Allosteric changes in HDM2 by the ATM phosphomimetic S395D mutation: implications on HDM2 function

Abstract

Allosteric changes imposed by post-translational modifications regulate and differentiate the functions of proteins with intrinsic disorder regions. HDM2 is a hub protein with a large interactome and with different cellular functions. It is best known for its regulation of the p53 tumour suppressor. Under normal cellular conditions, HDM2 ubiquitinates and degrades p53 by the 26S proteasome but after DNA damage, HDM2 switches from a negative to a positive regulator of p53 by binding to p53 mRNA to promote translation of the p53 mRNA. This change in activity is governed by the ataxia telangiectasia mutated kinase via phosphorylation on serine 395 and is mimicked by the S395D phosphomimetic mutant. Here we have used different approaches to show that this event is accompanied by a specific change in the HDM2 structure that affects the HDM2 interactome, such as the N-termini HDM2–p53 protein–protein interaction. These data will give a better understanding of how HDM2 switches from a negative to a positive regulator of p53 and gain new insights into the control of the HDM2 structure and its interactome under different cellular conditions and help identify interphases as potential targets for new drug developments.

MDM2 regulates RB levels during genotoxic stress

“…It has been shown that under DNA damage conditions, the kinase ATM is activated and phosphorylates MDM2 in Ser 395 (Maya et al , 2001). This post‐translational modification stimulates a change in MDM2 conformation that promotes a switch in its activity (Medina‐Medina et al , 2016; Uhrik et al , 2019); initially, a ubiquitin ligase for the tumour suppressor p53, it becomes a translation factor for p53 (Candeias et al , 2008; Medina‐Medina et al , 2016). We have previously shown that mutant MDM2(S395D) mimic the Ser 395 phosphorylation promoted by ATM under DNA damage conditions (Gajjar et al , 2012; Medina‐Medina et al , 2016; Uhrik et al , 2019).…”

“…Moreover, this mutant is also able to bind and ubiquitinate the RB protein. It has been reported that this phosphorylation changes the conformation and switches the activity of MDM2 from a ubiquitin ligase acting on p53 to a translation factor acting through a direct interaction between MDM2 and the p53 mRNA (Medina‐Medina et al , 2016; Uhrik et al , 2019). Although the parallelism is evident, the mechanism is not the same.…”

“…This post-translational modification stimulates a change in MDM2 conformation that promotes a switch in its activity (Medina-Medina et al, 2016;Uhrik et al, 2019); initially, a ubiquitin ligase for the tumour suppressor p53, it becomes a translation factor for p53 (Candeias et al, 2008;Medina-Medina et al, 2016). We have previously shown that mutant MDM2 (S395D) mimic the Ser 395 phosphorylation promoted by ATM under DNA damage conditions (Gajjar et al, 2012;Medina-Medina et al, 2016;Uhrik et al, 2019). Using this phospho-mimetic mutant, we evaluate the effect of MDM2(S395D) on the expression of the endogenous RB in the absence of doxorubicin in two cell lines, H1299 and C33.…”

Resistance mechanisms to inhibitors of p53-MDM2 interactions in cancer therapy: can we overcome them?

“…Moreover, activated ATM phosphorylates MDM2 on Ser395, which switches MDM2 from a negative to a positive regulator of TP53 mRNA translation. However, in the absence of the TP53 mRNA-MDM2 interaction, Ser395 phospho-MDM2 has a greater capacity to ubiquitinate p53, leading to its degradation [145][146][147]. In addition, the interplay between MDM2-ATM-p53 and TP53 mRNA plays a significant role in p53 stabilisation [148].…”

“…For the former, the N-terminal domain of MDM2 represents an important interaction interface for many proteins such as p73, p63, DP1, HAUSP, hTERT, NUMB and NOTCH [ 193 ], and MDM2 inhibitors that bind to this domain potentially abolish these interactions. Moreover, MDM2 exhibits structural plasticity and allosteric changes impact its functions [ 145 , 194 ], implying that not only N-terminal interacting proteins are influenced by MDM2 inhibitors. One of the first pieces of evidence for a p53-independent action of nutlin-3 came from Ambrosini et al in 2007.…”