MDM2 — master regulator of the p53 tumor suppressor protein

Gene 242 (2000) 15–29www.elsevier.com/locate/gene

Review

MDM2 — master regulator of the p53 tumor suppressor protein

Jamil Momand *, Hsiao-Huei Wu 1, Gargi Dasgupta 2California State University at Los Angeles, Department of Chemistry and Biochemistry, 5151 State University Dr., Los Angeles, CA 90032, USA

Received 14 August 1999; received in revised form 12 October 1999; accepted 4 November 1999Received by A.J. van Wijnen

Abstract

MDM2 is an oncogene that mainly functions to modulate p53 tumor suppressor activity. In normal cells the MDM2 proteinbinds to the p53 protein and maintains p53 at low levels by increasing its susceptibility to proteolysis by the 26S proteosome.Immediately after the application of cellular stress, the ability of MDM2 to bind to p53 is blocked or altered in a fashion thatprevents MDM2-mediated degradation. As a result, p53 levels rise, causing cell cycle arrest or apoptosis. In this review, we presentevidence for the existence of three highly conserved regions (CRs) shared by MDM2 proteins and MDMX proteins of differentspecies. These highly conserved regions encompass residues 42–94 (CR1), 301–329 (CR2), and 444–483 (CR3) on human MDM2.These three domains are respectively important for binding p53, for binding the retinoblastoma protein, and for transferringubiquitin to p53. This review discusses the major milestones uncovered in MDM2 research during the past 12 years and potentialuses of this knowledge in the fight against cancer. © 2000 Elsevier Science B.V. All rights reserved.

Keywords: Cell cycle; Checkpoint; E2F1; Gene amplification; Oncogene; p73; Rb; Transactivator; Ubiquitin ligase

1. Introduction laboratory were isolating proteins bound to the p53tumor suppressor protein with the rationale that such

Murine Double Minute Clone 2 (MDM2) was origi- proteins might regulate p53 activity (Momand et al.,nally cloned from purified acentric chromosomes har- 1992). In a cultured transformed rat fibroblast linebored within a spontaneously transformed Balb/c3T3 that overexpresses a temperature-sensitive mutantcell line called 3T3DM (Cahilly-Snyder et al., 1987). p53, a 90 kDa phosphoprotein was observed to co-The rationale for cloning genes from these abnormal immunoprecipitate with p53 at the permissive temper-chromosomes, also known as double minutes, is that ature. The p90 kDa protein turned out to be the productthey often contain amplified genes that contribute to of the MDM2 gene. It was subsequently found thatcellular proliferation and tumorigenesis. MDM2 was the overexpression of the MDM2 gene blockedsecond of two tandem genes cloned together from these p53-mediated transactivation of a reporter gene bearingamplified sequences. When a genomic clone of MDM2 a p53-responsive element. Thus, a potential mechanismwas amplified in rodent cells, it conferred high tumori- of MDM2-mediated oncogenicity was established-inacti-genic potential in nude mice (Fakharzadeh et al., 1991). vation of the p53 tumor suppressor gene. In a thirdThis observation gave the first suggestion that MDM2 laboratory, the human homologue of MDM2 wasis an oncogene. Independently, researchers in another mapped to chromosome 12q13–14 and was shown to

be amplified in approximately 30% of osteosarcomasand soft tissue tumors (Oliner et al., 1992). Therefore,* Corresponding author. Tel.: +1-323-343-2361;within a span of 2 years a new oncogene was discovered,fax: +1-323-343-6490.

E-mail address: [email protected] (J. Momand) a mechanism of its action was proposed, and its involve-1 Present address: Department of Anatomy and Neurobiology, ment in human cancers was established.

University of California at Irvine, 364 MedSurge II, Irvine, CA Since 1992, researchers have uncovered a clearer92697, USA.picture of how MDM2 modulates p53 activity and of2 Present address: Children’s Hospital at Orange County-Cancer

Research, 455 S. Main Street, Orange, CA 92868, USA. the prevalence of MDM2 abnormalities in human can-

0378-1119/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved.PII: S0378-1119 ( 99 ) 00487-4

16 J. Momand et al. / Gene 242 (2000) 15–29

cers. Several reviews have covered MDM2 (Haines, be found in the same species as p53 hints at the prospectthat MDM2 may specifically function to regulate p53.1997; Lane and Hall, 1997; Momand and Zambetti,

1997; Piette et al., 1997; Prives, 1998; Freedman et al.,1999; Juven-Gershon and Oren, 1999); however, the

2.2. MDM2 knockout studiesresearch pace has quickened and some major discoverieshave been made quite recently. The purpose of this

