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.Key words: p53, Mdm2, RING domain, ubiquitylation, ubiquitin ligase, E3     

Turning the RING Domain Protein MdmX into an Active Ubiquitin-Protein Ligase

The related RING domain proteins MdmX and Mdm2 are best known for their role as negative regulators of the tumor suppressor p53. However, although Mdm2 functions as a ubiquitin ligase for p53, MdmX does not have appreciable ubiquitin ligase activity. In this study, we performed a mutational analysis of the RING domain of MdmX, and we identified two distinct regions that, when replaced by the respective regions of Mdm2, turn MdmX into an active ubiquitin ligase for p53. Mdm2 and MdmX form homodimers as well as heterodimers with each other. One of the regions identified localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with the ubiquitin-conjugating enzyme UbcH5b. The second region contains the cryptic nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5b. Here, we show that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5b in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and ubiquitin ligase activity. Finally, evidence is provided to suggest that the RING domain of Mdm2 not only binds to UbcH5b but also acts as an allosteric activator of UbcH5b.     

Hetero-oligomerization with MdmX rescues the ubiquitin/Nedd8 ligase activity of RING finger mutants of Mdm2

Mdm2 is a member of the RING finger family of ubiquitin ligases and is best known for its role in targeting the tumor suppressor p53 for ubiquitination and degradation. Mdm2 can bind to itself and to the structurally related protein MdmX, and these interactions involve the RING finger domain of Mdm2 and MdmX, respectively. In this study, we performed a mutational analysis of the RING finger domain of Mdm2, and we identified several amino acid residues that are important for Mdm2 to exert its ubiquitin ligase function. Mutation of some of these residues interfered with the Mdm2-Mdm2 interaction indicating that a homomeric complex represents the active form of Mdm2. Mutation of other residues did not detectably affect the ability of Mdm2 to interact with itself but reduced the ability of Mdm2 to interact with UbcH5. Remarkably, MdmX efficiently rescued the ubiquitin ligase activity of the latter Mdm2 mutants in vitro and within cells. Because the interaction of Mdm2 with MdmX is more stable than the Mdm2-Mdm2 interaction, this suggests that Mdm2-MdmX complexes play a prominent role in p53 ubiquitination in vivo. Furthermore, we show that, similar to Mdm2, the Mdm2-MdmX complex has Nedd8 ligase activity and that all mutations that affect the ubiquitin ligase activity of Mdm2 also affect its Nedd8 ligase activity. From a mechanistic perspective, this suggests that the actual function of Mdm2 and Mdm2-MdmX, respectively, in p53 ubiquitination and in p53 neddylation is similar for both processes.     

MdmX is a RING finger ubiquitin ligase capable of synergistically enhancing Mdm2 ubiquitination

It has been well documented that Mdm2 and its homologue MdmX not only are critical negative regulators of the tumor suppressor p53 but that both Mdm2 and MdmX interact to affect the function of the other. The mechanisms through which these effects are manifested, however, remain unclear. Although Mdm2 has been established as a RING finger ubiquitin ligase, MdmX has not been shown to possess this activity despite the extensive sequence homology between their respective RING finger domains. Here we demonstrate that MdmX acts as a ubiquitin ligase in vitro, being capable of autoubiquitination, as well as mediating the ubiquitination of p53. The addition of Mdm2 to in vitro ubiquitination assays containing MdmX results in a synergistic increase of ubiquitin conjugation. Analysis of the resulting ubiquitin conjugates reveals that this observed synergy reflects an increase in Mdm2 ubiquitination. This study also suggests that ubiquitination of Mdm2 and MdmX may not serve as a signal for degradation, as we show that each are capable of synthesizing non-lysine 48 polyubiquitin chains and, in fact, utilize multiple lysine linkages. Taken together, these findings suggest a more active role for MdmX in the Mdm2-MdmX-p53 regulatory network than has been proposed previously.     

