Domain Of MDM2 Research Articles

S100 proteins interact with the N-terminal domain of MDM2

S100 proteins interact with the transactivation domain and the C-terminus of p53. Further, S100B has been shown to interact with MDM2, a central negative regulator of p53. Here, we show that S100B bound directly to the folded N-terminal domain of MDM2 (residues 2-125) by size exclusion chromatography and surface plasmon resonance experiments. This interaction with MDM2 (2-125) is a general feature of S100 proteins; S100A1, S100A2, S100A4 and S100A6 also interact with MDM2 (2-125). These interactions with S100 proteins do not result in a ternary complex with MDM2 (2-125) and p53. Instead, we observe the ability of a subset of S100 proteins to disrupt the extent of MDM2-mediated p53 ubiquitylation in vitro. Structured summary MINT- 7905256: MDM2 (uniprotkb: Q00987) binds (MI: 0407) to s100A6 (uniprotkb: P06703) by surface plasmon resonance (MI: 0107) MINT- 7905063: MDM2 (uniprotkb: Q00987) and s100A1 (uniprotkb: P23297) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905376: s100A4 (uniprotkb: P26447) and MDM2 (uniprotkb: Q00987) physically interact (MI: 0915) by competition binding (MI: 0405) MINT- 7905130: s100A6 (uniprotkb: P06703) and MDM2 (uniprotkb: Q00987) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905207: s100A6 (uniprotkb: P06703) and p53 (uniprotkb: P04637) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905043: s100B (uniprotkb: P04271) and MDM2 (uniprotkb: Q00987) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905196: p53 (uniprotkb: P04637) and s100A4 (uniprotkb: P26447) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905358: p53 (uniprotkb: P04637) and s100A4 (uniprotkb: P26447) physically interact (MI: 0915) by fluorescence polarization spectroscopy (MI: 0053) MINT- 7905220: MDM2 (uniprotkb: Q00987) binds (MI: 0407) to s100B (uniprotkb: P04271) by surface plasmon resonance (MI: 0107) MINT- 7905104: s100A4 (uniprotkb: P26447) and MDM2 (uniprotkb: Q00987) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905229: MDM2 (uniprotkb: Q00987) binds (MI: 0407) to s100A1 (uniprotkb: P23297) by surface plasmon resonance (MI: 0107) MINT- 7905317, MINT- 7905162: s100B (uniprotkb: P04271) and p53 (uniprotkb: P04637) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905238: MDM2 (uniprotkb: Q00987) binds (MI: 0407) to s100A2 (uniprotkb: P29034) by surface plasmon resonance (MI: 0107) MINT- 7905174, MINT- 7905308: s100A1 (uniprotkb: P23297) and p53 (uniprotkb: P04637) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905247: MDM2 (uniprotkb: Q00987) binds (MI: 0407) to s100A4 (uniprotkb: P26447) by surface plasmon resonance (MI: 0107) MINT- 7905090: s100A2 (uniprotkb: P29034) and MDM2 (uniprotkb: Q00987) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905142, MINT- 7905326: MDM2 (uniprotkb: Q00987) and p53 (uniprotkb: P04637) bind (MI: 0407) by molecular sieving (MI: 0071) MINT- 7905185, MINT- 7905347: s100A2 (uniprotkb: P29034) and p53 (uniprotkb: P04637) bind (MI: 0407) by molecular sieving (MI: 0071)

MDM2 Promotes Proteasomal Degradation of p21Waf1 via a Conformation Change

MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21(Waf1). The present study was designed to characterize the mechanism(s) by which MDM2 induces p21(Waf1) degradation. We first determined the regions of MDM2 required for p21(Waf1) degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21(Waf1) interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21(Waf1) degradation. Specifically, amino acids 226-250 of MDM2 were required for p21(Waf1) binding and degradation, and amino acids 251-260 were necessary for p21(Waf1) degradation. The latter region induced a conformation change in p21(Waf1), increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226-250 or aa 251-260), its capacity to promote p21(Waf1) degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21(Waf1)) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitin-independent manner, this mechanism may underlie part of its tumorigenic properties.

