An essential function of the extreme C-terminus of MDM2 can be provided by MDMX

MDM2 (HDM2) is a ubiquitin ligase that can target the p53 tumor suppressor protein for degradation. The RING domain is essential for the E3 activity of MDM2, and we show here that the extreme C-terminal tail of MDM2 is also critical for efficient E3 activity. Loss of E3 function in MDM2 mutants deleted of the C-terminal tail correlated with a failure of these mutants to oligomerize with MDM2, or with the related protein MDMX (HDMX). However, MDM2 containing point mutations within the C-terminus that inactivated E3 function retained the ability to oligomerize with the wild-type MDM2 RING domain and MDMX, and our results indicate that oligomers containing both wild-type MDM2 and a C-terminal mutant protein retain E3 function both in auto-degradation and degradation of p53. Interestingly, the E3 activity of C-terminal point mutants of MDM2 can also be supported by interaction with wild-type MDMX, suggesting that MDMX can directly contribute to E3 function.     

Structure of the MDM2/MDMX RING domain heterodimer reveals dimerization is required for their ubiquitylation in trans

MDM2, a ubiquitin E3-ligase of the RING family, has a key role in regulating p53 abundance. During normal non-stress conditions p53 is targeted for degradation by MDM2. MDM2 can also target itself and MDMX for degradation. MDMX is closely related to MDM2 but the RING domain of MDMX does not possess intrinsic E3-ligase activity. Instead, MDMX regulates p53 abundance by modulating the levels and activity of MDM2. Dimerization, mediated by the conserved C-terminal RING domains of both MDM2 and MDMX, is critical to this activity. Here we report the crystal structure of the MDM2/MDMX RING domain heterodimer and map residues required for functional interaction with the E2 (UbcH5b). In both MDM2 and MDMX residues C-terminal to the RING domain have a key role in dimer formation. In addition we show that these residues are part of an extended surface that is essential for ubiquitylation in trans. This study provides a molecular basis for understanding how heterodimer formation leads to stabilization of MDM2, yet degradation of p53, and suggests novel targets for therapeutic intervention.     

The RING finger domain of MDM2 is essential for MDM2-mediated TGF-beta resistance

In this study, we attempt to gain insights into the molecular mechanism underlying MDM2-mediated TGF-beta resistance. MDM2 renders cells refractory to TGF-beta by overcoming a TGF-beta-induced G1 cell cycle arrest. Because the TGF-beta resistant phenotype is reversible upon removal of MDM2, MDM2 likely confers TGF-beta resistance by directly targeting the cellular machinery involved in the growth inhibition by TGF-beta. Investigation of the structure-function relationship of MDM2 reveals three elements essential for MDM2 to confer TGF-beta resistance in both mink lung epithelial cells and human mammary epithelial cells. One of these elements is the C-terminal half of the p53-binding domain, which at least partially retained p53-binding and inhibitory activity. Second, the ability of MDM2 to mediate TGF-beta resistance is disrupted by mutation of the nuclear localization signal, but is restored upon coexpression of MDMX. Finally, mutations of the zinc coordination residues of the RING finger domain abrogates TGF-beta resistance, but not the ability of MDM2 to inhibit p53 activity or to bind MDMX. These data suggest that RING finger-mediated p53 inhibition and MDMX interaction are not sufficient to cause TGF-beta resistance and imply a crucial role of the E3 ubiquitin ligase activity of this domain in MDM2-mediated TGF-beta resistance.     

Regulation of MDM2 E3 ligase activity by phosphorylation after DNA damage

MDM2 is a major regulator of p53 by acting as a ubiquitin E3 ligase. The central acidic domain and C-terminal RING domain of MDM2 are both indispensable for ubiquitination of p53. Our previous study suggested that ATM phosphorylation of MDM2 near the C terminus inhibits RING domain oligomerization, resulting in p53 stabilization after DNA damage. We present here evidence that these modifications allosterically regulate the functions of both acidic domain and RING domain of MDM2. Using chemical cross-linking, we show that the MDM2 RING domain forms oligomers including dimer and higher-order complexes in vivo. RING domain dimerization efficiency is negatively regulated by upstream sequence. ATM-mediated phosphorylation of the upstream sequence further inhibits RING dimerization. Forced oligomerization of MDM2 partially overcomes the inhibitory effect of phosphorylation and stimulates p53 ubiquitination. Furthermore, the ability of MDM2 acidic domain to bind p53 core domain and induce p53 misfolding are also suppressed by the same C-terminal ATM sites after DNA damage. These results suggest that the acidic domain and RING domain of MDM2 are both allosterically coupled to the intervening ATM sites, which enables the same modification to regulate multiple MDM2 functions critical for p53 ubiquitination.     