Genetic studies strongly argue that MDM2 plays areview is to point out fundamental milestones reachedmajor role in regulating p53 function. Mouseduring the course of research into this interesting geneMDM2−/− embryos are inviable, dying between theand to present an in-depth analysis of more recenttime of inception and day E6.5 of gestation (Jones et al.,progress in our understanding of MDM2’s varied func-1995; Montes de Oca Luna et al., 1995). Deletion oftions. Finally, we will chart potential areas of futurethe p53 gene rescues MDM2−/− embryos, indicatingresearch that will likely be important in furthering ourthat p53 is lethal in the absence of its negative regulator,understanding of this critical growth-control molecule.MDM2, during mouse development. Although notAt the outset, we wish to apologize to the authors ofrequired for proper mouse development, the p53 genethose studies that could not be cited owing to spaceproduct is active during embryogenesis (Gottlieb et al.,constraints.1997; Komarova et al., 1997) and, apparently, must beproperly controlled by MDM2. To determine if MDM2has p53-independent effects the phenotype of p53−/−MDM2−/− mice was compared to that of p53−/−2. Genetic studiesmice. Mouse p53−/− embryos develop normally butform tumors within 3 months after birth (Donehower2.1. Sequence analysis and species representationet al., 1992). Interestingly, p53−/−MDM2−/− mice exhibit the same phenotype as p53−/− miceTo date, the MDM2 gene has been sequenced inwith regard to tumor formation (Jones et al., 1996).human, hamster, mouse, zebrafish and frog (Fig. 1).There is no significant difference in the rate of tumorBased on sequence similarity, a highly related gene,incidence or tumor type in the two mice. Studies of cellsMDMX, was cloned from human and mouse. Alignmentderived from these mice also failed to distinguish signifi-of the four MDM2 and two MDMX gene sequencescant biological differences due to the presence or absencehighlights three regions of high identity, dubbed CR1,of MDM2 in cell growth and DNA damage responseCR2 and CR3. Previously, these three conserved regionsassays (Jones et al., 1996; McMasters et al., 1996).were identified using fewer MDM2 gene sequencesTogether, the animal studies and cell culture studies(Piette et al., 1997). According to our analysis, theindicate that a major function of MDM2 is to controlpercent identity shared within CR1, CR2 and CR3 is,p53 activity. If MDM2 has p53-independent functionsrespectively, 45%, 37% and 48%, each of which isthere appear to be other genes that compensate for thesesignificantly higher than the overall 16% identity sharedfunctions in mice.amongst these genes. Shown in Fig. 2A is a schematic

diagram of the molecules that bind MDM2 and theMDM2 sequences required for interaction. CR1 (resi-dues 42–94) is responsible for binding p53, p73, E2F1and DP1, all of which are proteins that modulate cell 3. Control of p53 stabilitygrowth. CR2 (residues 301–329) codes for a putativezinc binding domain and partially overlaps a region How does MDM2 control p53 function? Before

answering this question it is necessary to briefly reviewrequired for binding the Retinoblastoma (Rb) tumorsuppressor protein. CR3 (residues 444–483) encodes the the basic functions of p53. An overwhelming amount of

evidence indicates that p53 acts as a checkpoint geneRING finger domain, binds two Zn atoms, and containsa cysteine residue (residue 464) required for ubiquitin ( Kastan et al., 1991; Giaccia and Kastan, 1998). In

response to DNA damage and other types of stress,conjugation of p53. The consensus sequences of CR1,CR2 and CR3 were used to search GenBank and EMBL such as heat shock, hypoxia and hyperoxia, p53 is

responsible for either blocking cell cycle progression ordatabases for other MDM2-like genes. No sequenceswith a high degree of identity were obtained from instigating programmed cell death (apoptosis). In

response to most stressors, the p53 protein level increasesDrosophila melanogaster, Saccharomyces cerevisiae orEscherichia coli genomes. Interestingly, the p53 gene is within 1 to 12 h after treatment (Maltzman and Czyzyk,

1984; Kastan et al., 1991; Lu and Lane, 1993). Theencoded by a wide variety of vertebrate animals andsquid (Soussi and May, 1996), but like MDM2, p53 has increased levels are due to a combination of an increase

in p53 translation rate (Fu et al., 1996) and a decreasenot been identified in D. melanogaster, S. cerevisiae orE. coli. The observation that MDM2 genes appear to in p53 degradation rate (Maltzman and Czyzyk, 1984).

17J. Momand et al. / Gene 242 (2000) 15–29

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Fig. 2. Mapped domains on MDM2 that interact with cellular molecules. (A) Schematic diagram of MDM2 domains that interact with cellularmolecules. References are as follows: Rb (Hsieh et al., 1999); p53 (Chen et al., 1993; Picksley et al., 1994); p73 (Zeng et al., 1999); p19Arf/p14Arf(Pomerantz et al., 1998; Zhang et al., 1998); DP1 (Martin et al., 1995); E2F1 (Martin et al., 1995); MDMX (Tanimura et al., 1999); RNA(Elenbaas et al., 1996); ribosomal L5 (Elenbaas et al., 1996); CBP/p300 (Grossman et al., 1998). (B) Sequence alignment of known and putativeMDM2 binding motifs of proteins that interact with CR1. Residues with black diamonds on top are those residues in p53 that make contact withMDM2, underlined residues represent conserved residues predicted to be required for binding to MDM2.

3.1. p53 is a substrate for MDM2 ubiquitin ligase et al., 1987); (2) cells microinjected with neutralizingantibodies against MDM2 or with peptides that blockactivityMDM2–p53 complex formation have elevated p53 pro-tein levels (Blaydes et al., 1997; Midgley and Lane,MDM2 appears to play a major role in directly

controlling p53 protein degradation rate (Haupt et al., 1997; Blaydes and Wynford-Thomas, 1998; Wasylyket al., 1999); (3) cells treated with antisense-expressing1997a; Honda et al., 1997; Kubbutat et al., 1997;

