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Somatic missense mutations of the CSNK1A1 gene encoding casein kinase 1 alpha (CK1α) occur in a subset of myelodysplastic syndrome (MDS) with del(5q) karyotype. The chromosomal deletion causes CSNK1A1 haplo-insufficiency. CK1α mutations have also been observed in a variety of solid and hematopoietic tumors at low frequency. The functional consequence of CK1α mutation remains unknown. Here we show that tumor-associated CK1α mutations exclusively localize to the substrate-binding cleft. Functional analysis of recurrent mutants E98K and D140A revealed enhanced binding to the p53 inhibitor MDMX, increased ability to stimulate MDMX-p53 binding, and increased suppression of p21 expression. Furthermore, E98K and D140A mutants have reduced ability to promote phosphorylation of β-catenin, resulting in enhanced Wnt signaling. The results suggest that the CK1α mutations observed in tumors cause gain-of-function in cooperating with MDMX and inhibiting p53, and partial loss-of-function in suppressing Wnt signaling. These functional changes may promote expansion of abnormal myeloid progenitors in del(5q) MDS, and in rare cases drive the progression of other tumors.

The tumor-suppressor protein p53 is tightly controlled in normal cells by its two negative regulators--the E3 ubiquitin ligase MDM2 and its homolog MDMX. Under stressed conditions such as DNA damage, p53 escapes MDM2- and MDMX-mediated functional inhibition and degradation, acting to prevent damaged cells from proliferating through induction of cell cycle arrest, DNA repair, senescence or apoptosis. Ample evidence suggests that stress signals induce phosphorylation of MDM2 and MDMX, leading to p53 activation. However, the structural basis of stress-induced p53 activation remains poorly understood because of the paucity of technical means to produce site-specifically phosphorylated MDM2 and MDMX proteins for biochemical and biophysical studies. Herein, we report total chemical synthesis, via native chemical ligation, and functional characterization of (24-108)MDMX and its Tyr99-phosphorylated analog with respect to their ability to interact with a panel of p53-derived peptide ligands and PMI, a p53-mimicking but more potent peptide antagonist of MDMX, using FP and surface plasmon resonance techniques. Phosphorylation of MDMX at Tyr99 weakens peptide binding by approximately two orders of magnitude. Comparative X-ray crystallographic analyses of MDMX and of pTyr99 MDMX in complex with PMI as well as modeling studies reveal that the phosphate group of pTyr99 imposes extensive steric clashes with the C-terminus of PMI or p53 peptide and induces a significant lateral shift of the peptide ligand, contributing to the dramatic decrease in the binding affinity of MDMX for p53. Because DNA damage activates c-Abl tyrosine kinase that phosphorylates MDMX at Tyr99, our findings afford a rare glimpse at the structural level of how stress-induced MDMX phosphorylation dislodges p53 from the inhibitory complex and activates it in response to DNA damage.

Inactivation of the retinoblastoma protein (RB) has a major role in the development of human malignancies. We have previously shown that MDM2, an ubiquitin E3 ligase and major negative regulator of p53, binds to and promotes proteasome-mediated degradation of RB. MDMX, a homolog of MDM2, also binds to and inhibits p53 transactivation activity, yet it does not possess intrinsic ubiquitin ligase activity. Here, we show that MDMX binds to and promotes RB degradation in an MDM2-dependent manner. Specifically, the MDMX C-terminal ring domain binds to the RB C-pocket and enhances MDM2-RB interaction. Silencing MDMX induces RB accumulation, cell cycle arrest and senescence-like phenotypes, which are reverted by simultaneous RB knockdown. Furthermore, MDMX ablation leads to significant retardation of xenograft tumor growth, concomitant with RB accumulation. These results demonstrate that MDMX exerts oncogenic activity via suppression of RB, and suggest that both MDM2 and MDMX could be chemotherapeutic targets.

