p53-mdm2--the affair that never ends

Carcinogenesis vol.23 no.4 pp.541–547, 2002

COMMENTARY

p53–Mdm2—the affair that never ends

Dania Alarcon-Vargas and Ze’ev Ronai1

Ruttenberg Cancer Center Mount Sinai School of Medicine,New York, NY 10029, USA

1To whom correspondence should be addressedEmail: [email protected]

The p53–Mdm2 paradigm represents the best-studiedrelationship between a tumor suppressor gene which func-tions as a transcription factor and an oncogene, whichfunctions primarily as an E3 protein ligase. The intimaterelationship between these two partners has expanded toinclude almost every cellular biological system – fromdevelopment, to growth control and programmed cell death.The affair between Mdm2 and p53 is closely controlled bya complex array of post-translational modifications, whichin turn dictates the stability and activity of p53 and Mdm2.Functional diversity depends on the association with alarge subset of partner proteins, which dictates the type ofactivity and corresponding selectivity. Here we summarizethe current understanding of post-translational modifica-tions and their effect on conformation-based functionalrelationship between Mdm2 and p53, as it pertains to theirdiverse cellular biological functions.

p53

The p53 tumor suppressor, is a nuclear phosphoprotein thatfunctions as a DNA damage-inducible sequence specific tran-scription factor, as reviewed in refs1–8. In the absence ofgenetic damage, p53 transcriptional activity is inert (9), andis activated through signalling pathways that induce phos-phorylation of the p53 protein in response to stress (reviewedin refs 10–13). p53 has the typical structural domains of atranscription factor, which are supplemented by a proline-richsignalling domain (aa61–94) and a regulatory domain (aa363–393) (14). Depending on the conditions of cell growth, thetype and duration of stress or DNA damage, p53 selectivelyactivates a different subset of target genes which can causeeither apoptosis, growth arrest, altered DNA repair, or altereddifferentiation (refs 15,16; Figure 1). p53 regulates the G1-Scheckpoint in response to relatively low doses of DNA damage,heat shock, hyperoxia, hypoxia and other forms of stress.Among the multiple targets for the transcriptionally active p53which mediates efficient G1 arrest are cyclin-dependent kinaseinhibitor p21WAF-1, 14-3-3, and reprimo, which enable a suffi-cient pause to allow for the repair of damaged DNA to takeplace (17–19). Under extreme stress and severe DNA damage,p53 triggers the activation of genes implicated in the apoptoticcascade, including bax, DR5, p53AIP, PIDD, NOXA, PUMA,Fas/APO-1 and redox related genes (20–23). The mechanismsthat enable p53 to selectively trigger one of the two distinctpathways are the subject of intense investigations (24). Theability of p53 to exert regulation of such a large subset ofgenes can be attributed to its selective association with different

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transcription factors, each of which results in distinct regulatoryoutput. Post-translational modifications are an important deter-minant in the association and transcriptional output of p53 byaltering its conformation and concomitant association withother transcription factors and regulatory proteins (24–26).

Over 18 phosphoacceptor sites were reported for p53. Mostof them are modified in response to damage or stress (27–41),although, several sites are phosphorylated under normal growthconditions (42–45). It is the complexity of the phosphorylationon multiple sites that appears to dictate the fate and functionof the protein. Phosphorylation of p53 differs during cell cycleprogression in normal growing cells, and coincides with theability of p53 to associate with some of its regulatory proteinsincluding p300, Mdm2, and JNK (34, 46). In vivo labeling ofp53 revealed multiple forms of the protein, indicative of itsdiverse and complex phosphorylation. While phosphorylatedon aa9 during G0, p53 is phosphorylated on residues 9, 15,20, 372 during G1 and on aa37 and 392 during G2/M phase(34). The pattern of phosphorylation is altered significantly inresponse to stress and DNA damage (27–42).