Midgley and Lane, 1997). MDM2 binds p53 close to constructs targeted to MDM2 transcripts also havehigher levels of p53 protein (Teoh et al., 1997; Chenp53’s N-terminus and acts as a ubiquitin ligase (Honda

et al., 1997). Ubiquitin is a 76 amino acid residue et al., 1998); (4) cells overexpressing recombinantMDM2 from transfected cDNA encoding the MDM2protein that covalently attaches to substrate proteins at

free primary amine groups (the N-terminus or lysine gene have reduced p53 levels (Haupt et al., 1997a;Kubbutat et al., 1997); (5) cells expressing MDM2–residues) (Hershko and Ciechanover, 1998). Ubiquitin

conjugation marks proteins for rapid proteolysis by a encoding cDNAs with mutations that corrupt thep53-binding domain of MDM2 fail to exhibit lower p5326S proteosome that resides in the cytoplasm. Many

proteins, including p53 (Maki et al., 1996), are degraded levels (Haupt et al., 1997a). These data build a compel-ling case in favor of MDM2 as the mediator of p53by the 26S proteosome. In one of the steps leading to

p53 proteolysis, a ubiquitin residue is transferred from degradation.an MDM2 cysteine residue to p53 (Honda et al., 1997).Mutagenesis studies demonstrate that the conserved 3.2. MDM2-mediated nuclear export of p53residue cys464 on MDM2 (see Fig. 1) is necessary forboth transferring ubiquitin to p53 (Honda et al., 1997) Mere complex formation between MDM2 and p53 is

not sufficient to mediate p53 degradation. A series ofand for mediating p53 degradation in cultured cells( Kubbatat et al., 1999). reports suggests a model in which the p53–MDM2

complex must be shuttled from the nucleus to theAside from ubiquitination of p53 by MDM2, fivelines of evidence suggest that MDM2 marks p53 for cytoplasm in order for p53 degradation to occur

(Freedman and Levine, 1998; Roth et al., 1998; Laındegradation: (1) cDNAs encoding p53 N-terminal dele-tion mutations expressed in cultured cells produce p53 et al., 1999; Tao and Levine, 1999a). According to this

model, MDM2 constantly shuttles between the cyto-with increased half-lives (Jenkins et al., 1985; Rovinski


plasm and the nucleus due to its encoded nuclear been several reports showing that p53 has activities asidefrom transactivation, including base-excision repairlocalization sequence (NLS) and nuclear export

sequence (NES) (Roth et al., 1998) (see Figs. 1 and (Ford and Hanawalt, 1997), binding to DNA with smallbulges (Lee et al., 1995), and DNA double-strand2A). Mutations in the NES of MDM2 increase the

stability of p53 and cause the p53 to accumulate in the exonuclease activity (Mummenbrauer et al., 1996).There is even evidence that p53 can mediate apoptosisnucleus (Tao and Levine, 1999a). One of the tools used

to explore this model of controlling p53 level is an in a transactivation-independent manner (Caelles et al.,1994; Haupt et al., 1997b). Thus, there may be situationsantibiotic called Leptomycin B. It binds and inhibits a

crucial nuclear exporting protein known as CRM1. when it is advantageous to remove the p53 transactiva-tion activity and leave other activities in place (seeIncubation of cells with Leptomycin B leads to increased

p53 protein levels inside the nucleus and activates section 7.3).p53-mediated transactivation (Freedman and Levine,1998; Laın et al., 1999). This suggests that CRM1 isalso involved in exporting p53 from the nucleus. Other 5. The p53 negative feedback loopresearchers have found that p53 contains its own NESthat is sufficient for nuclear export in the absence of Early studies of MDM2 showed that p53 overexpres-

sion roughly correlates with MDM2 protein upregula-MDM2 (Stommel et al., 1999). In a model ofMDM2-independent export regulation, retention of p53 tion (Barak and Oren, 1992). On the surface, this

observation seems at odds with studies showing thatin the nucleus occurs through a process in whichp53 binds to itself to form a tetramer, thus masking its MDM2 leads to p53 proteolysis. It is now clear that

MDM2 and p53 are involved in a negative feedbackNES. Export occurs when the p53 tetramer is convertedto a dimer or monomer leading to an unmasking of the loop. In this loop, p53 activates MDM2, which, in turn,

downregulates p53. p53 upregulates MDM2 at the tran-NES. It will be important to determine the conditionsthat dictate whether p53 can export from the nucleus in scription level (Barak et al., 1993; Wu et al., 1993).

Sequence analysis of the MDM2 promoter reveals thean MDM2-dependent or in an MDM2-independentfashion. existence of a p53 DNA-binding consensus sequence

near the 3∞ end of intron 1 ( Wu et al., 1993; Barak et al.,1994). p53 binds the consensus sequence and transacti-vates MDM2. Controlling the feedback loop is a central4. Transactivation blocktheme in p53 activity regulation.

One can envisage two possible biological scenarios inIncreasing the proteolytic susceptibility of p53 is onemechanism by which MDM2 can turn off p53, but there which it might be necessary for MDM2 to negatively

regulate p53 (Fig. 3). In one, stress appears to de-repressis another mechanism as well. The p53 protein bindsdirectly to DNA promoter sequences at a consensus p53 activity by preventing MDM2-mediated downregu-

lation of p53. As shown in Fig. 3A, in this scenario,sequence (El-Deiry et al., 1992) and transactivates avariety of genes to mediate cell cycle arrest and, in some p53-mediated transactivation of MDM2 might, under

normal conditions, constantly maintain p53 at low levels.cell types, to mediate apoptosis (Levine, 1997). Manytransactivation factors have domains rich in acidic The majority of p53 would be marked for proteolysis

by MDM2. Some p53, however, would escapeamino acid residues that appear to be required forincreasing RNA polymerase II-mediated transcription MDM2-mediated degradation to keep the feedback loop

active. The p53 that escapes would be available to(Ma and Ptashne, 1987). Similarly, p53 has a stretch ofacidic amino acid residues near its N-terminus required transactivate the MDM2 gene transcriptionally. Under

these conditions cells would proliferate normally. Whenfor this function (Fields and Jang, 1990; Raycroft et al.,1990). MDM2 directly binds this region of p53 and the cell receives the appropriate stress signal, MDM2

binding to p53 would be blocked or modified. Disruptionshuts down p53-mediated transactivation by forming acomplex with p53 (Chen et al., 1993; Oliner et al., 1993; of MDM2-mediated p53 proteolysis would lead to an

increase in p53 protein that, in turn, would lead toPicksley et al., 1994). There is no need for p53 degrada-tion for this process. upregulation of p53-dependent genes required for cell

cycle arrest ( p21WAF1/CIP1) (El-Deiry et al., 1993; HarperOne may ask why MDM2 would evolve to degradep53 when it can prevent p53 activity by binding to its et al., 1993) and/or activation of apoptosis (BAX )