The MDMX acidic domain competes with the p53 transactivation domain for MDM2 N-terminal domain binding

The tumor suppressor protein p53 governs many cellular pathways to control genome integrity, metabolic homeostasis, and cell viability. The critical roles of p53 highlight the importance of proper control over p53 in maintaining normal cellular function, with the negative regulators MDM2 and MDMX playing central roles in regulating p53 activity. The interaction between p53 and either MDM2 or MDMX involves the p53 transactivation domain (p53TD) and the N-terminal domains (NTD) of MDM2 or MDMX. Recently, the acidic domain (AD) of MDMX was found to bind to its own NTD, inhibiting the p53-MDMX interaction. Given the established structural and functional similarity between the MDM2 and MDMX NTDs, we hypothesized that the MDMX AD would also directly bind to MDM2 NTD to inhibit p53-MDM2 interaction. Through solution-state nuclear magnetic resonance (NMR) spectroscopy and isothermal titration calorimetry (ITC), we show that the MDMX AD can indeed directly interact with the MDM2 NTD and, as a result, can compete for p53 binding. The MDMX AD is thus able to serve as a regulatory domain to inhibit the MDM2-p53 interaction and may also play a direct role in p53 activation.     

MDM2 promotes ubiquitination and degradation of MDMX

The p53 tumor suppressor is regulated by MDM2-mediated ubiquitination and degradation. Mitogenic signals activate p53 by induction of ARF expression, which inhibits p53 ubiquitination by MDM2. Recent studies showed that the MDM2 homolog MDMX is also an important regulator of p53. We present evidence that MDM2 promotes MDMX ubiquitination and degradation by the proteasomes. This effect is stimulated by ARF and correlates with the ability of ARF to bind MDM2. Promotion of MDM2-mediated MDMX ubiquitination requires the N-terminal domain of ARF, which normally inhibits MDM2 ubiquitination of p53. An intact RING domain of MDM2 is also required, both to interact with MDMX and to provide E3 ligase function. Increase of MDM2 and ARF levels by DNA damage, recombinant ARF adenovirus infection, or inducible MDM2 expression leads to proteasome-mediated down-regulation of MDMX levels. Therefore, MDMX and MDM2 are coordinately regulated by stress signals. The ARF tumor suppressor differentially regulates the ability of MDM2 to promote p53 and MDMX ubiquitination and activates p53 by targeting both members of the MDM2 family.     

Stabilization of the MDM2 oncoprotein by interaction with the structurally related MDMX protein

The MDM2 oncoprotein has transforming potential that can be activated by overexpression, and it represents a critical regulator of the p53 tumor suppressor protein. To identify other factors with a potential role in influencing the expression and/or function of MDM2, we utilized a yeast two-hybrid screening protocol. Here we report that MDM2 physically interacts with a structurally related protein termed MDMX. The results obtained in these studies provide evidence that C-terminal RING finger domains, contained within both of these proteins, play an important role in mediating the association between MDM2 and MDMX. The interaction of these proteins interferes with MDM2 degradation, leading to an increase in the steady-state levels of MDM2. MDMX also inhibits MDM2-mediated p53 degradation, with subsequent accumulation of p53. Taken together, these data indicate that MDMX has the potential to regulate the expression and function of the MDM2 oncoprotein.     

MDM2 protein-mediated ubiquitination of numb protein: identification of a second physiological substrate of MDM2 that employs a dual-site docking mechanism

