Hypophosphorylation of Mdm2 Augments p53 Stability

Abstract

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.

Suppression of UV-induced apoptosis by the human DNA repair protein XPG

“…Similarly, extensive incubation with calf intestinal phosphatase had no effect on the migration of the Mdm2 isoforms from wild-type cells treated or untreated with UV (data not shown). This suggests that neither sumoylation 23 nor phosphorylation 24 is implicated in the accumulation of the 90/92 kDa Mdm2 isoforms in wild-type and XP-C fibroblasts, or in their failure to be accumulated in TC-NER defective cells ( Figure 5). …”

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

“…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). Hypophosphorylation of putative sites within the acidic domain of Mdm2 reduced or ablated the ability of Mdm2 to degrade p53 (Blattner et al, 2002). Together, the evidence suggests that, like phosphorylation of p53, Mdm2 phosphorylation inhibits Mdm2-directed turnover of p53.…”

P53 and radiation responses

“…These phosphorylations thus interrupt the binding of MDM2 with P53 and protect P53 from ubiquitination and degradation. Recently, hypophosphorylated MDM2 in response to IR was also found to contribute to P53 stabilization, which is distinct from MDM2's ubiquitin ligase activity to mediate P53 degradation (Blattner et al, 2002). Other post-translational modifications on P53 can modify its stability under certain circumstances.…”