(Miyashta and Reed, 1995). This scenario can be termedtransactivation domain. A trivial explanation is thatdegradation is simply a second step of a p53 inactivation ‘keeping p53 checkpoint function in check’.

In another scenario, cells expressing active p53 wouldprocess that ultimately rids the cell of unnecessaryinactive p53–MDM2 complexes. An alternative explana- require MDM2 to downregulate p53 to allow cell prolif-

eration to begin (Fig. 3B). This scenario can be termedtion is that the p53–MDM2 complex may not be com-pletely inactivated for all p53 functions. There have ‘relief of p53-checkpoint function’. Cells that have sus-

https://www.researchgate.net/publication/14172140_p53_the_Cellular_Gatekeeper_Review_for_Growth_and_Division?el=1_x_8&enrichId=rgreq-635bf4f9-1edd-4793-9e7e-b7ccd7348302&enrichSource=Y292ZXJQYWdlOzEyNTkzNjIyO0FTOjEwMzYzNDMxMDI3MDk4N0AxNDAxNzE5NzUzODAz



tained damage to its DNA would activate p53, which, modulate complex formation. Oligomerization of p53might also affect binding of MDM2 if oligomerizationin turn, would mediate cell cycle arrest by upregulation

of p21WAF1/CIP1 and other genes. The p53 protein would alters the conformation of the MDM2 binding domainof p53. Finally, evidence suggests that the tumor sup-also transcriptionally activate the MDM2 gene, but the

MDM2 gene product would fail to interact properly pressor protein p19Arf/p14Arf binds MDM2 and inhibitsits ability to regulate p53.with p53. Failure to interact would be due to a post-

translational modification of p53, MDM2 or both pro-teins. After the DNA is repaired, p53 and MDM2 would 6.1. Phosphorylation of p53receive a signal. The signal would result in modificationof the proteins that allows them to form a complex. Phosphorylation of p53 at sites within or near the

p53–MDM2 interaction domain has been shown toOnce the complex is formed, p53 levels would be loweredand transactivation of p53-responsive genes would cease. prevent binding of MDM2. A crystal structure of the

p53-binding domain of MDM2 with a p53 peptide fromThe cells would then resume progression through thecell cycle. the transactivation domain reveals that MDM2 and p53

principally bind each other through hydrophobic inter-When would such a scenario be required? Althoughit has been demonstrated that stress-activated p53 can actions ( Kussie et al., 1996). MDM2 has 14 amino

acids within residues 50–100 that make van der Waalscause permanent cell cycle arrest (Di Leonardo et al.,1994) or apoptosis (Clarke et al., 1993; Lowe et al., contacts to p53. A groove is naturally created within

MDM2 to accommodate a portion of the p53 peptide1993), it has also been shown, in some cell types, thatstress-treated cells can continue to divide after a short encompassing residues 15–29. The p53 peptide forms

an a-helix within this groove. Within the p53 peptideperiod of arrest ( Kastan et al., 1991; Flatt et al., 1998).Cultured tumor cells (or immortal cells) showing tran- are three potential protein phosphorylation sites at

Ser15, Thr18 and Ser20. Phosphorylation of Ser15 inhib-sient arrest have been treated with stress agents. In thesecells it is observed that p53 levels transiently increase its binding of MDM2 to p53 and blocks

MDM2-mediated inhibition of p53-mediated transacti-(Chen et al., 1994; Haupt et al., 1997a). The transientincrease is closely followed by a transient increase in vation (Shieh et al., 1997). Site-directed mutagenesis

studies also indicate that Ser15 is important for optimalMDM2 levels. To formally test the hypothesis thatMDM2 is critical for p53 decrease and resumption of p53-mediated apoptotic activity (Unger et al., 1999a).

Although DNA-dependent kinase (DNA-PK) can phos-cell cycle progression some questions will have to beaddressed. For example, is MDM2 required for the phorylate this particular serine residue in vitro (Shieh

et al., 1997), there appear to be other kinases thatobserved delayed decrease in p53 level after DNAdamage in some cell types? What is the molecular respond to DNA damage and are required for activating

p53 through phosphorylation at this site (Jimenez et al.,mechanism that allows MDM2 to reform a complexwith p53 after DNA is repaired? If the p53–MDM2 1999). Future characterization of the kinases responsible

for phosphorylation of p53 at Ser15 will help us under-complex reforms after DNA repair, which, if any,p53-responsive genes are switched off ? Do situations stand how and when p53–MDM2 complex formation is

modulated during cell stress.arise where MDM2 deactivates p53 prior to completionof DNA repair? There is also evidence that p53 phosphorylation at