Structural and functional comparison of the RING domains of two p53 E3 ligases, Mdm2 and Pirh2

The tumor suppressor p53 maintains genome stability and prevents malignant transformation by promoting cell cycle arrest and apoptosis. Both Mdm2 and Pirh2 have been shown to ubiquitylate p53 through their RING domains, thereby targeting p53 for proteasomal degradation. Using structural and functional analyses, here we show that the Pirh2 RING domain differs from the Mdm2 RING domain in its oligomeric state, surface charge distribution, and zinc coordination scheme. Pirh2 also possesses weaker E3 ligase activity toward p53 and directs ubiquitin to different residues on p53. NMR and mutagenesis studies suggest that whereas Pirh2 and Mdm2 share a conserved E2 binding site, the seven C-terminal residues of the Mdm2 RING directly contribute to Mdm2 E3 ligase activity, a feature unique to Mdm2 and absent in the Pirh2 RING domain. This comprehensive analysis of the Pirh2 and Mdm2 RING domains provides structural and mechanistic insight into p53 regulation by its E3 ligases.     

MdmX protein is essential for Mdm2 protein-mediated p53 polyubiquitination

Genetic evidence has implicated both Mdm2 and MdmX as essential in negative regulation of p53. However, the exact role of MdmX in this Mdm2-dependent protein degradation is not well understood. Most, if not all, previous Mdm2 studies used GST-Mdm2 fusion proteins in the in vitro assays. Here, we show that the p53 polyubiquitination activity of GST-Mdm2 is conferred by the GST tag and non-GST-tagged Mdm2 only catalyzes monoubiquitination of p53 even at extremely high concentrations. We further demonstrate that MdmX is a potent activator of Mdm2, facilitating dose-dependent p53 polyubiquitination. This activation process requires the RING domains of both MdmX and Mdm2 proteins. The polyubiquitination activity of Mdm2/MdmX is Mdm2-dependent. Unlike Mdm2 or MdmX overexpression alone, co-overexpression of MdmX and Mdm2 consistently triggered p53 degradation in cells. Moreover, cellular polyubiquitination of p53 was only observable in the cytoplasm where both Mdm2 and MdmX are readily detectable. Importantly, RNAi knockdown of MdmX increased levels of endogenous p53 accompanied by reduced p53 polyubiquitination. In conclusion, our work has resolved a major confusion in the field derived from using GST-Mdm2 and demonstrated that MdmX is the cellular activator that converts Mdm2 from a monoubiquitination E3 ligase to a polyubiquitination E3 ligase toward p53. Together, our findings provide a biochemical basis for the requirement of both Mdm2 and MdmX in the dynamic regulation of p53 stability.     

The Mdm2 RING domain C-terminus is required for supramolecular assembly and ubiquitin ligase activity

Mdm2, a key negative regulator of the p53 tumor suppressor, is a RING-type E3 ubiquitin ligase. The Mdm2 RING domain can be biochemically fractionated into two discrete species, one of which exists as higher order oligomers that are visible by electron microscopy, whereas the other is a monomer. Both fractions are ATP binding and E3 ligase activity competent, although the oligomeric fraction exhibits lower dependence on the E2 component of ubiquitin polymerization reactions. The extreme C-terminal five amino acids of Mdm2 are essential for E3 ligase activity in vivo and in vitro, as well as for oligomeric assembly of the protein. A single residue (phenylalanine 490) in that sequence is critical for both properties. Interestingly, the C-terminus of the Mdm2 homologue, MdmX (itself inert as an E3 ligase), can fully substitute for the equivalent segment of Mdm2 and restore its E3 activity. We further show that the Mdm2 C-terminus is involved in intramolecular interactions and can set up a platform for direct protein-protein interactions with the E2.     

Association of p19(ARF) with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumor suppressor p53.

