ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53 activation by DNA damage

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid a ctivation of p53 by ionizing radiation and radiomimetic agents is largely d ependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncop rotein is a pivotal negative regulator of p53. In response to ionizing radi ation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphoryl ation prior to p53 accumulation. This results in a decrease in its reactivi ty with the 2A10 monoclonal antibody. Phage display analysis identified a c onsensus 2A10 recognition sequence, possessing the core motif DYS. Unexpect edly, this motif appears twice within the human Mdm2 molecule, at positions corresponding to residues 258-260 and 393-395. Both putative 2A10 epitopes are highly conserved and encompass potential phosphorylation sites. Serine 395, residing within the carboxy-terminal 2A10 epitope, is the major targe t on Mdm2 for phosphorylation by ATM in vitro. Mutational analysis supports the conclusion that Mdm2 undergoes ATM-dependent phosphorylation on serine 395 in vivo in response to DNA damage. The data further suggests that phos phorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shu ttling of p53 and its subsequent degradation, thereby enabling p53 accumula tion. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.