Ser20 can control p53–MDM2 complex formation. Analanine (Ala) substitution at Ser20 in p53 leads toincreased p53 proteolysis ( Unger et al., 1999b). This6. Regulation of p53–MDM2 complex formationsame substitution appears to enhance binding of p53 toMDM2 in vivo in comparison with wild-type p53.To date, three post-translational events have been

demonstrated to affect p53–MDM2 complex formation: Interestingly, Ala20 mutant p53 and wild-type p53appear to bind to MDM2 with equal efficiency in vitro.phosphorylation, oligomerization and binding to other

proteins. Phosphorylation of sites within or near the These data can be interpreted to indicate that phosphor-ylation at Ser20 on wild-type p53 in vivo perturbs thisp53–MDM2 interaction domains might be expected to

Fig. 3. MDM2-mediated degradation of p53 may modulate p53 responses to cell stress in two scenarios. Scenario 1: keeping p53 checkpointfunction in check. I. During cellular proliferation p53 mediates MDM2 transcription. The MDM2 protein binds some p53 and enhances p53degradation. A fraction of p53 survives degradation to generate more MDM2. II. When DNA is damaged a signal is elicited that disrupts or altersthe p53–MDM2 complex. p53 degradation is inhibited and high levels of p53 accumulate. The p53 protein is free to activate genes to mediate cellcycle arrest or apoptosis. Scenario 2: relief of p53 checkpoint function. I. The DNA damage signal keeps p53 levels high, maintaining cells in thearrested state. MDM2 levels are also elevated due to p53 activity. However, MDM2 fails to bind to p53. II. Once the DNA is repaired a signaltriggers MDM2 to regain its p53 binding properties. MDM2 binding results in p53 proteolysis and genes activated by p53 are turned off. The cellsthen begin to progress through the cell cycle again.


domain to a greater extent than does an Ala substitution, undoubtedly help us understand the role of MDM2phosphorylation in modulating its activity.and that phosphorylation prevents p53–MDM2 complex

formation.Analysis of the structure of the p53–MDM2 complex 6.3. Oligomerization of p53 and its effect on MDM2

binding( Kussie et al., 1996; Blommers et al., 1997), suggeststhat phosphorylation at Ser15 or Ser20 may not beexpected to directly hinder van der Waals contacts Another type of post-translational event that controls

p53–MDM2 complex formation is p53 oligomerization.between MDM2 and p53. These residues do not appearto make direct contacts with MDM2. One clue as to Several reports have shown that p53 oligomerization is

necessary for efficient binding by MDM2 andhow p53–MDM2 complex formation can be blocked byp53 phosphorylation has come from in vitro p53 confor- MDM2-mediated degradation of p53 (Marston et al.,

1995; Kubbutat et al., 1998; Maki, 1999). p53 forms amation studies. Limited proteolysis of p53 indicates thatp53 phosphorylation at serine residues 15 and 37 alters tetramer naturally in solution (Friedman et al., 1993)

and, once activated, the tetramer form is more efficientits tertiary conformation (Shieh et al., 1997). In theunphosphorylated state, the MDM2 binding domain of than the monomer at binding DNA in a sequence-

dependent fashion (Hupp et al., 1992; Wang et al.,p53 has the propensity to adopt a two b-turn structure,which is very similar to an a-helix (Botuyan et al., 1995). The tetramer is an arrangement of two dimers

that interact along a distinct parallel helix–helix interface1997). This folded structure, inherent within the p53transactivation domain, is expected to increase the prob- spanning residues 320–356 (Clore et al., 1994; Lee et al.,

1994; Jeffrey et al., 1995). Efficient MDM2 binding toability of binding to MDM2 because it should requirevery little perturbation to convert it into an a-helix. One p53 can also be maintained when a foreign dimerization

domain from the transcription factor GCN4 is used tocan speculate that phosphorylation at Ser15 or Ser20may destabilize the two b-turn structure and, thereby, replace the p53 oligomerization domain (Maki, 1999).

Oligomerization of p53 may stabilize the N-terminusmake p53 less susceptible to binding by MDM2.into a conformation that increases its affinity forMDM2. This could account for both the greater levelof binding and the apparent higher rates of p53 ubiquitin6.2. Phosphorylation of MDM2conjugation when the oligomerization domain is present(Marston et al., 1995; Kubbutat et al., 1998; Maki,Phosphorylation of MDM2 might also control p53–

MDM2 complex formation. One report has shown that 1999). Oligomerization of p53 also increases the abilityof p53 to be phosphorylated by DNA-PK at Ser20MDM2 is phosphorylated by DNA-PK at Ser17 (Mayo

et al., 1997), a residue conserved in all MDM2-carrying (Shieh et al., 1999), indicating that other intracellularproteins have evolved to recognize the oligomeric formspecies except zebrafish. Phosphorylation at this site

inhibits p53–MDM2 complex formation in vitro. of p53.Interestingly, an Ala substitution at this site maintainsits capacity to bind p53 in vitro and, in transient 6.4. p19ARF/p14ARFtransfection assays, is a more effective inhibitor ofp53-mediated transactivation than wild-type MDM2. MDM2 can bind molecules that ultimately modulate

the p53-signaling pathway. One of these is p19ArfThis implies that wild-type MDM2 is phosphorylatedin the p53-transactivation measurement assay, whereas ( Kamijo et al., 1997; Chin et al., 1998; Pomerantz et al.,

1998; Zhang et al., 1998; Kurokawa et al., 1999). Thisthe mutant, Ala17MDM2, is not. In a recent study,MDM2 was shown to be phosphorylated in response to protein is unusual because it is translated from a tran-

script synthesized from an alternative reading frame,ionizing radiation ( Khosravi et al., 2000). Ionizingradiation-induced MDM2 phosphorylation requires the hence the abbreviated term ‘Arf ’. The p19Arf transcript

is derived from a common second exon shared by twopresence of the Ataxia Telangiectasia (ATM ) gene, agene frequently mutated in patients with a rare inherited genes encoded by the Ink4a/Arf murine locus, also

known as the CDKN2A gene in humans. When the genedisorder that makes them extremely sensitive to radia-tion. The ATM gene is required for efficient activation encoding the p19Arf transcript is specifically deleted mice

develop tumors early in life ( Kamijo et al., 1997).of p53 in response to ionizing radiation ( Kastan et al.,1992) and its product can phosphorylate p53 at Ser15 Specifically, signals emanating from the cellular onco-

gene Myc or the Adenovirus oncogene E1a require(Siliciano et al., 1997; Banin et al., 1998). Interestingly,the ATM gene product can phosphorylate MDM2 in p19Arf to activate p53 in mouse cells (de Stanchina et al.,

1998; Zindy et al., 1998). The p19Arf protein can bindvitro as well. This implies that ionizing radiation triggersATM to phosphorylate MDM2 and p53, which, in MDM2 within the N-terminal 284 amino acid residue

domain of MDM2, but the carboxyl terminus appearsturn, prevents MDM2 binding to p53. Future studieswith MDM2 phosphopeptide-specific antibodies will necessary to increase the efficiency of binding

https://www.researchgate.net/publication/14377086_The_MDM2_oncoprotein_binds_specifically_to_RNA_though_its_RING_finger_domain?el=1_x_8&enrichId=rgreq-635bf4f9-1edd-4793-9e7e-b7ccd7348302&enrichSource=Y292ZXJQYWdlOzEyNTkzNjIyO0FTOjEwMzYzNDMxMDI3MDk4N0AxNDAxNzE5NzUzODAz


(Pomerantz et al., 1998; Zhang et al., 1998). Binding of p53 in the nucleus. Studies revealing a clearer mechanismby which p14Arf regulates p53 activity will likely beMDM2 to p19Arf in vitro decreases the ubiquitin ligase

activity of MDM2 (Honda and Yasuda, 1999). In mouse brought to light in the near future.Aside from oncogene signaling, another layer offibroblasts, the p19Arf protein, in addition to binding

MDM2, sequesters MDM2 into the nucleolus (Tao and control is imposed on the p14Arf–MDM2–p53 signalingpathway. Interestingly, p53 downregulates p14Arf expres-Levine, 1999b; Weber et al., 1999). Nucleolar localiza-

tion appears necessary for MDM2 inactivation because sion, suggesting a second negative feedback controlmechanism on p53 (Stott et al., 1998). Downregulationp19Arf mutants that fail to accumulate in the nucleolus,

but that still bind MDM2, do not upregulate p53 protein of p14Arf is predicted to increase MDM2 activity andthereby reduce p53 activity. This hypothesis is strength-levels ( Weber et al., 1999). The released p53 protein

resulting from p19Arf overexpression resides in both the ened by the observation that cultured tumor cellsexpressing wild-type p53 do not express p14Arf, whereasnucleus and the cytoplasm, likely due to its rapid

shuttling between these two compartments. This obser- tumor cells with mutations in the p53 gene express highlevels of p14Arf (Stott et al., 1998). The mechanism byvation is consistent with a study showing that p19Arf

forms a complex with MDM2 but not with p53 which this inverse correlation occurs will undoubtedlybe studied in the near future. In sum, p53 appears( Kurokawa et al., 1999). Thus, in mouse cells, p19Arf

binds to MDM2 and sequesters MDM2 in the nucleolus, capable of upregulating its negative inhibitor, MDM2,by two mechanisms: (1) direct transcriptional activationreleasing p53 to transactivate downstream effector genes.

The human homologue of p19Arf, p14Arf acts, in most of MDM2 and (2) downregulation of the inhibitor ofMDM2, p14Arf.respects, like its mouse counterpart (Stott et al., 1998;

Zhang and Xiong, 1999). Like its mouse homologue, Two distinct signaling pathways regulate MDM2activity, both of which end up disrupting proper p53–nucleolar localization of p14Arf appears to be required

for p53 activation (Zhang and Xiong, 1999). Nucleolar MDM2 interactions, and culminate in p53 activation.One pathway is activated by DNA-damaging agents andlocalization of p14Arf appears to be responsible for

blocking p53 nuclear export. Presumably, this occurs by results in phosphorylation of p53 and MDM2. Thesecond pathway is activated by oncogenes and proceedssequestering MDM2 and protecting p53 from

MDM2-mediated degradation. Many mutations from through p14Arf. The details of p53 activation throughMDM2 inhibition may depend on modifying factorshuman tumors within the p14Arf gene prevent p14Arf

nucleolar localization and have a reduced ability to that vary according to species, cell type and whethercells express viral proteins. Tumors that have inactiva-block p53 nuclear export (Zhang and Xiong, 1999).