We have demonstrated previously that the oncoprotein Mdm2 has a ubiquitin ligase activity for the tumor suppressor p53 protein. In the present study, we characterize this ubiquitin ligase activity of Mdm2. We first demonstrate the ubiquitination of several p53 point mutants and deletion mutants by Mdm2. The point mutants, which cannot bind to Mdm2, are not ubiquitinated by Mdm2. The ubiquitination of the C-terminal deletion mutants, which contain so-called Mdm2-binding sites, is markedly decreased, compared with that of wild-type p53. The binding of Mdm2 to p53 is essential for ubiquitination, but p53's tertiary structure and/or C-terminal region may also be important for this reaction. DNA-dependent protein kinase is known to phosphorylate p53 on Mdm2-binding sites, where DNA damage induces phosphorylation, and p53 phosphorylated by this kinase is not a good substrate for Mdm2. This suggests that DNA damage-induced phosphorylation stabilizes p53 by inhibiting its ubiquitination by Mdm2. We further investigated whether the tumor suppressor p19(ARF) affects the ubiquitin ligase activity of Mdm2 for p53. The activity of p19(ARF)-bound Mdm2 was found to be lower than that of free Mdm2, suggesting that p19(ARF) promotes the stabilization of p53 by inactivating Mdm2.     

SUMO-1 modification of Mdm2 prevents its self-ubiquitination and increases Mdm2 ability to ubiquitinate p53

Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability.     

Auto-ubiquitination of Mdm2 Enhances Its Substrate Ubiquitin Ligase Activity

The RING domain E3 ubiquitin ligase Mdm2 is the master regulator of the tumor suppressor p53. It targets p53 for proteasomal degradation, restraining the potent activity of p53 and enabling cell survival and proliferation. Like most E3 ligases, Mdm2 can also ubiquitinate itself. How Mdm2 auto-ubiquitination may influence its substrate ubiquitin ligase activity is undefined. Here we show that auto-ubiquitination of Mdm2 is an activating event. Mdm2 that has been conjugated to polyubiquitin chains, but not to single ubiquitins, exhibits substantially enhanced activity to polyubiquitinate p53. Mechanistically, auto-ubiquitination of Mdm2 facilitates the recruitment of the E2 ubiquitin-conjugating enzyme. This occurs through noncovalent interactions between the ubiquitin chains on Mdm2 and the ubiquitin binding domain on E2s. Mutations that diminish the noncovalent interactions render auto-ubiquitination unable to stimulate Mdm2 substrate E3 activity. These results suggest a model in which polyubiquitin chains on an E3 increase the local concentration of E2 enzymes and permit the processivity of substrate ubiquitination. They also support the notion that autocatalysis may be a prevalent mode for turning on the activity of latent enzymes.     

Deconstructing nucleotide binding activity of the Mdm2 RING domain

Mdm2, a central negative regulator of the p53 tumor suppressor, possesses a Really Interesting New Gene (RING) domain within its C-terminus. In addition to E3 ubiquitin ligase activity, the Mdm2 RING preferentially binds adenine base nucleotides, and such binding leads to a conformational change in the Mdm2 C-terminus. Here, we present further biochemical analysis of the nucleotide–Mdm2 interaction. We have found that MdmX, an Mdm2 family member with high sequence homology, binds adenine nucleotides with similar affinity and specificity as Mdm2, suggesting that residues involved in nucleotide binding may be conserved between the two proteins and adenosine triphosphate (ATP) binding may have similar functional consequences for both Mdm family members. By generating and testing a series of proteins with deletions and substitution mutations within the Mdm2 RING, we mapped the specific adenine nucleotide binding region of Mdm2 to residues 429–484, encompassing the minimal RING domain. Using a series of ATP derivatives, we demonstrate that phosphate coordination by the Mdm2 P-loop contributes to, but is not primarily responsible for, ATP binding. Additionally, we have identified the 2′ and 3′ hydroxyls of the ribose and the C6 amino group of the adenine base moiety as being essential for binding.     

Effects of MdmX on Mdm2-mediated downregulation of pRB

Mdm2, a RING-finger type ubiquitin ligase, is overexpressed in a variety of human cancers. It promotes ubiquitination of the tumor suppressor p53 and can function as an oncogene by largely downregulating p53. Recently, we reported that Mdm2 degrades retinoblastoma tumor suppressor protein (pRB) via the ubiquitin-proteasome system. In the present study, we assessed the effects of MdmX, a structural homolog of Mdm2, on the Mdm2-mediated ubiquitination of pRB. MdmX is known to negatively regulate p53 function by enhancing the Mdm2-mediated ubiquitination and degradation of p53. Interestingly, MdmX inhibited the Mdm2-mediated pRB ubiquitination. Furthermore, an MdmX siRNA decreased the endogenous pRB level, while MdmX overexpression stimulated pRB functions in cultured cells. Therefore, MdmX may have different roles in the regulation of Mdm2 activity for ubiquitination of pRB and p53.     