Mdm2 facilitates the association of p53 with the proteasome

The ubiquitin ligase Mdm2 targets the p53 tumor suppressor protein for proteasomal degradation. Mutating phosphorylation sites in the central domain of Mdm2 prevents p53 degradation, although it is still ubiquitylated, indicating that Mdm2 has a post-ubiquitylation function for p53 degradation. We show that Mdm2 associates with several subunits of the 19S proteasome regulatory particle in a ubiquitylation-independent manner. Mdm2 furthermore promotes the formation of a ternary complex of itself, p53, and the proteasome. Replacing phosphorylation sites within the central domain with alanines reduced the formation of the ternary complex. The C-terminus of Mdm2 was sufficient for interaction with the proteasome despite an additional proteasome binding site in the Mdm2 N-terminus. In addition to binding to the proteasome, the C-terminus of Mdm2 bound to the central domain, possibly competing with, and therefore blocking, Mdm2/proteasome interaction. We propose that Mdm2 facilitates, or at least enhances, the association of p53 with the proteasome and that phosphorylation of the central domain of Mdm2 regulates this process.

Abstract 1109: Identification of a novel MDM2 splice transcript that lacks nuclear transport signals but harbors the p53 binding motif in human glioma cells

Abstract MDM2 a nuclear phosphoprotein binds with E3 ubiquitin ligase activity, binds and targets p53 for proteosomal degradation, hence is a negative regulator of p53. Consequently, over-expression of MDM2 results in the loss of p53 tumor suppressor function even in the absence of inactivating mutations in the p53 gene. To date, more than 40 different alternative spliced MDM2 transcript variants have been reported in both normal and tumor cells, with most of these lacking, in part or in full, the p53 binding domain of MDM2. In this study, we examined the levels of MDM2 gene expression in a panel of glioblastoma (GBM) cell lines and in vivo primary tumor xenografts by quantitative PCR /RT-PCR and examined the full length MDM2 cDNAs for the presence of MDM2 splice transcript variants using the nested PCR technique and a combination of direct sequencing and TA vector sequencing. The results showed a significant heterogeneity in MDM2 expression and in MDM2 splice variants among the tumors. Importantly, we identified a novel splice MDM2 transcript that was consistently associated with low MDM2 transcript levels in the tumors. Sequence analysis of this MDM2 spliced transcript showed it to harbor an internal deletion of three exons, namely 7, 8, and 9, containing the nuclear localization signal (NLS), the nuclear export signal (NES) and a portion of the acidic domain (AD). The p53 binding domain was fully conserved. The deletion caused a shift in the MDM2 open reading frame that created a stop codon (TGA) at codon 316. The spliced MDM2 transcript encoded a truncated MDM2 protein that harbors the functional p53 binding motif capable of interacting with the p53 protein but without the ability to localize to the nucleolus. Overall, our results with the 15 tumors showed no strong correlation between the presence of this novel splice MDM2 transcript and the expression levels of p53in the tumors. Studies are ongoing to determine the functional consequences of this novel MDM2 spliced variant on p53 downstream function in glioma cells. Supported by grants RO1CA112519, RO1CA127872, RO1CA91438, P30CA114236 and P5OCA108786 from the National Institutes of Health (USA). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1109.

Abstract 4516: A new class of MDM2 inhibitors cause growth inhibition and stabilize wt p53 in lymphoma cells but do not interfere with MDM2 E3 ligase activity

