DNA Damage-induced MDMX Degradation Is Mediated by MDM2

Hidehiko Kawai,Dmitri Wiederschain,Hiroyuki Kitao,Jeremy Stuart,Kelvin K.C Tsai,Zhi-Min Yuan

doi:10.1074/jbc.m308295200

Abstract

Although genetic studies have demonstrated that MDMX is essential to maintain p53 activity at low levels in non-stressed cells, it is unknown whether MDMX regulates p53 activation by DNA damage. We show here that DNA damage-induced p53 induction is associated with rapid down-regulation of the MDMX protein. Significantly, interference with MDMX down-regulation results in the suppression of p53 activation by genotoxic stress. We also demonstrate that DNA damage-induced MDMX reduction is mediated by MDM2, which targets MDMX for proteasomal degradation by a distinct mechanism that permits preferential MDMX degradation and therefore ensures optimal p53 activation.

Highlights

The tumor suppressor gene p53 encodes a transcription factor that controls the expression of a number of genes, the products of which mediate either cell cycle arrest or apoptosis [1]
Together with the failure to detect any significant effect of the dose of IR used on MDMX mRNA level (Fig. 1E), these results suggest that IR induces MDMX down-regulation via an increased proteolysis
Available evidence indicates that inhibition of MDM2-mediated p53 degradation is essential for adequate p53 activation in response to various forms of genotoxic stress

Summary

The abbreviations used are

E3, ubiquitin-protein isopeptide ligase; MEF, mouse embryo fibroblasts; DKO, double knock-out; GFP, green fluorescent protein; EGFP, enhanced GFP; RT, reverse transcriptase; PBS, phosphate-buffered saline; NES, nuclear export sequence; NLS, nuclear localization sequence; RNAi, RNA interference; RFD, Ring finger domain; Gy, grays. In contrast to MDM2, MDMX lacks ubiquitin E3 ligase activity in cells and is unable to target p53 for ubiquitin/proteasome-dependent proteolysis [4]. MDM2 is an extremely unstable protein since it is capable of self-ubiquitination that leads to rapid proteasomal degradation [8]. Regulation of MDM2 stability might be another important mechanism by which p53 activity can be controlled. We have recently shown that MDMX binds to and increases the stability of MDM2, directly contributing to the ability of MDM2 to maintain p53 at low levels [9]. We demonstrate here that MDMX contributes to the regulation of DNA damage-induced p53 activation. MDM2 utilizes distinct mechanisms to target MDMX and p53 for degradation, exerting differential control over the levels of these two proteins in response to DNA damage

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION