The tumor suppressor p53 and the oncoprotein simian virus 40 T antigen bind to overlapping domains on the MDM2 protein.

The oncogene mdm2 has been found to be amplified in human sarcomas, and the gene product binds to the tumor suppressor p53. In this report, we describe the dissection of the MDM2-binding domain on p53 as well as the p53-binding domain on MDM2. We also demonstrate that the oncoprotein simian virus 40 T antigen binds to the product of cellular oncogene mdm2. We have constructed several N- and C-terminal deletion mutants of p53 and MDM2, expressed them in vitro, and assayed their in vitro association capability. The N-terminal boundary of the p53-binding domain on MDM2 is between amino acids 1 and 58, while the C-terminal boundary is between amino acids 221 and 155. T antigen binds to an overlapping domain on the MDM2 protein. On the other hand, the MDM2-binding domain of p53 is defined by amino acids 1 and 159 at the N terminus. At the C terminus, binding is progressively reduced as amino acids 327 to 145 are deleted. We determined the effect of human MDM2 on the transactivation ability of wild-type human p53 in the Saos-2 osteosarcoma cell line, which does not have any endogenous p53. Human MDM2 inhibited the ability of human p53 to transactivate the promoter with p53-binding sites. Thus, human MDM2 protein, like the murine protein, can inactivate the transactivation ability of human p53. Interestingly, both the transactivation domain and the MDM2-binding domain of p53 are situated near the N terminus. We further show that deletion of the N-terminal 58 amino acids of MDM2, which eliminates p53 binding, also abolishes the capability of inactivating p53-mediated transactivation. This finding suggests a correlation of in vitro p53-MDM2 binding with MDM2's ability in vivo to interfere with p53-mediated transactivation.     

RYBP stabilizes p53 by modulating MDM2

The mouse double minute 2 (MDM2)–p53 interaction regulates the activity of p53 and is a potential target for human cancer therapy. Here, we report that RYBP (RING1‐ and YY1‐binding protein), a member of the polycomb group (PcG), interacts with MDM2 and decreases MDM2‐mediated p53 ubiquitination, leading to stabilization of p53 and an increase in p53 activity. RYBP induces cell‐cycle arrest and is involved in the p53 response to DNA damage. Expression of RYBP is decreased in human cancer tissues compared with adjacent normal tissues. These results show that RYBP is a new regulator of the MDM2–p53 loop and that it has tumour suppressor activity.     

Multiple murine double minute gene 2 (MDM2) proteins are induced by ultraviolet light

The mdm2 (murine double minute 2) oncogene encodes several proteins, the largest of which (p90) binds to and inactivates the p53 tumor suppressor protein. Multiple MDM2 proteins have been detected in tumors and in cell lines expressing high levels of mdm2 mRNAs. Here we show that one of these proteins (p76) is expressed, along with p90, in wild-type and p53-null mouse embryo fibroblasts, indicating that it may have an important physiological role in normal cells. Expression of this protein is induced, as is that of p90, by UV light in a p53-dependent manner. The p76 protein is synthesized via translational initiation at AUG codon 50 and thus lacks the N terminus of p90 and does not bind p53. In cells, p90 and p76 can be synthesized from mdm2 mRNAs transcribed from both the P1 (constitutive) and P2 (p53-responsive) promoters. Site-directed mutagenesis reveals that these RNAs give rise to p76 via internal initiation of translation. In addition, mdm2 mRNAs lacking exon 3 give rise to p76 exclusively, and such mRNAs are induced by p53 in response to UV light. These data indicate that p76 may be an important product of the mdm2 gene and a downstream effector of p53.     

Cell cycle regulatory functions of the human oncoprotein MDM2

The protein (MDM2) coded by the mouse double minute-2 (mdm2) gene or its human homologue is well known as an oncoprotein. Malignant human tumors particularly breast tumors and soft tissue sarcomas frequently overexpress MDM2. Artificial amplification of mdm2 gene derived from a transformed murine cell line enhances tumorigenic potential of murine cells. Consistent with its tumorigenic property, mouse or human MDM2 can inactivate several functions of the tumor suppressor p53 and can degrade p53. The protein also interacts with other tumor suppressors, and these interactions may contribute to its tumorigenic property. In spite of its oncogenic role, mouse or human MDM2 induces G(1) arrest in normal human or murine cells. Some cell lines bearing known genetic mutations are insensitive to MDM2-mediated growth arrest. This review is aimed to collect available information on the functions of MDM2 that could potentially regulate cell cycle and to discuss how this information may fit in one model that could explain the two apparently opposite G(1) arrest and oncogenic function of MDM2.     