Abstract The p53 tumor suppressor protein function is impaired in over 50% of all cancers via inactivating mutations. However, the frequency of p53 mutations is much lower in hematological malignancies. Most lymphomas have wild-type (wt) p53, mutant (mt) p53 is found in less than 30% of all cases. The ubiquitin E3 ligase MDM2 plays a central role in cancer development and progression and is typically overexpressed in lymphomas. MDM2 expression destabilizes functional wt p53 by causing its polyubiquitination and subsequent degradation in the proteasome. Small-molecule inhibitors (SMI) that can block the MDM2-p53 protein-protein interaction and restore p53 function are a promising therapeutic strategy for the treatment of cancers retaining wt p53 that is degraded by overexpression of MDM2. By utilizing two wt p53 and two mt p53 lymphoma cell lines whose p53 status was confirmed by DNA sequencing, we studied the growth inhibitory potential of two different classes of MDM2 SMIs: Nutlin-3, a cis-imidazoline, and MI-219, a new spiro-oxindole MDM2 SMI. Both trypan blue exclusion and MTT assays demonstrated IC50s between 2 and 4 µM in wt p53 cells in vitro. Mutant p53 cells displayed IC50s greater than 10 µM. Growth inhibition was time- and dose-dependent. Additional flow cytometric analyses empolying Annexin V and propidium iodide staining illustrated increasing apoptosis with cells decreasing in G2/M and S phase that also showed time- and dose-dependency. The MDM2 SMI MI-219 showed a much more prominent cell cycle arrest compared to Nutlin-3. Western blotting demonstrated a stabilization of wt p53 and proapoptotic proteins such as p21, Bcl-2, and Bax. In order to determine whether MDM2 SMIs affect the E3 ligase RING-finger domain of MDM2, in vitro autoubiquitination assays were performed using recombinant MDM2 with various concentration of drug. MDM2 autoubiquitination was not affected, even at a concentration as high as 50 µM. These results show that MDM2 autoubiquitination and thus, its degradation remains unaffected in the presence of MDM2 inhibitors that interfere with the p53-MDM2 binding site. This feature of MDM2 SMI is attractive since inhibition of MDM2 autoubiquitination would lead to its stabilization and could negate effects on p53. Our data suggest that MI-219 would be a useful treatment for lymphomas expressing wt p53 and that this should be explored in further preclinical and future clinical studies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4516.

Abstract 3162: MDM2 regulates MYCN mRNA stabilization and translation in human neuroblastoma cells

Abstract The MYCN gene plays a critical role in determining the clinical behavior of neuroblastoma. Genomic amplification occurs in high-risk subsets, but it remains unclear how MYCN expression is regulated in human neuroblastomas. Here, we report that the expression of MYCN is regulated by the oncoprotein MDM2 at the post-transcriptional level. Ectopic overexpression of MDM2 or an increase in the cytoplasmic levels of endogenous MDM2 by inhibition of PI3K/Akt activity enhanced both MYCN mRNA and protein expression. UV cross-linking and RNA-protein binding assays demonstrated that the C-terminal RING domain of MDM2 bound to AU-rich elements within the 3’-untranslated region (3’-UTR) of MYCN mRNA. Furthermore, gene transfection and reporter assay confirmed that MDM2 increased MYCN 3’-UTR-mediated mRNA stability and induced translation of reporter luciferase. Conversely, MDM2 silencing by specific siRNA rendered the MYCN mRNA unstable, reducing the abundant MYCN protein found in MYCN-overexpressing neuroblastoma cell lines, which are a consequence of gene amplification. Importantly, silencing of MDM2 in MYCN-amplified neuroblastoma cell lines resulted in a remarkable inhibition of cell growth and induction of cell death, through a p53-independent pathway. These results indicate that MDM2 plays a p53-independent role in regulating MYCN mRNA stabilization and protein translation, and suggest that MDM2-mediated MYCN expression is an important mechanism for tumor growth and for disease progression, specifically in MYCN-associated neuroblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3162.

The Effects of Phosphomimetic Lid Mutation on the Thermostability of the N-terminal Domain of MDM2

The multidomain E3 ubiquitin ligase MDM2 catalyzes p53 ubiquitination by a “dual-site” docking mechanism whereby MDM2 binding to at least two distinct peptide motifs on p53 promotes ubiquitination. One protein–protein interaction occurs between the N-terminal hydrophobic pocket of MDM2 and the transactivation motif of p53, and the second interaction occurs between the acidic domain of MDM2 and a motif in the DNA-binding domain of p53. A flexible N-terminal pseudo-substrate or “lid” adjacent to the N-terminal hydrophobic pocket of MDM2 has a phosphorylation site, and there are distinct models proposed on how the phosphorylated lid could affect MDM2 function. Biochemical studies have predicted that phosphomimetic mutation will stabilize the lid on the surface of MDM2 and will “open” the hydrophobic pocket and stabilize the MDM2–p53 complex, while NMR studies proposed that phosphomimetic mutation “closes” the lid over the MDM2 pocket and inhibits MDM2–p53 complex formation. To resolve these discrepancies, we utilized a quantitative fluorescence-based dye binding assay to measure the thermal unfolding of wild-type (wt), ΔLid, and S17D N-terminal domains of MDM2 as a function of increasing ligand concentration. Our data reveal that S17D lid mutation increases, rather than decreases, the thermostability of the N-terminal domain of MDM2 in the absence or in the presence of ligand. ΔLid mutation, by contrast, increases MDM2 thermoinstability. This is consistent with biochemical data, using full-length MDM2, showing that the S17D mutation stabilizes the MDM2–p53 complex and increases the specific activity of the E3 ubiquitin ligase function of MDM2. These data indicate that phosphomimetic lid mutation results in an “opening,” rather than a “closing,” of the pocket of MDM2 and highlight the ability of small intrinsically disordered or unstructured peptide motifs to regulate the specific activity of a protein.

