Dynamics in the p53-Mdm2 Ubiquitination Pathway

The tumor suppressor p53 is highly regulated under various states of cellular stress. p53 stability is predominantly regulated through the ubiquitin-proteasomal pathway by the E3 ligase Mdm2. p53 ubiquitination is a dynamic process with Mdm2 capable of catalyzing both mono- and polyubiquitination. Additionally, deubiquitination is an important step occurring in p53 and Mdm2 stabilities. Factors such as HAUSP, p14ARF, and MdmX play important regulatory roles in p53 ubiquitination/deubiquitination and their interplay with Mdm2 and p53 compound layers of complexity for regulating this important pathway.     

MdmX protects p53 from Mdm2-mediated degradation

The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.     

Regulation of the Mdm2–p53 pathway by the ubiquitin E3 ligase MARCH7

The tumor suppressor p53 plays a prominent role in the protection against cancer. The activity of p53 is mainly controlled by the ubiquitin E3 ligase Mdm2, which targets p53 for proteasomal degradation. However, the regulation of Mdm2 remains not well understood. Here, we show that MARCH7, a RING domain‐containing ubiquitin E3 ligase, physically interacts with Mdm2 and is essential for maintaining the stability of Mdm2. MARCH7 catalyzes Lys⁶³‐linked polyubiquitination of Mdm2, which impedes Mdm2 autoubiquitination and degradation, thereby leading to the stabilization of Mdm2. MARCH7 also promotes Mdm2‐dependent polyubiquitination and degradation of p53. Furthermore, MARCH7 is able to regulate cell proliferation, DNA damage‐induced apoptosis, and tumorigenesis via a p53‐dependent mechanism. These findings uncover a novel mechanism for the regulation of Mdm2 and reveal MARCH7 as an important regulator of the Mdm2–p53 pathway.     

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.     

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     

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.     

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.     

p53 ubiquitination: Mdm2 and beyond

Although early studies have suggested that the oncoprotein Mdm2 is the primary E3 ubiquitin ligase for the p53 tumor suppressor, an increasing amount of data suggests that p53 ubiquitination and degradation are more complex than once thought. The discoveries of MdmX, HAUSP, ARF, COP1, Pirh2, and ARF-BP1 continue to uncover the multiple facets of this pathway. There is no question that Mdm2 plays a pivotal role in downregulating p53 activities in numerous cellular settings. Nevertheless, growing evidence challenges the conventional view that Mdm2 is essential for p53 turnover.     

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.     

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.     

Targeting RING domains of Mdm2–MdmX E3 complex activates apoptotic arm of the p53 pathway in leukemia/lymphoma cells

Reactivation of tumor-suppressor p53 for targeted cancer therapy is an attractive strategy for cancers bearing wild-type (WT) p53. Targeting the Mdm2–p53 interface or MdmX ((MDM4), mouse double minute 4)–p53 interface or both has been a focus in the field. However, targeting the E3 ligase activity of Mdm2–MdmX really interesting new gene (RING)–RING interaction as a novel anticancer strategy has never been explored. In this report, we describe the identification and characterization of small molecule inhibitors targeting Mdm2–MdmX RING–RING interaction as a new class of E3 ligase inhibitors. With a fluorescence resonance energy transfer-based E3 activity assay in high-throughput screening of a chemical library, we identified inhibitors (designated as MMRis (Mdm2–MdmX RING domain inhibitors)) that specifically inhibit Mdm2–MdmX E3 ligase activity toward Mdm2 and p53 substrates. MMRi6 and its analog MMRi64 are capable of disrupting Mdm2–MdmX interactions in vitro and activating p53 in cells. In leukemia cells, MMRi64 potently induces downregulation of Mdm2 and MdmX. In contrast to Nutlin3a, MMRi64 only induces the expression of pro-apoptotic gene PUMA (p53 upregulated modulator of apoptosis) with minimal induction of growth-arresting gene p21. Consequently, MMRi64 selectively induces the apoptotic arm of the p53 pathway in leukemia/lymphoma cells. Owing to the distinct mechanisms of action of MMRi64 and Nutlin3a, their combination synergistically induces p53 and apoptosis. Taken together, this study reveals that Mdm2–MdmX has a critical role in apoptotic response of the p53 pathway and MMRi64 may serve as a new pharmacological tool for p53 studies and a platform for cancer drug development.Activation of tumor-suppressor p53 as a targeted non-genotoxic cancer therapy has been pursued for many years,^{1, 2} because p53 possesses potent tumor-suppressing activity in vivo.^{3, 4, 5} p53 can inhibit cancer cell growth by cell cycle arrest or terminate their proliferation by inducing apoptosis and senescence.⁶ The p53-based therapy is particularly attractive for cancer types including retinoblastoma, neuroblastoma and leukemia/lymphoma in which p53 is rarely mutated⁷ and p53-dependent apoptotic pathway is a predominant endpoint.^{8, 9, 10} Except for cancer-selected p53 mutations, the p53 activity is mainly inhibited by p53-binding proteins Mdm2 and MdmX ((MDM4), mouse double minute 4) in normal and cancer cells.^{11, 12} Prior focus of p53 reactivation strategy has been on targeting the Mdm2–p53 and/or MdmX–p53 interface. This has led to the discovery of a list of potent Mdm2–p53 inhibitors¹³ with several compounds of this class being advanced to phase I clinical trials in hematological neoplasia and solid tumors.² However, the therapeutic effects of these Mdm2–p53 inhibitors can be attenuated by MdmX overexpression.^{14, 15, 16} Although peptide inhibitors with dual functions of inhibiting both Mdm2–p53 and MdmX–p53 interactions will overcome this problem and enhance p53-dependent cancer killing;^{17, 18} these inhibitors will not inhibit Mdm2 E3 ligase activity toward non-p53 targets such as retinoblastoma protein (RB), p21 and DAXX (death domain-associated protein),^{19, 20, 21} which to a different extent contributes to the p53-dependent biological effects.Recent genetic studies indicated that really interesting new gene (RING) domains of Mdm2 and MdmX are required for in vivo inhibition of p53 activity during development.^{22, 23, 24} MdmX was reported to stimulate Mdm2-mediated p53 multiple monoubiquitination using glutathione S-transferase (GST) fusion Hdm2 proteins.^{25, 26} Using non-GST Hdm2 proteins in in vitro biochemical assays, we found that MdmX–Mdm2 RING–RING interaction is essential for p53 polyubiquitination and proteasome-dependent degradation.²⁶ These findings established that Mdm2–MdmX complex is the key regulator of p53 activity and Mdm2–MdmX RING–RING interaction is a critical but an unexplored interface for drug targeting.²⁷ Identification of E3 ligase inhibitors for cancer therapy presents a huge opportunity but with great challenges.²⁸ In this report, we describe successful identification and characterization of small molecule inhibitors for the E3 ligase activity of Mdm2–MdmX E3 complex. Among seven specific MMRis (Mdm2–MdmX RING domain inhibitors), MMRi64 was followed up in detail in this report. MMRi64 has several unique features that distinguish it from Mdm2–p53 inhibitor Nutlin3a. MMRi64 disrupts Mdm2–MdmX interaction in vitro and inhibits the E3 ligase activity of Mdm2–MdmX without affecting the E3 ligase activity of Mdm2 RING domain homodimers. MMRi64 induces p53 accumulation without induction of Mdm2 and p21 in lymphoma cells, which is distinct from the effects of Nutlin3a. Finally, MMRi64 induces PUMA (p53 upregulated modulator of apoptosis) but strongly downregulates MdmX and Mdm2, consequently activating the apoptotic arm of the p53 pathway in leukemia/lymphoma cells without the induction of growth arrest.     