The complex phosphorylation of p53 is mediated by multiplekinases. In many cases, more than one kinase demonstratedthe ability to phosphorylate the same phosphoacceptorresidue. Figure 2 summarizes the current knowledge of p53phosphorylation and the corresponding kinases implicated inthis phosphorylation. Since signaling cascades often exhibitactive cross-talk, it is possible to understand the nature ofthe cross reactivity reported to date. Among the better-characterized studies regarding the role of a given kinase inselective phosphorylation and function of p53 are those thatrelied on the use of knockouts, either by constitutive downregulation or, conversely, activation of the respective kinase.Along those lines, ATM and ATR were confirmed as kinasesthat contribute to p53 phosphorylation on serine 15 (27,28), andChk2 is among the kinases that contribute to phosphorylation ofp53 on serine 20 and possibly other sites (32,33). Both siteswere shown to play an important role in the ability of p53 toassociate with Mdm2, and in conferring p53 stability andtranscriptional activities (47). JNK phosphorylation of p53 onThr81 represents the recent addition of a site that appears toplay an important role in p53 stability and transcriptionalactivities (48).

The diverse sites and motifs that are phosphorylated requirethat multiple phosphatases will be engaged in the regulationof p53 phosphorylation. The corresponding phosphatases areyet to be identified and characterized, but are expected to playa key role in the regulation of p53 stability and activities (49).Phosphatases are likely to regulate p53 following stress, asrevealed from dephosphorylation of Ser376 within 30 minafter exposure to ionizing irradiation (40).

The pattern of p53 phosphorylation is altered in humantumors, which exhibit a substantially greater degree of overallphosphorylation, independent of tumor type, or the form (wildtype or mutant p53), which points towards a more general

D.Alarcon-Vargas and Z.Ronai

failure of one or more of the p53 phosphatases (50). Extensivephosphorylation, in contrast to what is seen in untransformedcells, is expected to alter the conformation of p53 and itscorresponding association with other transcription factors,thereby providing a mechanism by which p53 activities canbe altered in human tumors that do not harbor mutant formsof p53 (51). The latter may explain the increasing evidencefor malfunction of p53, despite its wt form in human tumors(52). Of interest to note is that a recently reported NMR-basedanalysis rules out the allosteric model for the regulation ofp53 (53).

In addition to its complex pattern of phosphorylation, p53is acetylated on at least 3 known residues, aa320, 373 and382. p53 acetylation is mediated by pCAF and CBP/p300 inresponse to DNA damage and stress, and is possibly dependenton its phosphorylation. Acetylation has been implicated intranscriptional activities of p53 and its association with mem-bers of the basal transcriptional machinery (54–57). The findingthat DNA damage inducible gene p33ING2 negatively regulatescell proliferation through acetylation of p53 highlights onemechanism that underlies the regulation of p53 acetylation (58).

In addition, p53 was shown to undergo conjugation to asmall ubiquitin like protein named SUMO. Although originallyimplicated in its sub-nuclear localization and possible transcrip-tional activities (59–61), a recent study rules out these functions(62). Furthermore, the relatively low level of sumoylated p53,which is not affected in response to stress or DNA damagesuggests that p53 in its sumoylated form may acquire othercellular functions, yet to be identified. p53 sumoylation isfacilitated by the activity of PIASI, which is implicated as aSumo ligase towards p53 (63).

About 50% of many human tumor types carry a p53

Fig. 1. Major activities implicated for p53 and Mdm2 are illustrated.

Fig. 2. Currently known phospho-acceptor sites on p53 and thecorresponding kinases implicated in their phosphorylation is illustrated.

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mutation. Most of the mutations are localized within the DNAbinding domain, thereby affecting p53 transcriptional activities.Such mutations can partially or completely abrogate the abilityof p53 to elicit transcriptional activities (64–66). As a result,the ability of p53 to elicit growth arrest, apoptosis, or both,is impaired. Some mutations were reported to render p53transcriptionally active towards a new subset of genes, whichresults in gain of function phenotypes (67,68). Characteristicof mutant p53 is its elevated expression due to greater stability,which is attributed to its inability to induce the expression ofits primary regulator, Mdm2 (69).

The growing number of proteins implicated in the regulationof p53 stability can be divided into targeting and protectingmolecules. Among proteins that protect p53 from degradationare HIF1α, WT1, p300, TAFII31, and the viral proteins, SV40,large T antigen, and E1B (70–75). It is the balance betweenstabilizing and degrading molecules that eventually dictatesthe outcome of p53 stability. This balance is likely to beaffected by the conformation of p53, which is modified uponits phosphorylation or mutation, thereby altering the affinityfor association with a select set of proteins (76). Cell cycle,stress, and DNA damage, are among the most well character-ized scenarios known to involve altered p53 phosphorylationand the subsequent change in its association with targeting orstabilizing molecules (Figure 3).

