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

Abstract

The p53 tumor suppressor protein, a key regulator of cellular responses to genotoxic stress, is stabilized and activated after DNA damage. The rapid activation of p53 by ionizing radiation and radiomimetic agents is largely dependent on the ATM kinase. p53 is phosphorylated by ATM shortly after DNA damage, resulting in enhanced stability and activity of p53. The Mdm2 oncoprotein is a pivotal negative regulator of p53. In response to ionizing radiation and radiomimetic drugs, Mdm2 undergoes rapid ATM-dependent phosphorylation prior to p53 accumulation. This results in a decrease in its reactivity with the 2A10 monoclonal antibody. Phage display analysis identified a consensus 2A10 recognition sequence, possessing the core motif DYS. Unexpectedly, 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 target 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 phosphorylated Mdm2 may be less capable of promoting the nucleo-cytoplasmic shuttling of p53 and its subsequent degradation, thereby enabling p53 accumulation. Our findings imply that activation of p53 by DNA damage is achieved, in part, through attenuation of the p53-inhibitory potential of Mdm2.

DNA double-strand break repair and development

“…Particularly in the nervous system, ATM-dependent apoptosis after DNA damage critically requires activation of p53 and Chk2 (Herzog et al, 1998;Chong et al, 2000;Takai et al, 2002). ATM indirectly affects p53 stabilization through downregulation of its ubiquitin ligase Mdm2 (Khosravi et al, 1999;Maya et al, 2001). COP1 is yet another E3 ligase of p53 that ATM phosphorylates, resulting in its degradation and subsequent p53 stabilization (Dornan et al, 2006).…”

Ribosome biogenesis surveillance: probing the ribosomal protein-Mdm2-p53 pathway

“…Mdm2 is also reported to be extensively modified through a number of posttranslational modifications including ubiquitination, sumoylation and phosphorylation (Meek and Knippschild, 2003). Although DNA damage is known to activate ATM kinase-induced phosphorylation of p53, it also targets Mdm2 at ser395 for phosphorylation in vitro (Maya et al, 2001). Furthermore, two multisite phosphorylation clusters were identified in vivo at the N-terminus of Mdm2, the region comprising the p53-binding domain and nuclear localization signal (Hay and Meek, 2000).…”

Myc pathways provoking cell suicide and cancer

“…Finally, Mdm2 then shuttles polyubiquitinated p53 to the cytosol for degradation by the proteasome (Freedman and Roth et al, 1998). Thus, p53 can be activated indirectly by signals that inhibit Mdm2, such as that which occurs when Mdm2 is phosphorylated by the ataxia telangiectasia serine/threonine kinase (Atm) following g-irradiation (Khosravi et al, 1999;Maya et al, 2001). Indeed, the Nand C-terminal domains of p53 are highly modified by post-translational modifications, including acetylation and sumoylation of residues in its C-terminus and regulatory phosphorylations at both ends of the protein (Gu and Roeder, 1997;Sakaguchi et al, 1998;Gostissa et al, 1999;Rodriguez et al, 1999;Muller et al, 2000;Kahyo et al, 2001;Schmidt and Muller, 2002).…”