The oncogene Mdmx is overexpressed in many human malignancies, and together with Mdm2, negatively regulates the p53 tumor suppressor. However, a p53-independent function of Mdmx that impacts genome stability has been described, but this function is not well understood. In the present study, we determined that of the thirteen different cancer types evaluated, 6–90% of those that had elevated levels of Mdmx had concurrent inactivation (mutated or deleted) of p53. We show elevated levels of Mdmx inhibited double-strand DNA break repair and induced chromosome and chromatid breaks independent of p53, leading to genome instability. Mdmx impaired early DNA damage response signaling, such as phosphorylation of the serine/threonine-glutamine motif, mediated by the ATM kinase. Moreover, we identified Mdmx associated with Nbs1 of the Mre11-Rad50-Nbs1 (MRN) DNA repair complex, and this association increased upon DNA damage and was detected at chromatin. Elevated Mdmx levels also increased cellular transformation in a p53-independent manner. Unexpectedly, all Mdmx-mediated phenotypes also occurred in cells lacking Mdm2 and were independent of the Mdm2-binding domain (RING) of Mdmx. Therefore, Mdmx-mediated inhibition of the DNA damage response resulted in delayed DNA repair and increased genome instability and transformation independent of p53 and Mdm2. Our results reveal a novel p53- and Mdm2-independent oncogenic function of Mdmx that provides new insight into the many cancers that overexpress Mdmx.

Mdm2 is a critical negative regulator of the tumor suppressor protein p53. Mdm2 is an E3 ligase whose overexpression leads to functional inactivation of p53. Mdm2 protein stability is regulated by several mechanisms including RING (Really Interesting New Gene) domain-mediated autoubiquitination. Here we report biochemical identification of NEDD4-1 as an E3 ligase for Mdm2 that contributes to the regulation of Mdm2 protein stability in cells. NEDD4-1 was identified from Jurkat cytosolic fractions using an enzyme-dead Mdm2 mutant protein as a substrate for in vitro E3 ligase assays. We show that lysates from Nedd4-1 knockout (KO) mouse embryonic fibroblasts (MEFs) have significantly diminished E3 ligase activity toward Mdm2 compared with lysates from wild-type (WT) MEFs. Recombinant NEDD4-1 promotes Mdm2 ubiquitination in vitro in a concentration- and time-dependent manner. In cells, NEDD4-1 physically interacts with Mdm2 via the RING domain of Mdm2. Overexpression of NEDD4-1, but not an enzyme-dead NEDD4-1CS mutant, increases ubiquitination of Mdm2. NEDD4-1 catalyzes the formation of K63-type polyubiquitin chains on Mdm2 that are distinct from K48-type polyubiquitination chains mediated by the Mdm2/MdmX complex. Importantly, K63-type polyubiquitination by NEDD4-1 competes with K48-type polyubiquitination on Mdm2 in cells. As a result, NEDD4-1-mediated ubiquitination stabilizes Mdm2. NEDD4-1 knockdown reduces the t1/2 (half-life) of endogenous Mdm2 from 20 to 12 min in U2OS cells. Nedd4-1 KO MEFs manifest increased p53 levels and activity, a more robust DNA damage response and increased G1 arrest compared with WT MEFs. Similarly, NEDD4-1 knockdown in WT-p53-bearing cells increases basal p53 levels and activity in an Mdm2-dependent manner, causes stronger p53 responses to DNA damage and results in p53-dependent growth inhibition compared with corresponding NEDD4-1-proficient control cells. This study identifies NEDD4-1 as a novel component of the p53/Mdm2 regulatory feedback loop that controls p53 activity during stress responses.

Over two decades of MDM2 research has resulted in the accumulation of a wealth of knowledge of many aspects of MDM2 regulation and function, particularly with respect to its most prominent target, p53. For example, recent knock-in mouse studies have shown that MDM2 heterooligomer formation with its homolog, MDMX, is necessary and sufficient in utero to suppress p53 but is dispensable during adulthood. However, despite crucial advances such as these, several aspects regarding basic in vivo functions of MDM2 remain unknown. In one such example, although abundant evidence suggests that MDM2 forms homooligomers and heterooligomers with MDMX, the function and regulation of these homo- and heterooligomers in vivo remain incompletely understood. In this review, we discuss the current state of our knowledge of MDM2 oligomerization as well as current efforts to target the MDM2 oligomer as a broad therapeutic option for cancer treatment.