The E3 ubiquitin ligase, MDM2, uses a dual-site mechanism to ubiquitinate and degrade the tumor suppressor protein p53, involving interactions with the N-terminal hydrophobic pocket and the acidic domain of MDM2. The results presented here demonstrate that MDM2 also uses this same dual-site mechanism to bind to the cell fate determinant NUMB with both the N-terminal hydrophobic pocket and the acidic domain of MDM2 also involved in forming the interaction with NUMB. Furthermore, the acidic domain interactions are crucial for MDM2-mediated ubiquitination of NUMB. Contrary to p53, where two separate domains form the interface with MDM2, only one region within the phosphotyrosine binding domain of NUMB (amino acids 113-148) mediates binding to both these regions of MDM2. By binding to both domains on MDM2, NUMB disrupts the MDM2-p53 complex and MDM2-catalyzed ubiquitination of p53. Therefore, we have identified the mechanism NUMB uses to regulate the steady-state levels of the p53 in cells. By targeting the acidic domain of MDM2 using acid domain-binding ligands we can overcome MDM2-mediated ubiquitination and degradation of NUMB impacting on the stabilization of p53 in cells. Furthermore, delivery of MDM2 acid domain-binding ligands to cancer cells promotes p53-dependent growth arrest and the induction of apoptosis. This highlights the dual-site mechanism of MDM2 on another physiological substrate and identifies the acid domain as well as N terminus as a potential target for small molecules that inhibit MDM2.     

Autoactivation of the MDM2 E3 Ligase by Intramolecular Interaction

The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels.     

Identification of a Catalytic Active but Non-Aggregating MDM2 RING Domain Variant

As a key regulator of the tumour suppressor protein p53, MDM2 is involved in various types of cancer and has thus been an attractive drug target. So far, small molecule design has primarily focussed on the N-terminal p53-binding domain although on-target toxicity effects have been reported. Targeting the catalytic RING domain of MDM2 resembles an alternative approach to drug MDM2 with the idea to prevent MDM2-mediated ubiquitination of p53 while retaining MDM2′s ability to bind p53. The design of RING inhibitors has been limited by the extensive aggregation tendency of the RING domain, making it challenging to undertake co-crystallization attempts with potential inhibitors. Here we compare the purification profiles of the MDM2 RING domain from several species and show that the MDM2 RING domain of other species than human is much less prone to aggregate although the overall structure of the RING domain is conserved. Through sequence comparison and mutagenesis analyses, we identify a single point mutation, G443T, which greatly enhances the dimeric fraction of human MDM2 RING domain during purification. Neither does the mutation alter the structure of the RING domain, nor does it affect E2(UbcH5B)–Ub binding and activity. Hence, MDM2-G443T facilitates studies involving binding partners that would be hampered by the low solubility of the wild-type RING domain. Furthermore, it will be valuable for the development of MDM2 RING inhibitors.     

Mutational analysis of Mdm2 C-terminal tail suggests an evolutionarily conserved role of its length in Mdm2 activity toward p53 and indicates structural differences between Mdm2 homodimers and Mdm2/MdmX heterodimers

Mdm2 can mediate p53 ubiquitylation and degradation either in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer. The ubiquitin ligase activity of these complexes resides mainly in their respective RING finger domains and also requires adjacent C-terminal tails. So far, structural studies have failed to show significant differences between Mdm2 RING homodimers and Mdm2/MdmX RING heterodimers. Here, we report that not only the primary amino acid sequence, but also the length of the C-terminal tail of Mdm2 is highly conserved through evolution and plays an important role in Mdm2 activity toward p53. Mdm2 mutants with extended C termini do not ubiquitylate p53 despite being capable of forming Mdm2 homodimers through both RING-acidic domain and RING-RING interactions. All extended mutants also retained the ability to interact with MdmX, and this interaction led to reactivation of their E3 ubiquitin ligase activity. In contrast, only a subset of extended Mdm2 mutants was activated by the interaction with Mdm2 RING domain, suggesting that Mdm2 homodimers and Mdm2/MdmX heterodimers may not be structurally and functionally fully equivalent.     

MDMX overexpression prevents p53 activation by the MDM2 inhibitor Nutlin

The p53 tumor suppressor plays a key role in maintaining genomic stability and protection against malignant transformation. MDM2 and MDMX are both p53-binding proteins that regulate p53 stability and activity. Recent development of the MDM2 inhibitor Nutlin 3 has greatly facilitated functional analysis of MDM2-p53 binding. We found that although MDMX is homologous to MDM2 and binds to the same region on p53 N terminus, Nutlin does not disrupt p53-MDMX interaction. The ability of Nutlin to activate p53 is compromised in tumor cells overexpressing MDMX. Combination of Nutlin with MDMX siRNA resulted in synergistic activation of p53 and growth arrest. These results suggest that MDMX is also a valid target for p53 activation in tumor cells. Development of novel compounds that are MDMX-specific or optimized for dual-inhibition of MDM2 and MDMX are necessary to achieve full activation of p53 in tumor cells.