intrinsic protein kinase activity and phosphorylated p53 on serine-15 in a manganese-dependent manner. Ionizing radiation, but not ultraviolet radiation, rapidly enhanced this p53-directed kinase activity of endogenous ATM. These observations, along with the fact that phosphorylation of p53 on serine-15 in response to ionizing radiation is reduced in ataxia telangiectasia cells, suggest that ATM is a protein kinase that phosphorylates p53 in vivo.

Data are presented demonstrating that DNA damage leads to specific post-translational modifications of p53 protein. Using two-dimensional peptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, recombinant truncated p53 protein, and synthetic p53 tryptic peptides, a unique p53 phosphopeptide was identified after exposure of ML-1 cells to ionizing irradiation. This peptide represents the first 24 amino acids of p53 and contains three phosphorylated serine residues. A specific p53 phosphopeptide antibody identified serine-15 as one of the two serines in p53 that becomes phosphorylated following DNA damage induced by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not affect the kinetics of p53 binding to or dissociating from DNA as assessed by electrophoretic mobility-shift assays. However, p53 phosphorylation induced by DNA damage correlates with enhanced transcription of downstream p53 target genes. Low levels of phosphoserine-15 p53 are detectable within 6 hr after IR in AT cells, whereas lymphoblasts from normal individuals exhibit this modification within 1 hr. In contrast, phosphorylation of p53 on serine-15 is similar in normal and AT cells after UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be involved in the subsequent induction and activation of p53, and that although ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-15.

Ataxia telangiectasia mutated (ATM) phosphorylates p53 protein in response to ionizing radiation, but the complex phenotype of AT cells suggests that it must have other cellular substrates as well. To identify substrates for ATM and the related kinases ATR and DNA-PK, we optimized in vitro kinase assays and developed a rapid peptide screening method to determine general phosphorylation consensus sequences. ATM and ATR require Mn 2؉ , but not DNA ends or Ku proteins, for optimal in vitro activity while DNA-PKCs requires Mg 2؉ , DNA ends, and Ku proteins. From p53 peptide mutagenesis analysis, we found that the sequence S/TQ is a minimal essential requirement for all three kinases. In addition, hydrophobic amino acids and negatively charged amino acids immediately NH 2 -terminal to serine or threonine are positive determinants and positively charged amino acids in the region are negative determinants for substrate phosphorylation. We determined a general phosphorylation consensus sequence for ATM and identified putative in vitro targets by using glutathione Stransferase peptides as substrates. Putative ATM in vitro targets include p95/nibrin, Mre11, Brca1, Rad17, PTS, WRN, and ATM (S440) itself. Brca2, phosphatidylinositol 3-kinase, and DNA-5B peptides were phosphorylated specifically by ATR, and DNA Ligase IV is a specific in vitro substrate of DNA-PK. Ataxia telangiectasia (AT)1 is a complex human autosomal recessive disease characterized by a pleiotropic phenotype including neuronal degeneration, oculocutaneous telangiectasias, immune dysfunction, cancer predisposition, and premature aging (1). Cells derived from AT patients exhibit a variety of abnormal responses to ionizing irradiation (IR), such as checkpoint alterations in the G 1 , S, and G 2 phases of the cell cycle and radiosensitivity with increased chromosomal breakage and telomere fusions (2, 3). The G 1 checkpoint abnormality results from a quantitative defect in p53 induction (4 -6), but the molecular mechanisms underlying the radiosensitivity and the S-phase and G 2 phase checkpoint defects have not yet been elucidated.The gene that is mutated in AT, ATM (ataxia telangiectasia, mutated), encodes a 370-kDa protein with a carboxyl-terminal sequence homologous to the catalytic domain of phosphotidylinositol 3-kinases (7). Although ATM is predominantly located in the nucleus (8 -11), a fraction of ATM protein also appears in cytoplasmic vesicles in certain cell types (10, 12) where it associates with ␤-adaptin (13). A family of gene products with similar sizes and sequences has been identified, including the yeast proteins rad3p, Mec1p, Tel1p, Tor1p, Tor2, and the mammalian proteins mTor/FRAP, DNA-PKcs (DNA-dependent protein kinase), ATR (ataxia telangiectasia and rad3-related kinase) and TRAP (7,14). In mammalian cells, ATR and DNA-PK also appear to be involved in DNA-damage response pathways (15-17). For example, several lines of evidence suggest primary roles of DNA-PK are in DNA-repair and V(D)J recombination (17). ATR is considered the mammalian c...