MDM2 RING Research Articles

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.

MDM2 interaction with nuclear corepressor KAP1 contributes to p53 inactivation

MDM2 is a RING domain ubiquitin E3 ligase and a major regulator of the p53 tumor suppressor. MDM2 binds to p53, inactivates p53 transcription function, inhibits p53 acetylation, and promotes p53 degradation. Here, we present evidence that MDM2 interacts with the nuclear corepressor KAP1. The binding is mediated by the N-terminal coiled-coil domain of KAP1 and the central acidic domain of MDM2. KAP1 stimulates formation of p53-HDAC1 complex and inhibits p53 acetylation by interacting with MDM2. Expression of KAP1 cooperates with MDM2 to promote p53 ubiquitination and degradation. The tumor suppressor ARF competes with KAP1 in MDM2 binding; oncogene induction of ARF expression reduces MDM2-KAP1 interaction. Depletion of endogenous KAP1 expression by RNAi stimulates p53 transcriptional activity, sensitizes p53 response to DNA damage, and increases apoptosis. Therefore, MDM2 interaction with KAP1 contributes to p53 functional regulation. ARF may regulate p53 acetylation and stability in part by inhibiting KAP1-MDM2 binding.

Regulation of p53 and MDM2 Activity by MTBP

p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not gamma-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells.

Mdm2-Mediated NEDD8 Conjugation of p53 Inhibits Its Transcriptional Activity

The only reported role for the conjugation of the NEDD8 ubiquitin-like molecule is control of the activity of SCF ubiquitin ligase complexes. Here, we show that the Mdm2 RING finger E3 ubiquitin ligase can also promote NEDD8 modification of the p53 tumor suppressor protein. Mdm2 is itself modified with NEDD8 with very similar characteristics to the autoubiquitination activity of Mdm2. By using a cell line (TS-41) with a temperature-sensitive mutation in the NEDD8 conjugation pathway and a p53 mutant that cannot be NEDDylated (3NKR), we demonstrate that Mdm2-dependent NEDD8 modification of p53 inhibits its transcriptional activity. These findings expand the role for Mdm2 as an E3 ligase, providing evidence that Mdm2 is a common component of the ubiquitin and NEDD8 conjugation pathway and indicating the diverse mechanisms by which E3 ligases can control the function of substrate proteins.

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 p14ARF-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.

Cellular stress and DNA damage invoke temporally distinct Mdm2, p53 and PML complexes and damage-specific nuclear relocalization.

Mdm2 is a nucleoplasmic and nucleolar protein interacting with p53 and alternative reading frame (ARF) tumor suppressor proteins. Here we demonstrate relocalization and novel interactions of Mdm2 with the promyelocytic leukemia (PML) protein following cellular stress and DNA damage. We show that Mdm2 and PML interact directly in vivo and in vitro depending on the Mdm2 RING finger domain and the PML C-terminus, and that Mdm2 is recruited to the PML nuclear bodies by overexpression of PML. Cellular stress and DNA damage caused by UV-radiation, downregulation of the proteasome and arsenic trioxide promoted Mdm2 and PML damage-specific nuclear relocalization and interaction in a p53-independent manner. However, in vitro analyses showed that PML, Mdm2 and p53 form trimeric complexes. UV-radiation caused rapid rearrangements of PML nuclear bodies and promoted PML-p53 and PML-Mdm2 complex formation, coinciding with p53 stabilization and preceding p53-Mdm2 interaction suggesting temporally distinct complexes. The results demonstrate novel associations between Mdm2 and PML and show the capacity of PML to participate in the activation and stabilization of p53 in response to cellular stress through PML interaction with Mdm2.

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.

The MDM2 RING finger is required for cell cycle-dependent regulation of its protein expression

The MDM2 oncoprotein is overexpressed in many human tumors and cancers. MDM2 functions as an E3 ligase for p53 and for itself. MDM2 also interacts with the retinoblastoma protein (RB) and the transcription factor E2F1 to promote cell cycle S-phase entry. Here, we report that MDM2 protein expression is cell cycle-regulated, which is dependent on its RING finger domain and requires Lys446. We show that MDM2 protein is stabilized at S phase. In addition, overexpression of MDM2 results in stimulation of E2F activity and accumulation of cells in S phase. These data suggest that ubiquitination of MDM2 is cell cycle-regulated and that MDM2 may play a role in cell cycle progression.

Overexpression of Mdm2 and MdmX fusion proteins alters p53 mediated transactivation, ubiquitination, and degradation.

Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Mdm2, a p53 inducible protein, negatively regulates p53 by inhibiting p53 transcriptional activity and promoting ubiquitin mediated proteasome degradation. The Mdm2 ring finger domain has been shown to possess E3 ligase activity and to be a necessary domain for targeting p53 degradation. MdmX, a p53 binding protein sharing a high degree of structural homology with Mdm2, has emerged as another negative regulator of the p53 tumor suppressor. MdmX has also been shown to block p53 transactivation but unlike Mdm2 cannot induce p53 degradation. Since MdmX also possesses a ring finger domain that allows MdmX to associate with Mdm2, this study focused on elucidating how the ring and zinc fingers of these two proteins affected p53 function. We have generated a series of fusion proteins between Mdm2 and MdmX by swapping the ring finger domains with or without the zinc finger domains and examined how these fusions regulated p53 induced transactivation, ubiquitination, and degradation. All fusions inhibited the transcriptional activity of p53. In the absence of Mdm2, none of the fusion proteins could trigger p53 ubiquitination or degradation. However, in a cell line with endogenous Hdm2, Mdm2:X fusions containing the ring finger domain with or without the zinc finger domain demonstrated p53 ubiquitination presumably through stabilization of Hdm2. Additionally, an Mdm2:XZFRF fusion also degraded p53 when endogenous Hdm2 was present. Results from immunofluorescence studies suggest that p53 is colocalized to the cytoplasm when coexpressed with a Mdm2:X fusion (Mdm2:XZFRF) and that this fusion is capable of stabilizing endogenous Hdm2. Since none of the fusions triggered p53 ubiquitination in cells lacking Mdm2, these results indicate that the E3 ligase domain within the ring finger of Mdm2 when part of MdmX and the MdmX ring finger fused to Mdm2 were not sufficient to trigger p53 ubiquitination, in vivo.

Mdmx stabilizes p53 and Mdm2 via two distinct mechanisms.

The p53 protein maintains genomic integrity through its ability to induce cell cycle arrest or apoptosis in response to various forms of stress. Substantial regulation of p53 activity occurs at the level of protein stability, largely determined by the activity of the Mdm2 protein. Mdm2 targets both p53 and itself for ubiquitylation and subsequent proteasomal degradation by acting as an ubiquitin ligase, a function that needs an intact Mdm2 RING finger. For efficient degradation of p53 nuclear export appears to be required. The Mdmx protein, structurally homologous to Mdm2, does not target p53 for degradation, but even stabilizes both p53 and Mdm2, an activity most likely mediated by heterodimerization of the RING fingers of Mdm2 and Mdmx. Here we show that Mdmx expression leads to accumulation of ubiquitylated, nuclear p53 but does not significantly affect the Mdm2-mediated ubiquitylation of p53. In contrast, Mdmx stabilizes Mdm2 by inhibiting its self-ubiquitylation.

The Mdm-2 amino terminus is required for Mdm2 binding and SUMO-1 conjugation by the E2 SUMO-1 conjugating enzyme Ubc9.

Covalent attachment of SUMO-1 to Mdm2 requires the activation of a heterodimeric Aos1-Uba2 enzyme (ubiquitin-activating enzyme (E1)) followed by the conjugation of Sumo-1 to Mdm2 by Ubc9, a protein with a strong sequence similarity to ubiquitin carrier proteins (E2s). Upon Sumo-1 conjugation, Mdm2 is protected from self-ubiquitination and elicits greater ubiquitin-protein isopeptide ligase (E3) activity toward p53, thereby increasing its oncogenic potential. Because of the biological implication of Mdm2 sumoylation, we mapped Ubc9 binding on Mdm2. Here we demonstrate that Ubc9 can associate with Mdm2 only if amino acids 40-59 within the N terminus of Mdm2 are present. Mdm2 from which amino acids 40-59 have been deleted can no longer be sumoylated. Furthermore, addition of a peptide that corresponds to amino acids 40-59 on Mdm2 to a sumoylation reaction efficiently inhibits Mdm2 sumoylation in vitro and in vivo. In UV-treated cells Mdm2 exhibits reduced association with Ubc9, which coincides with decreased Mdm2 sumoylation. Our findings regarding the association of Ubc9 with Mdm2, and the effect of UV-irradiation on Ubc9 binding, point to an additional level in the regulation of Mdm2 sumoylation under normal growth conditions as well as in response to stress conditions.

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.