There is an increasing interest in determining the role of ribosomal proteins (RPs) in the regulation of MDM2-p53 pathway in coordinating cellular response to stress. Herein, we report a novel regulatory role of ribosomal protein S25 (RPS25) in MDM2-mediated p53 degradation and a feedback regulation of S25 by p53. We demonstrated that S25 interacted with MDM2 and inhibited its E3 ligase activity, resulting in the reduction of MDM2-mediated p53 ubiquitination and the stabilization and activation of p53. S25, MDM2 and p53 formed a ternary complex following ribosomal stress. The nucleolar localization and MDM2-binding domains of S25 were critical for its role in MDM2-mediated p53 regulation. Knockdown of S25 by siRNA attenuated the induction and activation of p53 following ribosomal stress. S25 stabilized and cooperated with MDMX to regulate MDM2 E3 ligase activity. Furthermore, S25 was identified to be a transcriptional target of p53; p53 directly bound to S25 promoter and suppressed S25 expression. Our results suggest that there is a S25-MDM2-p53 regulatory feedback loop, which may have an important role in cancer development and progression.

Mdm2 is the major negative regulator of p53 tumor-suppressor activity. This oncoprotein is overexpressed in many human tumors that retain the wild-type p53 allele. As such, targeted inhibition of Mdm2 is being considered as a therapeutic anticancer strategy. The N-terminal hydrophobic pocket of Mdm2 binds to p53 and thereby inhibits the transcription of p53 target genes. Additionally, the C-terminus of Mdm2 contains a RING domain with intrinsic ubiquitin E3 ligase activity. By recruiting E2 ubiquitin-conjugating enzyme(s), Mdm2 acts as a molecular scaffold to facilitate p53 ubiquitination and proteasome-dependent degradation. Mdmx (Mdm4), an Mdm2 homolog, also has a RING domain and hetero-oligomerizes with Mdm2 to stimulate its E3 ligase activity. Recent studies have shown that C-terminal residues adjacent to the RING domain of both Mdm2 and Mdmx contribute to Mdm2 E3 ligase activity. However, the molecular mechanisms mediating this process remain unclear, and the biological consequences of inhibiting Mdm2/Mdmx co-operation or blocking Mdm2 ligase function are relatively unexplored. This study presents biochemical and cell biological data that further elucidate the mechanisms by which Mdm2 and Mdmx co-operate to regulate p53 level and activity. We use chemical and genetic approaches to demonstrate that functional inhibition of Mdm2 ubiquitin ligase activity is insufficient for p53 activation. This unexpected result suggests that concomitant treatment with Mdm2/Mdmx antagonists may be needed to achieve therapeutic benefit.

MDMX and MDM2 are two nonredundant essential regulators of p53 tumor suppressor activity. MDM2 controls p53 expression levels, whereas MDMX is predominantly a negative regulator of p53 trans-activity. The feedback loops between MDM2 and p53 are well studied and involve both negative and positive regulation on transcriptional, translational and post-translational levels but little is known on the regulatory pathways between p53 and MDMX. Here we show that overexpression of p53 suppresses mdmx mRNA translation in vitro and in cell-based assays. The core domain of p53 binds the 5' untranslated region (UTR) of the mdmx mRNA in a zinc-dependent manner that together with a trans-suppression domain located in p53 N-terminus controls MDMX synthesis. This interaction can be visualized in the nuclear and cytoplasmic compartment. Fusion of the mdmx 5'UTR to the ovalbumin open reading frame leads to suppression of ovalbumin synthesis. Interestingly, the transcription inactive p53 mutant R273H has a different RNA-binding profile compared with the wild-type p53 and differentiates the synthesis of MDMX isoforms. This study describes p53 as a trans-suppressor of the mdmx mRNA and adds a further level to the intricate feedback system that exist between p53 and its key regulatory factors and emphasizes the important role of mRNA translation control in regulating protein expression in the p53 pathway.