Runt-Related Transcription Factor RUNX3 Is a Target of MDM2-Mediated Ubiquitination

The p14(ARF)-MDM2-p53 pathway constitutes an effective mechanism for protecting cells from oncogenic stimuli such as activated Ras and Myc. Importantly, Ras activation induces p14(ARF) and often occurs earlier than p53 inactivation during cancer development. Here, we show that RUNX3, a tumor suppressor in various tumors including stomach, bladder, colon, and lung, is stabilized by Ras activation through the p14(ARF)-MDM2 signaling pathway. RUNX3 directly binds MDM2 through its Runt-related DNA-binding domain. MDM2 blocks RUNX3 transcriptional activity by interacting with RUNX3 through an acidic domain adjacent to the p53-binding domain of MDM2 and ubiquitinates RUNX3 on key lysine residues to mediate nuclear export and proteasomal degradation. Our data indicate that the lineage-specific tumor suppressor RUNX3 and the ubiquitous p53 protein are both principal responders of the p14(ARF)-MDM2 cell surveillance pathway that prevents pathologic consequences of abnormal oncogene activation.

MDM2 Controls the Timely Expression of Cyclin A to Regulate the Cell Cycle

Overexpression of MDM2 has been related to oncogenesis. In this communication, we present evidence to show that MDM2 controls the cell cycle-dependent expression of cyclin A by using a pathway that ensures its timely expression. MDM2 does not inhibit cyclin D or E expression. Silencing of endogenous MDM2 expression elevates cyclin A expression. The p53-binding domain of MDM2 harbors a SWIB region homologous to a conserved domain of a chromosome remodeling factor BRG1-associated protein. The SWIB domain of MDM2 inhibits cyclin A expression in a p53- and BRG1-dependent fashion, suggesting that MDM2 interferes with p53 binding of the BRG1 complex freeing it to repress cyclin A expression. Silencing of cyclin-dependent kinase (cdk) inhibitor p16 prevents MDM2-mediated inhibition of cyclin A expression, implicating its role in the process. MDM2-mediated repression of cyclin A expression induces G(1)-S arrest, which can be rescued by ectopic expression of cyclin A. Cancer cells lacking p53, p16, or BRG1 escape MDM2-mediated repression of cyclin A expression and growth arrest. Our data propose a novel mechanism by which MDM2 controls the cell cycle in normal cells and how cancer cells may escape this important safety barrier.

Regulation of XIAP Translation and Induction by MDM2 following Irradiation

Increases in protein levels of XIAP in cancer cells have been associated with resistance to apoptosis induced by cellular stress. Herein we demonstrate that the upregulation of XIAP protein levels is regulated by MDM2 at the translational level. MDM2 was found to physically interact with the IRES of the XIAP 5'-UTR, and to positively regulate XIAP IRES activity. This XIAP IRES-dependent translation was significantly increased in MDM2-transfected cells where MDM2 accumulated in the cytoplasm. Cellular stress and DNA damage triggered by irradiation induced the dephosphorylation and cytoplasmic localization of MDM2, which also led to an increase in IRES-dependent XIAP translation. Upregulation of XIAP in MDM2-overexpressing cancer cells in response to irradiation resulted in resistance of these cells to radiation-induced apoptosis.