However, it is unclear if p14Arf nucleolar localization ting mutations in the kinases responsible for disruptingthe p53–MDM2 complex or in p14Arf may benefit fromper se is sufficient for p53 upregulation. This issue might

be clarified by creating a fusion protein with the MDM2 anti-MDM2 therapy.binding domain of p14Arf and a heterologous nucleolarsequence and testing of this chimera protein for p53upregulation. 7. Other molecules that bind MDM2

The fate of MDM2 after p14Arf activation maydepend on the genetic background of the cell. For 7.1. p73example, in human cells expressing viral proteins (HeLacells) p14Arf promotes MDM2 destabilization (Zhang Proteins that bear an MDM2 binding motif similar

to that of p53 are strong candidates for regulation byet al., 1998), whereas in non-viral protein expressingcells p14Arf expression leads to an increase in MDM2 MDM2. Recently, proteins have been discovered that

share significant sequence identity with p53 ( Kaghadlevels (Stott et al., 1998; Zhang and Xiong, 1999).When p14Arf and p53 are overexpressed by cDNA et al., 1997; Osada et al., 1998; Yang et al., 1998). These

proteins, named p73, p63 and p51, encode N-terminalco-transfection in non-viral protein expressing cells,p14Arf is located in the nucleolus and p53 is located in sequences that are very similar to the region of p53 that

interacts with MDM2 (Fig. 2B). One of these p53-likethe nucleus (Zhang and Xiong, 1999). By analogy withp19Arf, p53 nuclear accumulation would occur by proteins, p73, is capable of upregulating p53-responsive

genes ( Kaghad et al., 1997) and mediating apoptosisp14Arf-mediated sequestration of MDM2, leaving p53free in the nucleus. However, there is also evidence to (Jost et al., 1997). MDM2 is able to inhibit p73-mediated

transactivation and apoptosis but does not destabilizesuggest that, instead of releasing p53 from MDM2,p14Arf enters into a ternary complex with p53 and p73 (Dobbelstein et al., 1999; Zeng et al., 1999).

Furthermore, p73 upregulates MDM2 protein levels inMDM2 (Stott et al., 1998; Zhang and Xiong, 1999).Like its mouse homologue, it is possible that p14Arf cultured cells, suggesting a negative feedback loop sim-

ilar, in some respects, to p53 (Zeng et al., 1999). It isforms a complex with some MDM2 alone and sequestersit into the nucleolus to leave a subpopulation of active interesting to consider the biological significance of these


results in light of the fact that p53–MDM2 double 7.3. Retinoblastoma protein (Rb)knockout mice are indistinguishable from p53 knockoutmice. One might expect p73 to be free to induce growth The Rb tumor suppressor protein binds MDM2

within CR2 of MDM2 (Fig. 2A) ( Xiao et al., 1995;arrest or apoptosis in the p53−/−MDM−/− mice. Itis therefore likely that other molecules (perhaps Hsieh et al., 1999). Rb binding blocks MDM2’s ability

to destabilize p53 (Hsieh et al., 1999). However, theMDMX ) can also negatively regulate p73.Rb–MDM2 complex remains bound to p53 as it inhibitsp53-mediated transactivation. Interestingly, Rb-boundMDM2 fails to prevent p53-mediated apoptosis in7.2. E2F1cultured human tumor cells. p53, in this instance, appa-rently mediates apoptosis in a transactivation-indepen-MDM2 appears to bind other molecules that regulate

cell cycle progression. One of these is E2F1 (Martin dent manner. This indicates that Rb binding can allowp53-mediated apoptosis through MDM2 but inhibitset al., 1995), a transcription factor that activates

S-phase-specific genes. E2F1 forms a heterodimer with p53-mediated transactivation. This is a demonstrationof how two MDM2 functions (inhibition of p53 transac-DP1 and together these activate transcription of genes

through a specific DNA sequence called E2F. Whereas tivation and destabilization of p53) can be dissociated.As part of a critical step in Rb-mediated tumor suppres-MDM2 inhibits the activation of p53 and p73, MDM2

stimulates E2F1/DP1-dependent activation of the E2F sor activity, Rb binds and inhibits E2F1 (Qin et al.,1992). The domain of Rb that binds E2F1, called thepromoter (Martin et al., 1995). MDM2 stimulation

occurs independently of p53 and, in some cancer cells, pocket domain, also binds MDM2 ( Xiao et al., 1995).Thus, MDM2 can drive cells into S-phase by two distinctendogenous MDM2 is observed to form a complex with

E2F1 (Teoh et al., 1997). Based on sequence compari- mechanisms; one is by direct interaction with E2F1 andactivation of E2F1/DP1-mediated transactivation. Thesons, p53, p73, p63, p51 and E2F1 share a common

consensus sequence for MDM2 binding centered on a second mechanism is through inhibition of Rb bindingto E2F1 by competition. This suggests that MDM2 maylarge hydrophobic residue such as tryptophan (Trp) or

phenylalanine (Phe) (Fig. 2B). Three residues upstream augment E2F1-mediated upregulation of S-phase genes.This correlates quite nicely with earlier work showingof the Trp/Phe residue is another hydrophobic residue

[either phenylalanine (Phe) or valine (Val )] shared that MDM2 protein levels are upregulated after stimula-tion of quiescent cells (Olson et al., 1993; Mosner andwithin these MDM2-binding domains. Located three

residues downstream of the Trp/Phe residue is a leucine Deppert, 1994). It was previously shown that E2F1 andp53 cooperate to mediate apoptosis ( Wu and Levine,(Leu). Finally, a proline (Pro) is located within one to

three residues downstream of Leu. Within this 1994). Thus, if MDM2 can activate E2F1, and, at thesame time, be prevented from degrading p53, it isMDM2-binding consensus sequence are three residues

that make van der Waals contacts between p53 and possible that MDM2 could, in fact, be the ultimateregulator of p53-mediated apoptosis.MDM2 ( Kussie et al., 1996). This consensus sequence

can be written as Trp/Phe–X–X–Trp/Phe–X–X–Leu– It should be noted that MDM2 can also increase thepercentage of cells in S-phase independently of E2F1.Pro/X–Pro/X–Pro/X. It is likely that other proteins will

be discovered bearing this consensus sequence. It will When MDM2 is overexpressed in murine mammarygland tissue in a transgenic mouse model, a higher thanbe important to determine whether the activities of such

proteins will be regulated by MDM2. normal percentage of mammary cells reside in S-phase(Lundgren et al., 1997). At the same time, the totalBecause of MDM2’s ability to increase the proteolytic

sensitivity of p53, investigators have attempted to deter- number of surviving mammary cells in these mice is lessthan in normal mice and they are often polyploid. Thismine whether E2F1 protein levels are modulated by