MDMX is a heterodimeric partner of MDM2 and a critical regulator of p53. The MDMX level is generally elevated in tumors with wild-type p53 and contributes to p53 inactivation. MDMX degradation is controlled in part by MDM2-mediated ubiquitination. Here, we show that MDMX turnover is highly responsive to changes in MDM2 level in non-transformed cells, but not in tumor cells. We found that loss of alternate reading frame (ARF) expression, which occurs in most tumors with wild-type p53, significantly reduces MDMX sensitivity to MDM2. Restoration of ARF expression in tumor cells enables MDM2 to degrade MDMX in a dose-dependent manner. ARF binds to MDM2 and stimulates a second-site interaction between the central region of MDM2 and MDMX, and thus increases MDMX-MDM2 binding and MDMX ubiquitination. These results reveal an important abnormality in the p53-regulatory pathway as a consequence of ARF deficiency. Loss of ARF during tumor development not only prevents p53 stabilization by proliferative stress but also causes accumulation of MDMX that compromises p53 activity. This phenomenon may reduce the clinical efficacy of MDM2-specific inhibitors by preventing MDMX downregulation.

The transcriptional activity of the tumour suppressor, p53, requires direct binding between its transactivation domain (TAD, 1-57) and the transcriptional coactivator, p300. We systematically assessed the role of TAD phosphorylation on binding of the p300 domains CH3, Taz1, Kix and IBiD. Thr18 phosphorylation increased the affinity up to sevenfold for CH3 and Taz1, with smaller increases from phosphorylation of Ser20, Ser15, Ser37, Ser33, Ser46 and Thr55. Binding of Kix and IBiD was less sensitive to phosphorylation. Strikingly, hepta-phosphorylation of all Ser and Thr residues increased binding 40- and 80-fold with CH3 and Taz1, respectively, but not with Kix or IBiD. Substitution of all phospho-sites with aspartates partially mimicked the effects of hepta-phosphorylation. Mdm2, the main negative regulator of p53, competes with p300 for binding to TAD. Binding of Mdm2 to TAD was reduced significantly only on phosphorylation of Thr18 (sevenfold) or by hepta-phosphorylation (24-fold). The relative affinities of Mdm2 and p300 for p53 TAD can thus be changed by up to three orders of magnitude by phosphorylation. Accordingly, phosphorylation of Thr18 and hepta-phosphorylation dramatically shifts the balance towards favouring the binding of p300 with p53, and is thus likely to be an important factor in its regulation.

Previous studies have suggested that the mdmX gene is constitutively transcribed, and that MdmX protein activity is instead controlled by cellular localization and DNA damage induced Mdm2-mediated ubiquitination leading to proteasomal degradation. In these studies, we report that the human mdmX (hdmX) mRNA is reproducibly decreased in various human cell lines following treatment with various DNA-damaging agents. Repression of hdmX transcripts is observed in DNA-damaged HCT116 colon cancer cells and in isogenic p53(-/-) cells, suggesting that this effect is p53-independent. Reduction in the amount of hdmX transcript occurs in both human tumor cell lines and primary human diploid fibroblasts, and results in a significant reduction of HdmX protein. Examination of hdmX promoter activity suggests that damage-induced repression of hdmX mRNA is not significantly impacted by transcription initiation. In contrast, changes in hdmX mRNA splicing appear to partly explain the reduction in full-length hdmX mRNA levels in tumor cell lines with the destabilization of full-length hdmX transcripts, potentially through microRNA miR-34a regulation, also impacting transcript levels. Taken together, this study uncovers previously unrecognized cellular mechanisms by which hdmX mRNA levels are kept low following genotoxic stress.

Mdm2 overexpression confers a growth promoting activity upon cells primarily by downregulating the p53 tumor suppressor protein. Nevertheless, Mdm2 deregulation has also been implicated in inhibiting TGF-beta growth repression in a p53 independent manner. Our goal in this study was to examine whether overexpression of Mdm2 or MdmX, a Mdm2-related protein, could affect Smad-induced transactivation. As downstream signaling elements of the TGF-beta pathway, Smads represent one potential target for Mdm2 and MdmX. Here we show that MdmX but not Mdm2 is capable of inhibiting Smad induced transactivation. Based on deletion mutant analysis, MdmX inhibition of Smad transactivation was independent of the p53 and Mdm2 interaction domains, yet required amino acid residues 128-444. Using TGF-beta sensitive HepG2 cells, MdmX overexpression was shown to inhibit TGF-beta induced Smad transactivation. Additionally, mouse embryo fibroblasts (MEFs) lacking p53 and MdmX showed enhanced Smad transactivation when compared to MEFs lacking either p53 or p53 and Mdm2. Interestingly, the inhibition of Smad transactivation by MdmX could be reversed by p300, a functional co-activator of Smads and a necessary factor for Mdm2 nuclear export and did not result from altered Smad localization. In vitro studies demonstrate that MdmX binds to p300 as well as Smad3 and Smad4. Taken together, these results suggest that inhibition of Smad-induced transactivation by MdmX occurs by altering Smad interaction with its coactivator p300.