Length matters: C-terminal tails regulate Mdm2–MdmX complexes

Comment on: Dolezelova P, et al. Cell Cycle 2012; 11:953–62Mechanisms controlling the p53 regulatory network remain the focus of numerous investigations in hopes of identifying more robust cancer therapies. Both Mdm2 and MdmX are found overexpressed in tumors with wild-type p53 and represent a key molecular device modulating p53 function. Thus, examining the interplay between these three proteins becomes highly relevant in the search for new pharmacological interventions in oncology.Mdm2 is a RING-type E3 ubiquitin ligase capable of forming homo-oligomers and hetero-oligomerization with MdmX via the extreme C termini of their RING domains. Since its discovery 15 years ago, MdmX has been assigned many roles in the regulation of p53, either on its own or in concert with Mdm2. While clearly an essential negative regulator or p53 in development, its lack of intrinsic ubiquitin ligase activity has made the mechanism of p53 regulation more elusive than in the case of Mdm2. The capacity of MdmX to stimulate Mdm2-mediated p53 ubiquitination was first reported in 2003.¹ Subsequent biochemical comparisons of the activity of Mdm2–MdmX complexes showed that not only does the presence of MdmX in the complex alter the substrate specificity of the holo-enzyme, it also allows for poly-ubiquitin chain formation on p53 (modification required for nuclear exclusion and degradation of p53).^2-4In vitro observations describing the importance of the MdmX RING domain in regulation of p53 turnover have now gained in vivo experimental support from the two knock-in animal models.^5,6 Consistent with the notion that MdmX is an essential component of p53 polyubiquitination/proteasomal degradation pathway, mice expressing either a point mutant in the MdmX RING domain or a RING domain deletion mutant succumbed to a p53-dependent embryonic lethality. These data implicate the RING domain of MdmX as the sole region of importance in the ability of MdmX to regulate p53 and, by extension, the Mdm2-MdmX complex (and not the Mdm2 homodimer), as the principle negative regulator of p53 activity during development.The growing body of evidence describing the presence of MdmX in the complex as crucial for target selectivity as well as the processivity of the holoezyme somewhat flies in the face of the existing structural data. Two published structures of the Mdm2 homodimer and Mdm2/MdmX heterodimer indicate virtually no difference in the complexes.^7,8 In the absence of structural differences, how then are such significant differences in function accomplished? A hypothesis unifying structural and functional data is brought forth by a very intriguing study from the Uldrijan group, which systematically looks at the differences between complex formation and activity of Mdm2 and MdmX.⁹ Phylogenetic analysis showed that the last cystein of the RING domain is followed by exactly 13 amino acids in all Mdm orthologs of vertebrate origin. Based on this, the authors hypothesized that not only the sequence of the C-terminal tails, but also their exact length are of central importance to the function of the complexes. Subsequent investigation of the ability of Mdm2 and MdmX proteins, which have been extended at the C terminus by 5, 14 or 18 amino acids, was designed to test the importance of the length of the C-terminal extensions. To the researchers surprise, when examined based on their ability to hetero-oligomerize and ubiquitinate p53, Mdm2 proteins behaved differently depending on whether the oligomeric partner was Mdm2 or MdmX. Dolezelova et al. present unexpected experimental evidence for the heterocomplex being structurally and functionally distinct from the Mdm2 homodimer, while providing a mechanism for the observed in vivo functional differences between the complexes. Although the work casts slight doubt on the complete accuracy of the existing structures, it nicely aligns with the above-mentioned results, showing the singular importance of the MdmX RING domain in the activity of the holoenzyme. In light of these results, additional structural studies that will take in to account reported differences between the complexes will undoubtedly be informative and contribute to our understanding of the biochemistry of RING-type ubiquitin ligases and the mechanisms regulating p53 in cells.