Polymorphisms of p53 codon 72 and MDM2 promoter 309 and the risk of endometrial cancer

Genetic polymorphisms of p53 and its negative regulator murine double minute 2 homolog (MDM2) have been shown to be closely associated with tumorigenesis in a variety of human cancers. In the present study, single nucleotide polymorphism (SNP) at p53 codon 72 and MDM2 promoter 309 was examined for germline DNA samples from 102 endometrial cancer cases and 95 controls using polymerase chain reaction-based fragment analysis. There were no significant differences in the genotype and allele prevalence between control subjects and endometrial cancer patients for p53 codon 72. The GG genotype frequency of MDM2-SNP309 was statistically higher in endometrial cancer patients than that in normal healthy women when compared with the TG genotype ( P = 0.0088). However, no statistically significant differences were found between the TT and TG or GG genotype frequencies and allele prevalence. Interestingly, the combination of the homozygous Arg/Arg genotype of p53 codon 72 and homozygous GG genotype of MDM2 SNP309 polymorphisms was significantly associated with the risk of endometrial cancer (odds ratio = 3.28, 95% confidence interval = 1.13 to 9.53, P = 0.0212). The homozygous variants of wild p53 codon 72 and mutant MDM2 promoter 309 may cooperatively increase the risk of endometrial cancer in a Japanese population.     

MDM2, an introduction

The murine double minute 2 (mdm2) gene encodes a negative regulator of the p53 tumor suppressor. Amplification of mdm2 or increased expression by unknown mechanisms occurs in many tumors. Thus, increased levels of MDM2 would inactivate the apoptotic and cell cycle arrest functions of p53, as do deletion or mutation of p53, common events in the genesis of many kinds of tumors. MDM2 functions as an E3 ubiquitin ligase to degrade p53. MDM2 also binds another tumor suppressor, ARF. This interaction sequesters MDM2 in the nucleolus away from p53, thus activating p53. Many additional MDM2 interacting proteins have been identified. Functions of MDM2 independent of p53 have also been identified. This article is an introduction to MDM2, its structure and biological functions, as well as its relationship to its binding partners.     

MDM2 mediates p300/CREB-binding protein-associated factor ubiquitination and degradation

We recently reported that MDM2, a negative feedback regulator of the tumor suppressor p53, inhibits p300/CREB-binding protein-associated factor (PCAF)-mediated p53 acetylation. Our further study showed that MDM2 also regulates the stability of PCAF. MDM2 ubiquitinated PCAF in vitro and in cells. PCAF ubiquitination occurred at the N terminus and in the nucleus, as the nuclear localization signal sequence-deletion mutant of MDM2, which localized in the cytoplasm and degraded p53, was unable to degrade nuclear PCAF. Restriction of PCAF in the nucleus by leptomycin B did not affect MDM2-mediated PCAF degradation. Consistently, overexpression of MDM2 in p53 null cells caused the reduction of the protein level of PCAF, but not the mRNA level. Conversely, PCAF levels were higher in MDM2-deficient mouse p53(-/-)/mdm2(-/-) embryonic fibroblast (MEF) cells than that in MDM2-containing MEF cells. Furthermore, MDM2 reduced the half-life of PCAF by 50%. These results demonstrate that MDM2 regulates the stability of PCAF by ubiquitinating and degrading this protein.     

MDM2: life without p53

The MDM2 protein suppresses the ability of p53 to inhibit cellular proliferation or to induce cell death. This property underlies the oncogenic potential of MDM2, which is overexpressed in various human tumours. However, MDM2 also has p53-independent activities, which we focus on here. Similar to other oncogenes, surveillance pathways might counteract the deleterious effects of deregulated MDM2 expression. These pathways need to be inactivated for MDM2 oncogenic activity, which targets p53 but also other proteins.     

Stabilization of the MDM2 oncoprotein by mutant p53

MDM2 is a short-lived protein that regulates p53 degradation. We report here that transient coexpression of MDM2 and several p53 hotspot mutants resulted in stabilization and increased expression of MDM2. Ectopic expression of the mutant p53(175H) allele by recombinant adenovirus infection or stable transfection also stabilized endogenous MDM2 in p53-null cells. A panel of human tumor cell lines expressing different endogenous mutant p53 alleles also contained stabilized nuclear MDM2 at elevated levels when compared with p53-null cells. MDM2 was present in complexes with mutant p53 in tumor cells, and stabilization of MDM2 required direct binding to mutant p53. These results reveal a novel property of mutant p53 and a unique feature of tumors with p53 missense mutations. Accumulation of stable MDM2 may contribute to tumorigenesis through its p53-independent transforming functions.