Critical role for a central part of Mdm2 in the ubiquitylation of p53

The stability of the p53 protein is regulated by Mdm2. By acting as an E3 ubiquitin ligase, Mdm2 directs the ubiquitylation of p53 and its subsequent degradation by the 26S proteasome. In contrast, the Mdmx protein, although structurally similar to Mdm2, cannot ubiquitylate or degrade p53 in vivo. To ascertain which domains determine this functional difference between Mdm2 and Mdmx and consequently are essential for p53 ubiquitylation and degradation, we generated Mdm2-Mdmx chimeric constructs. Here we show that, in addition to a fully functional Mdm2 RING finger, an internal domain of Mdm2 (residues 202 to 302) is essential for p53 ubiquitylation. Strikingly, the function of this domain can be fulfilled in trans, indicating that the RING domain and this internal region perform distinct activities in the ubiquitylation of p53.     

A critical role for noncoding 5S rRNA in regulating Mdmx stability

Both p53 and Mdmx are ubiquitinated and degraded by the same E3 ligase Mdm2; interestingly, however, while p53 is rapidly degraded by Mdm2, Mdmx is a stable protein in most cancer cells. Thus, the mechanism by which Mdmx is degraded by Mdm2 needs further elucidation. Here, we identified the noncoding 5S rRNA as a major component of Mdmx-associated complexes from human cells. We show that 5S rRNA acts as a natural inhibitor of Mdmx degradation by Mdm2. RNAi-mediated knockdown of endogenous 5S rRNA, while not affecting p53 levels, significantly induces Mdmx degradation and, subsequently, activates p53-dependent growth arrest. Notably, 5S rRNA binds the RING domain of Mdmx and blocks its ubiquitination by Mdm2, whereas Mdm2-mediated p53 ubiquitination remains intact. These results provide insights into the differential effects on p53 and Mdmx by Mdm2 in vivo and reveal a critical role for noncoding 5S rRNA in modulating the p53-Mdmx axis.     

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.     

Mdm2 is a RING finger-dependent ubiquitin protein ligase for itself and p53

Mdm2 has been shown to regulate p53 stability by targeting the p53 protein for proteasomal degradation. We now report that Mdm2 is a ubiquitin protein ligase (E3) for p53 and that its activity is dependent on its RING finger. Furthermore, we show that Mdm2 mediates its own ubiquitination in a RING finger-dependent manner, which requires no eukaryotic proteins other than ubiquitin-activating enzyme (E1) and an ubiquitin-conjugating enzyme (E2). It is apparent, therefore, that Mdm2 manifests an intrinsic capacity to mediate ubiquitination. Mutation of putative zinc coordination residues abrogated this activity, as did chelation of divalent cations. After cation chelation, the full activity could be restored by addition of zinc. We further demonstrate that the degradation of p53 and Mdm2 in cells requires additional potential zinc-coordinating residues beyond those required for the intrinsic activity of Mdm2 in vitro. Replacement of the Mdm2 RING with that of another protein (Praja1) reconstituted ubiquitination and proteasomal degradation of Mdm2. However, this RING was ineffective in ubiquitination and proteasomal targeting of p53, suggesting that there may be specificity at the level of the RING in the recognition of heterologous substrates.     

c-Abl phosphorylation of Mdm2 facilitates Mdm2-Mdmx complex formation

Mdm2 and Mdmx are oncoproteins that have essential yet nonredundant roles in development and function as part of a multicomponent ubiquitinating complex that targets p53 for proteasomal degradation. However, in response to DNA damage, Mdm2 and Mdmx are phosphorylated and protect p53 through various mechanisms. It has been predicted that Mdm2-Mdmx complex formation modulates Mdm2 ligase activity, yet the mechanism that promotes formation of Mdm2-Mdmx complexes is unknown. Here, we show that optimal Mdm2-Mdmx complex formation requires c-Abl phosphorylation of Mdm2 both in vitro and in vivo. In addition, Abl phosphorylation of Mdm2 is required for efficient ubiquitination of Mdmx in vitro, and eliminating c-Abl signaling, using c-Abl(-/-) knock-out murine embryonic fibroblasts, led to a decrease in Mdmx ubiquitination. Further, p53 levels are not induced as efficiently in c-Abl(-/-) murine embryonic fibroblasts following DNA damage. Overall, these results define a direct link between genotoxic stress-activated c-Abl kinase signaling and Mdm2-Mdmx complex formation. Our results add an important regulatory mechanism for the activation of p53 in response to DNA damage.     

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.     

Molecular Basis for the Inhibition of p53 by Mdmx

The oncoprotein Mdm2, and the recently intensely studied, homologues protein Mdmx, are principal negative regulators of the p53 tumor uppressor. The mechanisms by which they regulate the stability and activity of p53 are not fully established. We have determined the crystal structure of the N-terminal domain of Mdmx bound to a 15-residue p53 peptide. The structure reveals that although the principle features of the Mdm2-p53 interaction are preserved in the Mdmx-p53 complex, the Mdmx hydrophobic cleft on which the p53 peptide binds is significantly altered: a part of the cleft is blocked by sidechains of Met and Tyr of the p53-binding pocket of Mdmx. Thus specific inhibitors of Mdm2-p53 would not be optimal for binding to Mdmx. Our binding assays show indeed that nutlins, the newly discovered, potent antagonists of the Mdm2-p53 interaction, are notcapable to efficiently disrupt the Mdmx-p53 interaction. To achieve full activation of p53 in tumor cells, compounds that are specific for Mdmx are necessary to complement the Mdm2 specific binders.     

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.     

Interaction of regulators Mdm2 and Mdmx with transcription factors p53, p63 and p73

The negative regulation of p53, a major human tumor suppressor, by Mdm2 and Mdmx is crucial for the survival of a cell, whereas its aberrant function is a common feature of cancer. Both Mdm proteins act through the spatial occlusion of the p53 transactivation (TA) domain and by the ubiquitination of p53, resulting in its degradation. Two p53 homologues, p63 and p73, have been described in humans. Unlike p53, these proteins regulate developmental processes rather than genome stability. Both p63 and p73 contain TA domains homologous to that of p53, but relatively little is known about their regulation by Mdm2 or Mdmx. Here, we present a detailed characterization of the interaction of Mdm2 and Mdmx with the TA domains of p63 and p73. Earlier reports of Mdm2 and Mdmx interactions with p73 are substantiated by the detailed quantitative characterization reported in this study. Most importantly, earlier contradictions concerning the presumed interaction of the Mdm proteins with p63 are convincingly resolved and for the first time, the affinities of these interactions are determined. Finally, the contribution of these findings to our understanding of the physiological role of these interactions is discussed.