DNA-DAMAGE INDUCES PHOSPHORYLATION OF THE AMINO-TERMINUS OF P53

Data are presented demonstrating that DNA damage leads to specific pos t-translational modifications of p53 protein. Using two-dimensional pe ptide mapping of in vivo radiolabeled p53 tryptic phosphopeptides, rec ombinant 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 ac ids of p53 and contains three phosphorylated serine residues. A specif ic p53 phosphopeptide antibody identified serine-15 as one of the two serines in p53 that becomes phosphorylated following DNA damage induce d by either ionizing irradiation (IR) or ultraviolet (UV) irradiation in multiple cell types. IR-induced phosphorylation of p53 does not aff ect the kinetics of p53 binding to or dissociating from DNA as assesse d by electrophoretic mobility-shift assays. However, p53 phosphorylati on induced by DNA damage correlates with enhanced transcription of dow nstream p53 target genes. Low levels of phosphoserine-15 p53 are detec table within 6 hr after IR in AT cells, whereas lymphoblasts from norm al individuals exhibit this modification within 1 hr. In contrast, pho sphorylation of p53 on serine-15 is similar in normal and AT cells aft er UV irradiation. Our results indicate that p53 is phosphorylated in response to DNA damage, that this de novo phosphorylation may be invol ved in the subsequent induction and activation of p53, and that althou gh ATM affects the kinetics of p53 phosphorylation after IR, it is not absolutely required for phosphorylation of p53 on serine-15.