The tumour-suppressor p53 is a short-lived protein that is maintained at low, often undetectable, levels in normal cells. Stabilization of the protein in response to an activating signal, such as DNA damage, results in a rapid rise in p53 levels and subsequent inhibition of cell growth. Tight regulation of p53 function is critical for normal cell growth and development, and one mechanism by which p53 function is controlled is through interaction with the Mdm2 protein. Mdm2 inhibits p53 cell-cycle arrest and apoptic functions and we show here that interaction with Mdm2 can also result in a large reduction in p53 protein levels through enhanced proteasome-dependent degradation. Endogenous levels of Mdm2 are sufficient to regulate p53 stability, and overexpression of Mdm2 can reduce the amount of endogenous p53. Because mdm2 is transcriptionally activated by p53, this degradative pathway may contribute to the maintenance of low p53 concentrations in normal cells. Furthermore, mechanisms regulating the Mdm2-induced degradation of p53 may play a role in controlling the extent and duration of the p53 response.

The HDM2 protein plays an important role in regulating the stability and function of the p53 tumor suppressor protein. In this report, we show that the ribosomal protein L11 can interact with HDM2 and inhibit HDM2 function, thus leading to the stabilization and activation of p53. The inhibition of HDM2 activity by L11 shows some similarity to the previously described activity of ARF, and expression of either ARF or L11 can induce a p53 response. Enhancement of the interaction between endogenous L11 and HDM2 following treatment of cells with low levels of actinomycin-D suggests that the HDM2/L11 interaction represents a novel pathway for p53 stabilization in response to perturbations in ribosome biogenesis.

The p53 tumor suppressor protein can be phosphorylated at several sites within the N- and C-terminal domains, and several protein kinases have been shown to phosphorylate p53 in vitro. In this study, we examined the activity of p53 proteins with combined mutations at all of the reported N-terminal phosphorylation sites (p53N-term), all of the C-terminal phosphorylation sites (p53C-term), or all of the phosphorylation sites together (p53N/C-term). Each of these mutant proteins retained transcriptional transactivation functions, indicating that phosphorylation is not essential for this activity of p53, although a subtle contribution of the C-terminal phosphorylation sites to the activation of expression of the endogenous p21(Waf1/Cip1)-encoding gene was detected. Mutation of the phosphorylation sites to alanine did not affect the sensitivity of p53 to binding to or degradation by Mdm2, although alteration of residues 15 and 37 to aspartic acid, which could mimic phosphorylation, resulted in a slight resistance to Mdm2-mediated degradation, consistent with recent reports that phosphorylation at these sites inhibits the p53-Mdm2 interaction. However, expression of the phosphorylation site mutant proteins in both wild-type p53-expressing and p53-null lines showed that all of the mutant proteins retained the ability to be stabilized following DNA damage. This indicates that phosphorylation is not essential for DNA damage-induced stabilization of p53, although phosphorylation could clearly contribute to p53 stabilization under some conditions.

The p53 tumor suppressor protein is activated in cells in response to DNA damage and prevents the replication of cells sustaining genetic damage by inducing a cell cycle arrest or apoptosis. Activation of p53 is accompanied by stabilization of the protein, resulting in accumulation to high levels within the cell. p53 is normally degraded through the proteasome following ubiquitination, although the mechanisms which regulate this proteolysis in normal cells and how the p53 protein becomes stabilized following DNA damage are not well understood. We show here that p53 can also be a substrate for cleavage by the calcium-activated neutral protease, calpain, and that a preferential site for calpain cleavage exists within the N terminus of the p53 protein. Treatment of cells expressing wild-type p53 with an inhibitor of calpain resulted in the stabilization of the p53 protein. By contrast, in vitro or in vivo degradation mediated by human papillomavirus E6 protein was unaffected by the calpain inhibitor, indicating that the stabilization did not result from inhibition of the proteasome. These results suggest that calpain cleavage plays a role in regulating p53 stability.