Human epidermal growth factor receptor 4 (Her4) Suppresses p53 Protein via Targeting the MDMX-MDM2 Protein Complex

Abstract

Edited by Eric FearonDeregulated receptor tyrosine kinase (RTK) signaling is frequently associated with tumorigenesis and therapy resistance, but its underlying mechanisms still need to be elucidated. In this study, we have shown that the RTK human epidermal growth factor receptor 4 (Her4, also known as Erbb4) can inhibit the tumor suppressor p53 by regulating MDMX-mouse double minute 2 homolog (MDM2) complex stability. Upon activation by either overexpression of a constitutively active vector or ligand binding (Neuregulin-1), Her4 was able to stabilize the MDMX-MDM2 complex, resulting in suppression of p53 transcriptional activity, as shown by p53-responsive element-driven luciferase assay and mRNA levels of p53 target genes. Using a phospho-proteomics approach, we functionally identified a novel Her4-induced posttranslational modification on MDMX at Ser-314, a putative phosphorylation site for the CDK4/6 kinase. Remarkably, inhibition of Ser-314 phosphorylation either with Ser-to-Ala substitution or with a specific inhibitor of CDK4/6 kinase blocked Her4-induced stabilization of MDMX-MDM2 and rescued p53 activity. Our study offers insights into the mechanisms of deregulated RTK-induced carcinogenesis and provides the basis for the use of inhibitors targeting RTKmediated signals for p53 restoration. p53 is either mutated or functionally inactive in most human cancers. Several tumor types that retain wild-type p53 (i.e. breast cancer) display higher protein levels of MDMX and/or MDM2 2 (1-8), the primary inhibitors of p53, representing alternative mechanisms of p53 inactivation. Typically, p53 protein levels are tightly controlled and modulated by its principle negative regulator, the MDMX-MDM2 complex. Given that MDMX and MDM2 are amplified in many tumors, modulation of their stability offers an attractive therapeutic strategy to restore p53 function and so improve tumor responsiveness to DNA-damaging therapy.Although MDM2 is the main negative regulator of p53, there is now growing evidence supporting the notion that MDMX also plays a key role in modulating p53, mainly through its stabilization of MDM2 (9). In fact, the MDMX-MDM2 heterocomplex is the main form in which the two proteins are found in the cell (10, 11), and loss of this formation leads to p53 activation and embryonic lethality (9, 11-13).Under non-stressed conditions, MDMX and MDM2 suppress p53 activity. However, under stress conditions, MDMX and MDM2 are posttranslationally modified and disabled for their inhibition of p53. As a result, p53 can then respond to the stress and repair any damage. MDM2 and MDMX are structural homologs, with the major distinction that MDM2 contains an E3 ligase domain, a nuclear localization signal domain, and a nuclear export signal domain (14). There are two major ways in which p53 activity can be suppressed by the complex. First, MDM2 and MDMX can cooperatively bind to the transactivational domain of p53 and suppress its transcriptional activity. Second, MDM2 can act as an E3 ligase and induce p53 ubiquitination an...

Pulsatile MAPK Signaling Modulates p53 Activity to Control Cell Fate Decisions at the G2 Checkpoint for DNA Damage

“…Furthermore, we confirmed that HRG but not EGF decreases p53 expression ( Figures 2H, 2I, and S5A-S5D) through a similar mechanism involving the MDM2-MDMX axis (Gerarduzzi et al, 2016; data not shown), again suggesting that MAPK dynamics is crucial to establish a balance between cell-cycle arrest and progression in a p53-dependent manner. The p53 levels could be rescued by treating the cells with U0126 in the presence of HRG and DNA damage ( Figures S5E and S5F).…”

“…The abrogation of MAPK signaling enhances checkpoint stringency, whereas its sustained stimulation with HRG or light-inducible RAF increases checkpoint negligence. Mechanistically, sustained MAPK signaling during checkpoint activation results in the Lahav et al (2004), subsequently modeled with the addition of the WIP1 phosphatase role in Batchelor et al (2011), where ERK activity discovered in this work is exemplified with the reported MAPK-MDM2 interaction (Gerarduzzi et al, 2016). (C) A simple competitive model for the DNA damage checkpoint.…”

CDK4 inhibition diminishes p53 activation by MDM2 antagonists

“…We show that the complexes of CDK4 and Cyclin D1 on the one hand, and MDM2 and p53 on the other hand, associate with each other. These findings are in line with a previous report that MDMX, another close binding partner of MDM2 and p53, is phosphorylated by CDK4 37 . According to this report, CDK4-mediated phosphorylation of MDMX stabilizes its interaction with MDM2 to antagonize p53.…”

MDMX under stress: the MDMX-MDM2 complex as stress signals hub

“…Phosphorylation of MDMX on Ser314 was able to favor MDMX nuclear import and increase its stability by protecting it from MDM2-mediated degradation (12,13). The identification of the Ser314 site as a target of multiple tyrosine kinases signaling point at a role of MDMX as the converging point, or “hub”, of oncogenic signaling aimed at suppressing p53, given the prosurvival role of tyrosine kinases in cancer development and the fact that p53 functional deactivation is required for tumorigenesis.…”

“…We propose that MDMX represents a “signaling hub” transducing mitogenic signals as well as stress signals to the MDMX-MDM2 complex and therefore to p53. In a cancer context, our lab has shown that at least two receptor tyrosine kinases can inhibit p53 activation via modulation of the MDMX-MDM2 complex stability and, specifically, through post-translational modifications of MDMX (12,13). These and other studies (14) suggest that MDMX could be a promising and rather safe therapeutic target for pharmacological restoration of p53 in tumors harboring wild-type p53.…”