Predicted Functions of MdmX in Fine-Tuning the Response of p53 to DNA Damage

Tumor suppressor protein p53 is regulated by two structurally homologous proteins, Mdm2 and MdmX. In contrast to Mdm2, MdmX lacks ubiquitin ligase activity. Although the essential interactions of MdmX are known, it is not clear how they function to regulate p53. The regulation of tumor suppressor p53 by Mdm2 and MdmX in response to DNA damage was investigated by mathematical modeling of a simplified network. The simplified network model was derived from a detailed molecular interaction map (MIM) that exhibited four coherent DNA damage response pathways. The results suggest that MdmX may amplify or stabilize DNA damage-induced p53 responses via non-enzymatic interactions. Transient effects of MdmX are mediated by reservoirs of p53∶MdmX and Mdm2∶MdmX heterodimers, with MdmX buffering the concentrations of p53 and/or Mdm2. A survey of kinetic parameter space disclosed regions of switch-like behavior stemming from such reservoir-based transients. During an early response to DNA damage, MdmX positively or negatively regulated p53 activity, depending on the level of Mdm2; this led to amplification of p53 activity and switch-like response. During a late response to DNA damage, MdmX could dampen oscillations of p53 activity. A possible role of MdmX may be to dampen such oscillations that otherwise could produce erratic cell behavior. Our study suggests how MdmX may participate in the response of p53 to DNA damage either by increasing dependency of p53 on Mdm2 or by dampening oscillations of p53 activity and presents a model for experimental investigation.     

MdmX binding to ARF affects Mdm2 protein stability and p53 transactivation

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.     

UBE4B,a ubiquitin chain assembly factor,is required for MDM2-mediated p53 polyubiquitination and degradation

Although MDM2 is known to be a critical negative regulator of p53, MDM2 only catalyzes p53 mono- or multiple monoubiquitination in vitro and in vivo, which is insufficient for the initiation of proteasomal degradation. MDM2 does not polyubiquitinate p53 in vitro, however, which indicates that the activity of other ubiquitin ligase(s) or cofactor(s) is required for MDM2-mediated p53 polyubiquitination and degradation. In our recent study, we demonstrated that UBE4B, an E3 and E4 ubiquitin ligase with a U-box domain, interacts physically with both p53 and MDM2. Our findings revealed that UBE4B negatively regulates the level of p53 and inhibits p53-dependent transactivation and apoptosis. We propose that inhibition of MDM2 binding to UBE4B may provide another approach to inhibit MDM2 E3 ligase activity for tumor suppressor p53. It could lead to novel anticancer therapies, with the possibility of reducing the public health burden from cancer.Key words: ubiquitination, MDM2, UBE4B, p53, degradation     

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.     

Regulation of p53 by Mdm2: fate is in the numbers

The p53 tumor suppressor protein is normally restrained by the Mdm2 oncoprotein, which promotes p53 ubiquitination. In a recent issue of Science, report that p53 may face two alternative fates, depending on Mdm2 levels: high Mdm2 drives p53 polyubiquitination and degradation within the cell nucleus, whereas low Mdm2 promotes p53 monoubiquitination and nuclear exclusion.