High affinity interaction of the p53 peptide-analogue with human Mdm2 and Mdmx

The Mdm2 and Mdmx proteins are the principal negative regulators of the p53 tumor suppressor. Reactivation of p53 activity by disrupting the Mdm2/Mdmx-p53 interactions offers new possibilities for anticancer therapeutics. Here, we present crystal structures of two complexes, a p53-like mutant peptide with the N-terminal domains of Mdm2 and Mdmx, respectively. The structures reveal that the p53 mutant peptide (amino acid sequence: LTFEHYWAQLTS) assumes virtually identical conformations in both complexes despite the different shapes of the p53-binding pockets in these two proteins, has a more extended helical nature compared to the Mdm2-bound wild-type p53 peptide, and does not disturb the native folds of Mdm2 or Mdmx. The extension of the helical structure in the mutant p53 peptide greatly improves its binding to Mdm2 and Mdmx. The fluorescence polarization assay that we have developed using this peptide indicates the affinities towards Mdm2 of 3.6 nM and for Mdmx of 6.1 nM, compared to the low micromolar binding of a similar length wild-type p53 peptide to Mdm2/Mdmx. Our assay does not require expensive non-native amino acids, and allows measurements of the interaction with both Mdm2 and Mdmx in identical conditions - without modification of experimental conditions or setups between the two proteins. The structural information presented here, coupled with the robust fluorescence polarization assay, should enable development of a simple pharmacophore model of cross-selective Mdm2-Mdmx/p53 inhibitors.

A Function for the RING Finger Domain in the Allosteric Control of MDM2 Conformation and Activity

The MDM2 oncoprotein plays multiple regulatory roles in the control of p53-dependent gene expression. A picture of MDM2 is emerging where structurally discrete but interdependent functional domains are linked through changes in conformation. The domain structure includes: (i) a hydrophobic pocket at the N terminus of MDM2 that is involved in both its transrepressor and E3-ubiqutin ligase functions, (ii) a central acid domain that recognizes a ubiquitination signal in the core DNA binding domain of p53, and (iii) a C-terminal C2H2C4 RING finger domain that is required for E2 enzyme-binding and ATP-dependent molecular chaperone activity. Here we show that the binding affinity of MDM2s hydrophobic pocket can be regulated through the RING finger domain and that increases in pocket affinity are reflected by a gain in MDM2 transrepressor activity. Thus, mutations within the RING domain that affect zinc coordination, but not one that inhibits ATP binding, produce MDM2 proteins that have a higher affinity for the BOX-I transactivation domain of p53 and a reduced I(0.5) for p53 transrepression. An allosteric model for regulation of the hydrophobic pocket is supported by differences in protein conformation and pocket accessibility between wild-type and the RING domain mutant MDM2 proteins. Additionally the data demonstrate that the complex relationship between different domains of MDM2 can impact on the efficacy of anticancer drugs directed toward its hydrophobic pocket.

Epstein-Barr Virus Nuclear Antigen 3C Augments Mdm2-Mediated p53 Ubiquitination and Degradation by Deubiquitinating Mdm2

Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27(KIP) proteins, by recruitment of the SCF(Skp2) E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S proteasomes. Surprisingly, in lymphoblastoid cell lines, EBNA3C is extremely stable, and the mechanism for this stability is unknown. In this report we show that EBNA3C can function as a deubiquitination enzyme capable of deubiquitinating itself in vitro as well as in vivo. Functional mapping using deletion and point mutational analysis showed that both the N- and C-terminal domains of EBNA3C contribute to the deubiquitination activity. We also show that EBNA3C efficiently deubiquitinates Mdm2, an important cellular proto-oncogene, which is known to be overexpressed in several human cancers. The data presented here further demonstrate that the N-terminal domain of EBNA3C can bind to the acidic domain of Mdm2. Additionally, the N-terminal domain of EBNA3C strongly stabilizes Mdm2. Importantly, EBNA3C simultaneously binds to both Mdm2 and p53 and can form a stable ternary complex; however, in the presence of p53 the binding affinity of Mdm2 toward EBNA3C was significantly reduced, suggesting that p53 and Mdm2 might share a common overlapping domain of EBNA3C. We also showed that EBNA3C enhances the intrinsic ubiquitin ligase activity of Mdm2 toward p53, which in turn facilitated p53 ubiquitination and degradation. Thus, manipulation of the oncoprotein Mdm2 by EBNA3C potentially provides a favorable environment for transformation and proliferation of EBV-infected cells.

