Mutual Dependence of MDM2 and MDMX in Their Functional Inactivation of p53

Abstract

MDMX, an MDM2-related protein, has emerged as yet another essential negative regulator of p53 tumor suppressor, since loss of MDMX expression results in p53-dependent embryonic lethality in mice. However, it remains unknown why neither homologue can compensate for the loss of the other. In addition, results of biochemical studies have suggested that MDMX inhibits MDM2-mediated p53 degradation, thus contradicting its role as defined in gene knockout experiments. Using cells deficient in either MDM2 or MDMX, we demonstrated that these two p53 inhibitors are in fact functionally dependent on each other. In the absence of MDMX, MDM2 is largely ineffective in down-regulating p53 because of its extremely short half-life. MDMX renders MDM2 protein sufficiently stable to function at its full potential for p53 degradation. On the other hand, MDMX, which is a cytoplasmic protein, depends on MDM2 to redistribute into the nucleus and be able to inactivate p53. We also showed that MDMX, when exceedingly overexpressed, inhibits MDM2-mediated p53 degradation by competing with MDM2 for p53 binding. Our findings therefore provide a molecular basis for the nonoverlapping activities of these two p53 inhibitors previously revealed in genetic studies.The tumor suppressor gene p53 encodes a transcription factor that is activated in response to various forms of stress, leading to the induction of a number of genes whose products mediate either cell cycle arrest or apoptosis (1). Under most physiological conditions, p53 activity is tightly controlled, primarily through the ability of MDM2 to target p53 for degradation, which ensures cell survival. Current model of p53 activation suggests that diverse stress signals converge on a single regulatory node, namely the p53-MDM2 module, and interfere with the ability of MDM2 to target p53 for degradation (2). Analogous to MDM2, MDMX ablation is also associated with p53-dependent embryonic death in mice, placing MDMX in the category of essential p53 negative regulators (3). In contrast to MDM2, however, MDMX lacks ubiquitin E3 ligase activity and is unable to target p53 for ubiquitin-proteasome-dependent proteolysis (4). Moreover, MDMX was reported to inhibit MDM2-mediated p53 degradation (4 -6), contradicting the role of MDMX as defined by the genetic study. To resolve these conflicting results and gain better understanding of why neither gene product can compensate for the loss of the other, we generated MDMX-deficient cells using small interference RNA (siRNA) 1 and carried out biochemical analysis of MDM2 in these cells. In conjunction with the use of MEFs derived from either single or double knock-out mice, our loss-of-function approach allowed us to obtain compelling evidence at the molecular level to highlight mutual dependence of MDM2 and MDMX in their functional inhibition of p53 and provide support for the findings obtained in genetic studies. /MDM2Ϫ/Ϫ MEFs (Dr. Carl Maki, Harvard School of Public Health), were maintained in minimal essential medium supplemented with 10% fetal bovin...

Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4

“…The results show that in the absence of Mdm4, the transactivation of mdm2 is stimulated, leading to increased Mdm2 protein levels. In contrast to previous transfection studies, 40 these data suggest that Mdm4 does not affect Mdm2 protein stability significantly. This was indeed directly tested and the half-life of Mdm2 was found to be similar in p53 DP/DP cells and p53 DP/DP mdm4 À/À cells, as well as in p53 LSL/-and p53 LSL/-mdm4 À/À MEFs (Toledo et al, unpublished data).…”

“…One possible scenario, based on the observation that Mdm2 and Mdm4 heterodimerize, is that the two proteins work together to inhibit p53. 39,40 However, the molecular mechanisms underlining the abilities of both Mdm proteins to regulate p53 activity have yet to be clarified in vivo. 10 Conditional alleles have now been developed that yield further insight into how and in what cell types Mdm2 and Mdm4 regulate p53 (Figure 1).…”

“…Indeed, transfection studies suggest that Mdm4 stabilizes Mdm2, perhaps by interfering with its autoubiquitination. 40,51 Another report, however, suggested that Mdm4 stimulates not only Mdm2-mediated ubiquitination of p53, but also Mdm2 selfubiquitination. 52 In vivo, p53 levels stay below the limit of detection in both Western blotting and immunohistochemistry assays, when its expression was restored in progenitor and in postmitotic neuronal cells lacking mdm4.…”

Telomere attrition induces a DNA double-strand break damage signal that reactivates p53 transcription in HTLV-I leukemic cells

“…The p53 repressor MDMX is overexpressed in HTLV-1-transformed cells and in ex vivo ATL samples MDM2-related protein MDMX is a negative regulator of p53 function, which is overexpressed in a wide spectrum of human tumors that retain wild-type p53 (Gu et al, 2002). We investigated the expression levels of MDMX in both HTLV-I cell lines and ATL patients and found that, except for C10MJ, HTLV-I-transformed cells overexpressed MDMX (Figure 4a).…”

MdmX is a substrate for the deubiquitinating enzyme USP2a

“…It has also been suggested that MdmX may regulate the stability of p53 indirectly. Mdm2 and MdmX interact through their RING fingers and this interaction can stabilize Mdm2 and promote Mdm2-mediated degradation of p53 (Tanimura et al, 1999;Gu et al, 2002). However, in mice MdmX knockout increases p53 transcriptional activity but has little or no effect on p53 levels (Marine et al, 2006).…”