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.     

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     

Nucleotide binding by the Mdm2 RING domain facilitates Arf-independent Mdm2 nucleolar localization

The RING domain of Mdm2 contains a conserved Walker A or P loop motif that is a characteristic of nucleotide binding proteins. We found that Mdm2 binds adenine-containing nucleotides preferentially and that nucleotide binding leads to a conformational change in the Mdm2 C terminus. Although nucleotide binding is not required for Mdm2 E3 ubiquitin ligase activity, we show that nucleotide binding-defective P loop mutants are impaired in p14(ARF)-independent nucleolar localization both in vivo and in vitro. Consistent with this, ATP-bound Mdm2 is preferentially localized to the nucleolus. Indeed, we identify a unique amino acid substitution in the P loop motif (K454A) that uncouples nucleolar localization and E3 ubiquitin ligase activity of Mdm2 and leads to upregulation of the E3 activity both in human cells and in Caenorhabditis elegans. We propose that nucleotide binding-facilitated nucleolar localization of Mdm2 is an evolutionarily conserved regulator of Mdm2 activity.     

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.     

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.     

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.     

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.     

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.     

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.     

Ubiquitin ligase parkin promotes Mdm2-arrestin interaction but inhibits arrestin ubiquitination

Numerous mutations in E3 ubiquitin ligase parkin were shown to associate with familial Parkinson's disease. Here we show that parkin binds arrestins, versatile regulators of cell signaling. Arrestin-parkin interaction was demonstrated by coimmunoprecipitation of endogenous proteins from brain tissue and shown to be direct using purified proteins. Parkin binding enhances arrestin interactions with another E3 ubiquitin ligase, Mdm2, apparently by shifting arrestin conformational equilibrium to the basal state preferred by Mdm2. Although Mdm2 was reported to ubiquitinate arrestins, parkin-dependent increase in Mdm2 binding dramatically reduces the ubiquitination of both nonvisual arrestins, basal and stimulated by receptor activation, without affecting receptor internalization. Several disease-associated parkin mutations differentially affect the stimulation of Mdm2 binding. All parkin mutants tested effectively suppress arrestin ubiquitination, suggesting that bound parkin shields arrestin lysines targeted by Mdm2. Parkin binding to arrestins along with its effects on arrestin interaction with Mdm2 and ubiquitination is a novel function of this protein with implications for Parkinson's disease pathology.     

Assay for ubiquitin ligase activity: high-throughput screen for inhibitors of HDM2

An assay for the autoubiquitination activity of the E3 ligase HDM2 (Mdm2) was developed and adapted to a high-throughput format to identify inhibitors of this activity. The assay can also be used to measure the activity of other E3s and may be useful in finding both inhibitors and activators of a wide range of different ubiquitin ligases.     

Ribosomal Proteins RPL37, RPS15 and RPS20 Regulate the Mdm2-p53-MdmX Network

Changes to the nucleolus, the site of ribosome production, have long been linked to cancer, and mutations in several ribosomal proteins (RPs) have been associated with an increased risk for cancer in human diseases. Relevantly, a number of RPs have been shown to bind to MDM2 and inhibit MDM2 E3 ligase activity, leading to p53 stabilization and cell cycle arrest, thus revealing a RP-Mdm2-p53 signaling pathway that is critical for ribosome biogenesis surveillance. Here, we have identified RPL37, RPS15, and RPS20 as RPs that can also bind Mdm2 and activate p53. We found that each of the aforementioned RPs, when ectopically expressed, can stabilize both co-expressed Flag-tagged Mdm2 and HA-tagged p53 in p53-null cells as well as endogenous p53 in a p53-containing cell line. For each RP, the mechanism of Mdm2 and p53 stabilization appears to be through inhibiting the E3 ubiquitin ligase activity of Mdm2. Interestingly, although they are each capable of inducing cell death and cell cycle arrest, these RPs differ in the p53 target genes that are regulated upon their respective introduction into cells. Furthermore, each RP can downregulate MdmX levels but in distinct ways. Thus, RPL37, RPS15 and RPS20 regulate the Mdm2-p53-MdmX network but employ different mechanisms to do so.     

Mammalian Numb is a target protein of Mdm2, ubiquitin ligase

Drosophila Numb protein functions as an antagonist against Notch signal. The expression of this protein is asymmetrical in divided cells and thought to be involved in the neural cell differentiation and/or cell fate. Human homologue of Numb (hNumb) was cloned as Mdm2-binding protein by yeast two-hybrid screening. Since Mdm2 is an oncoprotein and has ubiquitin ligase activity toward tumor suppressor p53, we assessed to find out whether Mdm2 ubiquitinylates the hNumb protein. The recombinant hNumb expressed in Sf-9 cells using baculovirus protein expression system bound to Mdm2 in vitro. When hNumb was subjected to in vitro ubiquitinylation assay system, which contains E1, E2, or UbcH5c, and Mdm2, hNumb was ubiquitinylated as efficiently as the p53 protein. However, when the Ring-finger domain mutant of Mdm2 was used in place of wild-type Mdm2, hNumb was not ubiquitinylated. Furthermore, when U2OS cells were co-transfected with hNumb and Mdm2, the hNumb protein was ubiquitinylated and degraded. These data strongly suggest that Mdm2 functions as the ubiquitin ligase toward hNumb and that it induces its degradation in intact cells.     

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.