Mdm2 and MDMX are two structurally related p53-binding proteins which show the highest level of sequence similarity in the N-terminal p53-binding domains. Apart from its ability to inhibit p53 mediated transcription, a feature it shares with mdm2, very little is known about the physiological functions of MDMX. It is clearly distinct from mdm2 since its expression appears not to be regulated by p53 and it cannot compensate for lack of mdm2 in early development. We present data on the structural similarity between the p53 binding pockets of mdm2 and MDMX using p53- and phage-selected peptides. From the results we conclude that our recently devised innovative approach to reverse the mdm2-mediated inhibition of p53's transactivation function in vivo would probably target MDMX as well. Strategies for selectively targeting mdm2 and MDMX are suggested and a possible mechanism for regulating the p53-mdm2/MDMX interactions by protein phosphorylation is discussed.

In this study, we show that interfering with the splicing machinery results in activation of the tumour-suppressor p53. The spliceosome was targeted by small interfering RNA-mediated knockdown of proteins associated with different small nuclear ribonucleoprotein complexes and by using the small-molecule splicing modulator TG003. These interventions cause: the accumulation of p53, an increase in p53 transcriptional activity and can result in p53-dependent G(1) cell cycle arrest. Mdm2 and MdmX are two key repressors of p53. We show that a decrease in MdmX protein level contributes to p53 activation in response to targeting the spliceosome. Interfering with the spliceosome also causes an increase in the rate of degradation of Mdm2. Alterations in splicing are linked with tumour development. There are frequently global changes in splicing in cancer. Our study suggests that p53 activation could participate in protection against potential tumour-promoting defects in the spliceosome. A number of known p53-activating agents affect the splicing machinery and this could contribute to their ability to upregulate p53. Preclinical studies indicate that tumours can be more sensitive than normal cells to small-molecule spliceosome inhibitors. Activation of p53 could influence the selective anti-tumour activity of this therapeutic approach.

It has previously been shown that ubiquitin-specific protease 2a (USP2a) is a regulator of the Mdm2/p53 pathway. USP2a binds to Mdm2 and can deubiquitinate Mdm2 without reversing Mdm2-mediated p53 ubiquitination. Overexpression of USP2a causes accumulation of Mdm2 and promotes p53 degradation. We now show that MdmX is also a substrate for USP2a. MdmX associates with USP2a independently of Mdm2. Ectopic expression of wild-type USP2a but not a catalytic mutant prevents Mdm2-mediated degradation of MdmX. This correlates with the ability of wild-type USP2a to deubiquitinate MdmX. siRNA-mediated knockdown of USP2a in NTERA-2 testicular embryonal carcinoma cells and MCF7 breast cancer cells causes destabilization of MdmX and results in a decrease in MdmX protein levels, showing that endogenous USP2a participates in the regulation of MdmX stability. The therapeutic drug, cisplatin decreases MdmX protein expression. USP2a mRNA and protein levels were also reduced after cisplatin exposure. The magnitude and time course of USP2a downregulation suggests that the reduction in USP2a levels could contribute to the decrease in MdmX expression following treatment with cisplatin. Knockdown of USP2a increases the sensitivity of NTERA-2 cells to cisplatin, raising the possibility that suppression of USP2a in combination with cisplatin may be an approach for cancer therapy.

Persistent inhibition of telomerase induces a severe telomere shortening in human T-cell leukemia virus type-1-infected cells which signals a DNA double-strand break damage response, formation of telomere dysfunction-induced foci and activates the ATM pathway. In turn, activation of ATM and its downstream effectors led to an increased phosphorylation and acetylation on specific residues of p53 known to be involved in transcriptional activation. Disruption of Mdm2-p53 complexes coupled with increased proteasomal degradation of MDMX further enhanced reactivation of p53 transcription, ultimately leading to senescence of tumor cells. Induction of senescence in these T-cells was associated with an increased expression of p21, p16 and activation of GSK3beta. Our results support the cancer-aging model and demonstrate that the halt of aging in cancer cells can be reversed through reactivation of p53.