MDM2. Overexpression of MDM2 by transfection is phenotype does not depend on p53 or E2F1, indicatingthat MDM2 mediates these effects through other mole-not able to alter the expression of cotransfected E2F1

( Kubbatat et al., 1999). Interestingly, inhibition of cules (Lundgren et al., 1997; Reinke et al., 1999).endogenous MDM2 by antisense or antibody microin-jection in cultured cells does elicit E2F1 protein upregu- 7.4. CREB-binding protein and p300lation (Blattner et al., 1999), suggesting that endogenousE2F1 may be sensitive to MDM2 protein. Upregulation CREB binding protein (CBP) and p300 are transcrip-

tional co-activators that bind and enhance p53-mediatedof E2F1 appears to require wild-type p53 and theincreased E2F1 protein expression does not correlate transactivation (Avantaggiati et al., 1997; Gu et al.,

1997; Lill et al., 1997). These two proteins share greaterwith increased E2F transcription activity. The mecha-nism of E2F1 upregulation by MDM2 inhibition and than 63% identity overall and act identically in many

biological assays. CBP/p300 binds p53 at the p53 trans-the purpose of this upregulation will likely be a fruitfularea of future research. activation domain. Therefore, CBP/p300 may enhance


p53-mediated transactivation by competing MDM2 logic for going forward with this approach is that anti-MDM2 therapy might not only re-establish p53 activityaway from the p53 transactivation domain. Indeed,

using a mammalian two-hybrid assay MDM2 was shown in tumors with amplified MDM2 genes, but it mightalso re-establish p53 activity in wild-type p53-expressingto inhibit p53 binding to CBP/p300 ( Wadgaonkar and

Collins, 1999). Another study has shown that CBP/p300 tumors with normal levels of MDM2. For example,anti-MDM2 therapy might prove useful in cancersalso binds MDM2 and appears to sequester most of the

MDM2 under normal circumstances (Grossman et al., where p14Arf activity is abrogated. Anti-MDM2 therapymight also be useful in p53-mutant cells if it can block1998). Interestingly, CBP/p300 appears to be required

for MDM2-mediated p53 degradation (Grossman et al., the p53-independent growth-promoting activities ofE2F1. At the moment, tools for knocking out MDM21998). MDM2 protein that lacks its p300-binding

domain loses its ability to destabilize p53 protein even activity in human cancer cells are in a developmentalstage. One approach is to use antisense transcripts tothough binding between MDM2 and p53 remains

unaffected. The site on p53 necessary for destabilize MDM2 transcripts (Teoh et al., 1997; Chenet al., 1998). Using this approach, investigators haveCBP/p300-enhanced p53 destabilization is different from

the site on p53 necessary for CBP/p300-mediated demonstrated that MDM2 protein levels can be reducedand, depending on cell type, MDM2 protein level reduc-co-activation. The CBP/p300 destabilization site on p53

was determined to be within residues 90–160 (called the tion correlates with cell cycle arrest or increased sensitiv-ity to pro-apoptotic factors. A second approach is tocore domain), located downstream of the activation

domain. A p53 protein with a point mutation was use peptide domains that interfere with MDM2-bindingto p53 (Blaydes et al., 1997; Bottger et al., 1997; Wasylykcreated within the core domain and found to be unable

to bind p300. This same mutant p53 protein was found et al., 1999). It is likely, that, in the near future, smallmolecules will be designed to block MDM2 interactionsto be resistant to MDM2-mediated degradation. These

results pose the question: how can CBP/p300 increase with p53. In the beginning, cancers that stand to benefitfrom this therapy may be those that have a highp53 transactivation activity and also be responsible for

MDM2-mediated degradation of p53? It is possible that, percentage of cases where p53 is wild-type, such asleukemia, neuroblastomas and breast cancer.under normal conditions, during cellular proliferation,

the CBP/p300–MDM2 complex forms a transient com- The discovery of MDM2 and the myriad of ways inwhich this protein modulates p53 activity will likelyplex with p53, and degrades p53. Under conditions of

cell stress, MDM2 might lose its ability to bind p53 but establish a precedent for other negative feedback loops.This is especially true in light of the fact that p53the ability of CBP/p300 to bind p53 might remain

unaffected. This scenario would suggest that the level of homologues, such as p73, p63 and p51, are likely beregulated similarly. The hope, in the future, is to harnessCBP/p300 is sufficiently high to sequester MDM2 as

well as independently bind p53. Further investigations this knowledge to re-establish growth control inneoplasias.are required to determine the role of CPB/p300 in

MDM2 regulation of p53.

Acknowledgements8. MDM2 in human tumors

The authors gratefully acknowledge the support ofWhen MDM2 gene amplifications were first detectedthe University of California Breast Cancer Researchin soft tissue tumors and osteosarcomas many investiga-Program (1KB-0102) and critical reading of the manu-tors analyzed other tumors and malignancies for MDM2script by Dr Susan Kane and Ms. Saori Furuta. Thegene amplification and MDM2 protein overexpression.authors also thank Dr Yosef Shiloh and Dr Moshe OrenTo date, the overall gene amplification frequency isfor sharing data prior to publication.known to be 7%, with the highest frequency of amplifi-

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MDM2 — master regulator of the p53 tumor suppressor protein

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