Increased Radioresistance and Accelerated B Cell Lymphomas in Mice with Mdmx Mutations that Prevent Modifications by DNA-Damage-Activated Kinases

Abstract

Summary Mdmx is a critical negative regulator of the p53 pathway that is stoichiometrically limiting in some tissues. Post-translational modification and degradation of Mdmx after DNA damage have been proposed to be essential for p53 activation. We tested this model in vivo, where critical stoichiometric relationships are preserved. We generated an Mdmx mutant mouse in which three conserved serines (S341, S367, S402) targeted by DNA damage-activated kinases were replaced by alanines to investigate whether modifications of these residues are important for Mdmx degradation and p53 activation. The mutant mice were remarkably resistant to radiation, and very susceptible to Myc-induced lymphomagenesis. These data demonstrate that Mdmx down-regulation is crucial for effective p53-mediated radiation responses and tumor suppression in vivo. Significance P53 function is sensitive to the levels of its negative regulators, Mdm2 and Mdmx. Cell culture studies have suggested the importance of post-translational modifications in Mdm2 and Mdmx for p53 activation, but this has not been rigorously tested in vivo. This work shows that DNA damage and activated c-Myc both require phosphorylation of Mdmx in residues targeted by the damage kinases ATM and Chk2 for robust p53 activation. Preventing Mdmx post-translational modification stabilizes this negative regulator, which mitigates p53 activation, and presumably enables c-Myc to drive tumor cells with defective genomes into cycle in vivo. The data also stress the relevance of Mdmx as a potential therapeutic target.

14-3-3γ Inhibition of MDMX-mediated p21 Turnover Independent of p53

“…This pathway was later confirmed by an elegant knock-in study in mice (32). Because both p21 and 14-3-3␥ bind to the extended central region of MDMX (26,29,33), we speculated that 14-3-3␥ might suppress MDMX-mediated p21 degradation by influencing the p21 binding to MDMX.…”

Limiting the power of p53 through the ubiquitin proteasome pathway

“…In fact, no change in the stability of a temperature-sensitive mutant p53 was observed upon deletion of Mdm4 (Barboza et al 2008). Similarly, no noticeable difference in p53 ubiquitination pattern or p53 stability was observed in two other Mdm4 mutant mice, Mdm4DRING or MdmX-3SA (Wang et al 2009;Pant et al 2011). However, it is possible that the presence of Mdm2 masked the effect on p53 stability.…”

“…To date, it has not been possible to genetically distinguish RING ligase activity from Mdm4 binding. Physiological disruption of the interaction between p53 and Mdm2/Mdm4 also occurs upon post-translational modifications and impacts p53 ubiquitination (Prives and Hall 1999;Ito et al 2002;Li et al 2002;Chao et al 2003Chao et al , 2006Xirodimas et al 2004;Wang et al 2009;Gannon et al 2012). Thus, the activity of the Mdm2 E3 ligase toward p53 is regulated by multiple parameters: the RING domain, the C terminus, and interactions with Mdm4.…”

Fine-tuning p53 activity through C-terminal modification significantly contributes to HSC homeostasis and mouse radiosensitivity

“…Previous studies of the impact of Mdm2 and Mdmx expression on p53 activity revealed that the p53 pathway is exquisitely sensitive to p53 protein levels and basal activity, which can be modulated by p53-negative regulators (Mendrysa et al 2003;Terzian et al 2007;Wang et al 2009). Since Mdm2 heterozygous mice are known to have elevated p53 activity (Terzian et al 2007), we compared their radiosensitivity with p53 7KR mice.…”