The tumour suppressor genes, TP53 and RB1, and four genes involved in their regulation, INK4a, ARF, MDM2 and MDMX, were analysed in a series of 36 post-radiotherapy radiation-induced sarcomas. One-third of the tumours developed in patients carrying a germline mutation of RB1 that predisposed them to retinoblastoma and radiation-induced sarcomas. The genetic inactivation of RB1 and/or TP53 genes was frequently observed in these sarcomas. These inactivations were owing to an interplay between point mutations and losses of large chromosome segments. Radiation-induced somatic mutations were observed in TP53, but not in RB1 or in the four other genes, indicating an early role of TP53 in the radio-sarcomagenesis. RB1 and TP53 genes were biallelically coinactivated in all sarcomas developing in the context of the predisposition, indicating that both genes played a major role in the formation of these sarcomas. In the absence of predisposition, TP53 was biallelically inactivated in one-third of the sarcomas, whereas at least one allele of RB1 was wild type. In both genetic contexts, the TP53 pathway was inactivated by genetic lesions and not by the activation of the ARF/MDM2/MDMX pathway, as recently shown in retinoblastomas. Together, these findings highlight the intricate tissue- and aetiology-specific relationships between TP53 and RB1 pathways in tumorigenesis.

Using various mutants of p53 and mdm2, we demonstrate here that both the DNA binding and transactivation function of p53 are required for ASPP1 and ASPP2 to stimulate the apoptotic functions of p53. Mdm2 and mdmx prevent ASPP1 and ASPP2 from stimulating the apoptotic function of p53 by binding and inhibiting the transcriptional activity of p53. Importantly, mdm2 and mdmx can prevent the stimulatory effects of ASPP1 and ASPP2 without targeting p53 for degradation. These data provide a novel mechanism by which mdm2 and mdmx act as potent inhibitors of p53.

Mdm2 and MdmX function as cellular regulators of the p53 tumor suppressor protein. Intriguingly, the activities of these proteins are interdependent; MdmX stabilizes Mdm2, enabling its activities towards p53, but it also requires Mdm2 for its nuclear localization. Here we demonstrate that via its phosphorylation by CDK2/Cdc2p34, MdmX regulates nuclear export of Mdm2. Cdc2p34 phosphorylates MdmX on Ser 96 in vitro. Mutation within this site (MdmX(S96A)) impairs, whereas phosphomimic substitution (MdmX(S96D)) increases the cytoplasmic localization of MdmX, suggesting that CDK2/Cdc2p34 phosphorylation is required for export of MdmX from the nucleus. Consequently, cells that express MdmX(S96A) retain Mdm2 in their nuclei, suggesting that export of Mdm2 to the cytoplasm is MdmX-dependent. Similarly, treatment of cells with the pharmacological inhibitor of CDK2/Cdc2p34 or with a dominant-negative Cdc2 results in nuclear localization of MdmX and Mdm2 and decreases the level of Mdm2 expression. Since Cdc2p34 is active in nonstressed conditions, our finding provides a novel insight into the signaling cascade involved in the regulation of MdmX localization and for regulation of Mdm2 localization and stability.

Mdm4 (MdmX) is a p53-binding protein that shares structural similarities with Mdm2 and has been proposed to be a negative regulator of p53 function. Like Mdm2, the absence of Mdm4 has recently been found to induce embryonic lethality in mice that is rescued by p53 deletion. Mdm4-null embryos are reduced in size and die at mid-gestation, and Mdm4-deficient embryos and embryonic fibroblasts displayed reduced rates of cell proliferation. The p53-induced, cyclin-dependent kinase inhibitor p21 is strongly upregulated in Mdm4-null embryos and cells. Here, we report that deletion of p21 delays the mid-gestation lethality observed in Mdm4-null mice, suggesting that Mdm4 downregulates p53-mediated suppression of cell growth. Surprisingly, the absence of p21 also uncovers an antiproliferative effect of Mdm4 on cell growth in vitro and in Mdm4-heterozygous mice. These results indicate that p21 is a downstream modifier of Mdm4, and provides genetic evidence that Mdm4 can function to regulate cell growth both positively and negatively.