Role of Mdm2 acid domain interactions in recognition and ubiquitination of the transcription factor IRF-2

Mdm2 (murine double minute 2)-mediated ubiquitination of the p53 tumour suppressor requires interaction of the ligase at two distinct binding sites that form general multiprotein-docking sites for the p53 protein. The first Mdm2-binding site resides in the transactivation domain of p53 and is an allosteric effector site for Mdm2-mediated p53 ubiquitination; the second site requires the acid domain of Mdm2 to recognize a 'ubiquitination signal' within p53's DNA-binding core. In order to expand on fundamental requirements for a protein to function as an Mdm2 substrate and the role of the acid domain in recognition, we have carried out a bioinformatics search for open reading frames that have homology with the Mdm2-docking sites in p53. IRF-2 [IFN (interferon) regulatory factor-2], an IFN-regulated transcription factor, has been identified as an Mdm2-binding protein and substrate requiring interactions with both the hydrophobic pocket and the acid domain of Mdm2. Mutation of either of the two Mdm2-binding sites on IRF-2 can attenuate substrate ubiquitination, confirming the requirement of a dual-site substrate interaction mechanism. Ligands that bind to the hydrophobic pocket are not sufficient to inhibit Mdm2 E3-ligase activity. Rather, acid domain-binding ligands act as E3-ligase inhibitors, lending additional support to the idea that the acid domain of Mdm2 is key to understanding its mechanism of action. The ability of Mdm2 and IRF-2 to form a complex in cells complements the biochemical assays and together establishes a novel substrate with which to develop insights into E3-ubiquitin ligase-substrate interactions in vitro and in cells.

Targeting the Conformational Transitions of MDM2 and MDMX: Insights into Dissimilarities and Similarities of p53 Recognition

MDM2 and MDMX are oncogenic homologue proteins that regulate the activity and stability of p53, a tumor suppressor protein involved in more than 50% of human cancers. While the large body of experiments so far accumulated has validated MDM2 as a therapeutically important target for the development of anticancer drugs, it is only recently that MDMX has also become an attractive target for the treatment of tumor cells expressing wild type p53. The availability of structural information of the N-terminal domain of MDM2 in complex with p53-derived peptides and inhibitors, and the very recent disclosure of the crystal structure of the N-terminal domain of MDMX bound to a p53 peptide, offer an unprecedented opportunity to provide insight into the molecular basis of p53 recognition and the identification of discriminating features affecting the binding of the tumor suppressor protein at MDM2 and MDMX. By using coarse graining simulations, in this study we report the exploration of the conformational transitions featured in the pathway leading from the apo-MDM2 and apo-MDMX states to the p53-bound MDM2 and p53-bound MDMX states, respectively. The results have enabled us to identify a pool of diverse conformational states of the oncogenic proteins that affect the binding of p53 and the presence of conserved and non-conserved interactions along the conformational transition pathway that may be exploited in the design of selective and dual modulators of MDM2 and MDMX activity.

P53 mRNA controls p53 activity by managing Mdm2 functions

The E3 ubiquitin ligase Mdm2 is a focal regulator of p53 tumour suppressor activity. It binds p53, promoting its polyubiquitination and degradation, and also controls p53 synthesis. However, it is not known how this dual function of Mdm2 on p53 synthesis and degradation is achieved. Here we show that the p53 mRNA region encoding the Mdm2-binding site interacts directly with the RING domain of Mdm2. This impairs the E3 ligase activity of Mdm2 and promotes p53 mRNA translation. We also show that introduction of cancer-derived single silent point-mutations in the p53 mRNA weakens its binding to Mdm2 and results in reduced p53 activity. These data are consistent with a mechanism by which changes in silent nucleotides can affect the function of the encoded protein, and indicate that Mdm2-mediated control of p53 synthesis and degradation has evolved in the p53 mRNA sequence and its encoded amino acids.

