Functions of MDMX in the modulation of the p53-response

Abstract

The MDM family proteins MDM2 and MDMX are two critical regulators of the p53 tumor suppressor protein. Expression of both proteins is necessary for allowing the embryonal development by keeping the activity of p53 in check. Upon stresses that need to activate p53 to perform its function as guardian of the genome, p53 has to be liberated from these two inhibitors. In this review, we will discuss the various mechanisms by which MDMX protein levels are downregulated upon various types of stress, including posttranslational modifications of the MDMX protein and the regulation of mdmx mRNA expression, including alternative splicing. In addition, the putative function(s) of the described MDMX splice variants, particularly in tumor development, will be discussed. Lastly, in contrast to common belief, we have recently shown the existence of a p53-MDMX feedback loop, which is important for dampening the p53-response at later phases after genotoxic stress.

Figure 1

Schematic representation of MDMX protein structure in comparison with MDM2. Most indicated protein modification sites and protein-protein interactions shown are discussed in the text. Modifications of MDMX by sumoylation (K254, K379) have not yet shown to affect function or regulation of MDMX [5]. Phosphorylation of S298 stimulates the association of MDMX to p53 [6], and phosphorylation of S96 has been reported to affect the regulation of subcellular localization of MDM2 [7].

Figure 2

Schematic representation of the MDMX protein, the mdmx mRNA and the reported mdmx splicing variants.

Figure 3

The ratio between mdmx-S and mdmx-fl mRNA is increased by various types of DNA damage and by inhibition of nuclear export. RT-PCR analysis of RNAs extracted from various cell lines, treated for the indicated time periods with a variety of agents. The mdmx-specific PCR primers are located in exon 3 and exon 8. The doses genotoxic/drug treatments used: UV-C: 15 J/m²; cisplatin: 30 μM; LMB (Leptomycin B): 10 nM; Eto (Etoposide): 20 μM; Nutlin-3: 10 μM.

Figure 4

Expression of Mdmx-S protein is much lower compared to full-length Mdmx and increased upon MG132 treatment, whereas mRNA levels are even higher in transfection studies. MCF7 cells were transfected with the indicated constructs (6-well plates; 1 μg of each construct/well; all well were also transfected with 250 ng of CMV-eGFP expression vector as transfection control). Twenty four  hrs after transfection, the cells were either mock treated or treated with 20 μM MG132 for 7 hours. Cell lysates were analyzed by Western Blotting with the indicated antibodies (6658 = MDMX-S specific antibody) and extracted RNA was analyzed by quantitative real-time RT-PCR.