The p53 protein is one of the major tumor suppressor proteins. In response to DNA damage, p53 is prevented from degradation and accumulates to high levels. Ionizing radiation leads to hypophosphorylation of the p53 ubiquitin ligase Mdm2 at sites where phosphorylation is critical for p53 degradation and to the phosphorylation and activation of Akt/PKB, a kinase that phosphorylates and inhibits GSK-3. GSK-3, which normally phosphorylates Mdm2, is inactivated in response to ionizing radiation. We show that p53 accumulates in lymphoblasts from patients with the hereditary disorder ataxia telangiectasia in response to ionizing radiation despite the absence of a functional ATM kinase. Also, knockdown of ATR did not prevent p53 accumulation in response to ionizing radiation. Instead, p53 stabilization in response to ionizing radiation depended on the inactivation of GSK-3 and the presence of Akt/PKB. Akt/PKB is a target of DNA-PK, a kinase that is activated after ionizing radiation. Correspondingly, down-regulation of DNA-PK prevented phosphorylation of Akt/PKB and GSK-3 after ionizing radiation and strongly reduced the accumulation of p53. We therefore propose a signaling cascade for the regulation of p53 in response to ionizing radiation that involves activation of DNA-PK and Akt/PKB and inactivation of GSK-3 and Mdm2.

The Mdm2 protein mediates ubiquitylation and degradation of p53 and is a key regulator of this tumor suppressor. More recently, it has been shown that Mdm2 is highly phosphorylated within its central acidic domain. In order to address the issue of how these modifications might regulate Mdm2 function, putative phosphorylation sites within this domain were substituted, individually or in pairs, with alanine residues. Mutants with serine-to-alanine substitutions between residues 244 and 260 abolished or at least reduced the capacity of Mdm2 to promote p53 degradation. In each case, loss of degradation function was independent of the ability to bind to p53 or p14ARF. Moreover, each of the Mdm2 mutants completely retained the capacity to act as a ubiquitin ligase in vivo. Thus, ubiquitylation and degradation can be uncoupled. Two-dimensional phosphopeptide mapping coupled with the use of phospho-specific antibodies revealed that Mdm2 is phosphorylated physiologically at several sites within this region, consistent with the idea that phosphorylation is important for Mdm2 activity. Strikingly, treatment of cells with ionizing radiation resulted in a significant decrease in the phosphorylation of residues that are important for p53 turnover. This hypophosphorylation preceded p53 accumulation. These findings indicate that Mdm2 contributes an additional function toward the degradation of p53 that is distinct from its ubiquitin ligase activity and is regulated by phosphorylation. Our model suggests that hypophosphorylation of Mdm2 in response to ionizing irradiation inactivates this novel function, thereby contributing to p53 stabilization.

The Mdm2 oncoprotein regulates abundance and activity of the p53 tumor suppressor protein. For efficient degradation of p53, Mdm2 needs to be phosphorylated at several contiguous residues within the central conserved domain. We show that glycogen synthase kinase 3 (GSK-3) phosphorylated the Mdm2 protein in vitro and in vivo in the central domain. Inhibition of GSK-3 rescued p53 from degradation in an Mdm2-dependent manner while its association with Mdm2 was not affected. Likewise, inhibition of GSK-3 did not alter localization of p53 and Mdm2 or the interaction of Mdm2 and MdmX. Ionizing radiation, which leads to p53 accumulation, directed phosphorylation of GSK-3 at serine 9, which preceded and overlapped with the increase in p53 levels. Moreover, expression of a GSK-3 mutant where serine 9 was replaced with an alanine reduced the accumulation of p53 and induction of its target p21^WAF-1. We therefore conclude that inhibition of GSK-3 contributes to hypophosphorylation of Mdm2 in response to ionizing rays, and in consequence to p53 stabilization.

The p53 protein is one of the most important tumor suppressor proteins. Normally, the p53 protein is in a latent state. However, when its activity is required, e.g. upon DNA damage, nucleotide depletion or hypoxia, p53 becomes rapidly activated and initiates transcription of pro-apoptotic and cell cycle arrest-inducing target genes. The activity of p53 is regulated both by protein abundance and by post-translational modifications of pre-existing p53 molecules. In the 30 years of p53 research, a plethora of modifications and interaction partners that modulate p53’s abundance and activity have been identified and new ones are continuously discovered. This review will summarize our current knowledge on the regulation of p53 abundance and activity.

Background: P53 is a key tumor suppressor protein. In response to DNA damage, p53 accumulates to high levels in differentiated cells and activates target genes that initiate cell cycle arrest and apoptosis. Since stem cells provide the proliferative cell pool within organisms, an efficient DNA damage response is crucial. Results: In proliferating embryonic stem cells, p53 is localized predominantly in the cytoplasm. DNA damage-induced nuclear accumulation of p53 in embryonic stem cells activates transcription of the target genes mdm2, p21, puma and noxa. We observed bi-phasic kinetics for nuclear accumulation of p53 after ionizing radiation. During the first wave of nuclear accumulation, p53 levels were increased and the p53 target genes mdm2, p21 and puma were transcribed. Transcription of noxa correlated with the second wave of nuclear accumulation. Transcriptional activation of p53 target genes resulted in an increased amount of proteins with the exception of p21. While p21 transcripts were efficiently translated in 3T3 cells, we failed to see an increase in p21 protein levels after IR in embryonal stem cells. Conclusion: In embryonic stem cells where (anti-proliferative) p53 activity is not necessary, or even unfavorable, p53 is retained in the cytoplasm and prevented from activating its target genes. However, if its activity is beneficial or required, p53 is allowed to accumulate in the nucleus and activates its target genes, even in embryonic stem cells.

The ubiquitin ligase Mdm2 targets the p53 tumor suppressor protein for proteasomal degradation. Mutating phosphorylation sites in the central domain of Mdm2 prevents p53 degradation, although it is still ubiquitylated, indicating that Mdm2 has a post-ubiquitylation function for p53 degradation. We show that Mdm2 associates with several subunits of the 19S proteasome regulatory particle in a ubiquitylation-independent manner. Mdm2 furthermore promotes the formation of a ternary complex of itself, p53, and the proteasome. Replacing phosphorylation sites within the central domain with alanines reduced the formation of the ternary complex. The C-terminus of Mdm2 was sufficient for interaction with the proteasome despite an additional proteasome binding site in the Mdm2 N-terminus. In addition to binding to the proteasome, the C-terminus of Mdm2 bound to the central domain, possibly competing with, and therefore blocking, Mdm2/proteasome interaction. We propose that Mdm2 facilitates, or at least enhances, the association of p53 with the proteasome and that phosphorylation of the central domain of Mdm2 regulates this process.