Mdm2 Promotes Genetic Instability and Transformation Independent of p53

Mdm2, a regulator of the tumor suppressor p53, is frequently overexpressed in human malignancies. Mdm2 also has unresolved, p53-independent functions that contribute to tumorigenesis. Here, we show that increased Mdm2 expression induced chromosome/chromatid breaks and delayed DNA double-strand break repair in cells lacking p53 but not in cells with a mutant form of Nbs1, a component of the Mre11/Rad50/Nbs1 DNA repair complex. A 31-amino-acid region of Mdm2 was necessary for binding to Nbs1. Mutation of conserved amino acids in the Nbs1 binding domain of Mdm2 inhibited Mdm2-Nbs1 association and prevented Mdm2 from delaying phosphorylation of H2AX and ATM-S/TQ sites, repair of DNA breaks, and resolution of DNA damage foci. Similarly, the mutation of eight amino acids in the Mdm2 binding domain of Nbs1 inhibited Mdm2-Nbs1 interaction and blocked the ability of Mdm2 to delay DNA break repair. Both Nbs1 and ATM, but not the ubiquitin ligase activity of Mdm2, were necessary to inhibit DNA break repair. Only Mdm2 with an intact Nbs1 binding domain was able to increase the frequency of chromosome/chromatid breaks and the transformation efficiency of cells lacking p53. Therefore, the interaction of Mdm2 with Nbs1 inhibited DNA break repair, leading to chromosome instability and subsequent transformation that was independent of p53.

Acidic domain is indispensable for MDM2 to negatively regulate the acetylation of p53

MDM2 is the most important negative regulator of tumor suppressor p53. Both RING finger domain and acidic domain of MDM2 contribute to the ubiquitination of p53. The crosstalk between ubiquitination and acetylation of p53 prompts us to examine whether acidic domain is essential for MDM2 to regulate the acetylation of p53. We find that the acidic domain of MDM2 is necessary to inhibit p300-mediated acetylation of p53 as well as to mediate the deacetylation of p53. Our results indicate that acidic domain of MDM2 provides essential information for acetyltransferase p300 and deacetylase HDAC1 and is indispensable for MDM2 to negatively regulate the acetylation of p53.

Aberrant Expression of Nucleostemin Activates p53 and Induces Cell Cycle Arrest via Inhibition of MDM2

The nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis. Both depletion and overexpression of NS reduce cell proliferation. However, the mechanisms underlying this regulation are still unclear. Here, we show that NS regulates p53 activity through the inhibition of MDM2. NS binds to the central acidic domain of MDM2 and inhibits MDM2-mediated p53 ubiquitylation and degradation. Consequently, ectopic overexpression of NS activates p53, induces G(1) cell cycle arrest, and inhibits cell proliferation. Interestingly, the knockdown of NS by small interfering RNA also activates p53 and induces G(1) arrest. These effects require the ribosomal proteins L5 and L11, since the depletion of NS enhanced their interactions with MDM2 and the knockdown of L5 or L11 abrogated the NS depletion-induced p53 activation and cell cycle arrest. These results suggest that a p53-dependent cell cycle checkpoint monitors changes of cellular NS levels via the impediment of MDM2 function.

Transcription factor TAFII250 phosphorylates the acidic domain of Mdm2 through recruitment of protein kinase CK2

Induction and activation of the p53 tumour suppressor protein occurs in response to a number of cellular stresses, including disruption of RNA polymerase II-mediated transcription. Both p53 itself and its principle negative regulator, the E3 ubiquitin ligase Mdm2, are substrates for phosphorylation by the protein kinase CK2 in vitro. CK2 phosphorylates Mdm2 within its central acidic domain, a region that is critical for making a second point of contact with p53 and mediating p53 ubiquitylation and turnover. Additionally, there is evidence that CK2 interacts with, and regulates, both p53 and Mdm2 within the cell but the molecular mechanisms through which CK2-mediated regulation of the p53 response can occur are only poorly understood. Previously, we showed that the basal transcription factor TAFII250, a critical component of TFIID, can interact with Mdm2 and promote the association of the Mdm2 acidic domain with p53. In the present study, we show that immunoprecipitates of TAFII250, either from mammalian cell extracts or from baculovirus-infected cells expressing elevated levels of HA-tagged TAFII250, can phosphorylate Mdm2 in vitro within its acidic domain. We show that CK2 is tightly associated with TAFII250 and is the principal activity responsible for TAFII250-mediated phosphorylation of Mdm2. Our data fit with recent observations that phosphorylation of the acidic domain of Mdm2 stimulates its association with p53 and are consistent with a model in which recruitment of CK2 by TAFII250 may play a contributory role in the maintenance of Mdm2 phosphorylation and function.