Mdm2

The mdm2 gene is a cellular proto-oncogene that is oftenamplified in ~7% of all human cancers, but is more frequentlyobserved in soft-tissue sarcomas (77–79). Over-expression ofMDM2 protein can also occur by increased transcription orenhanced translation (80). In combination with a p53 mutation,the prognosis is worse than either event alone (81,82). Experi-ments with knock-out mice revealed that deletion of the mdm2gene results in embryonic lethality, which can be rescued bythe deletion of the p53 gene (83,84). Inhibition of cell growthand marked cell death are often seen in the absence of p53regulation by MDM2, further emphasizing the importance ofthe p53–Mdm2 auto-regulatory loop in the control of cellgrowth and death. MDM2 protein regulates the activity of p53protein by blocking its transcriptional activity, exporting ofp53 protein into the cytoplasm, and/or by promoting thedegradation of the p53 protein.

MDM2 possesses activities of a ubiquitin ligase, capable of

Fig. 3. Outlined are proteins implicated in the stabilization (right) versus thedegradation of p53 (left).

The p53–Mdm2 relationship

self-ubiquitination (85,86), as well as targeted ubiquitinationof its substrates, among which is p53 (87–90). The E3 activityof MDM2 is centered within the RING finger domain (86).The balance between self- and targeted-ubiquitination of Mdm2is modulated by its post translational modifications, includingphosphorylation and sumoylation. Upon Sumo conjugation toMdm2, its E3 ligase activities are shifted towards p53, whileits self-ubiquitination is minimized. DNA damage and stressreduce Mdm2 sumoylation, which renders more of Mdm2subject to self-ubiquitination and degradation. Consequently,the degree of p53 ubiquitination decreases and the amount ofp53 readily available for mediating its transcriptional activitiesincreases (91,92). Marking p53 for degradation is only onepart of MDM2’s role in promoting degradation. The MDM2protein contains nuclear localization and nuclear export signalswithin its structure (93), and as a result MDM2 constantlyshuttles between the nucleus and the cytoplasm. The p53protein also contains its own export signals, located within theamino and carboxy terminal regions (94). The Mdm2 protein,through binding p53 protein, shuttles p53 protein out of thenucleus, into the cytoplasm where it is degraded (95). Nuclearexport of p53 independent of Mdm2 was also shown to exist(96). Entry of Mdm2 into the nucleus was recently shownto be dependent on its phosphorylation by the PI3K/AKTkinases (97).

The ability of Mdm2 to associate with, and target, p53degradation highly depends on the phosphorylation status ofp53, as well as on the association of p53 with other cellularproteins. For example, Mdm2 binding can be outcompeted bya member of the basal transcriptional machinery, TAFII31,which associates with p53 in the same region as that utilizedby Mdm2 for binding, within the amino terminal domainof p53 (73). In response to stress and damage, when p53phosphorylation takes place on multiple residues, includingthose spanning the Mdm2 binding sites, Mdm2 no longerassociates with p53 (47,90).

Whereas Mdm2 is a major regulator of p53 stability, otherproteins are implicated in the regulation of p53 stabilityincluding JNK (46), human papilloma virus E6 (98), andCOP9 signalosome complex (45). Of interest to note is thatall non-Mdm2 regulators require the proline rich domain ofp53 for the ability to affect p53 stability. The JNK associationsite (aa97–106) was also found to be important in JAB1 andE6 association (45,99). Phosphorylation of p53 by JNK, which

Fig. 4. Outline of genes that are implicated as effectors (targets) of p53 orMdm2 or both.

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stabilizes p53 and enables it to be transcriptionally active, ismapped to T81, which is found within the proline rich domainrequired for non-Mdm2 based regulation of p53 stability (48).

Mdm2 phosphorylation is an emerging area of developmentsubject to growing complexity (100). To date, the kinasesimplicated in Mdm2 phosphorylation and function are DNA-PK, ATM, AKT, p38, and Cdk (refs 97,101–104; Z.Ranai,unpublished data). The finding that the same kinase canphosphorylate both Mdm2 and p53 further adds to the closelyconnected regulation between the two proteins. Whereas p38was found to phosphorylate p53 at least on two residues,resulting in its greater stability (105,106), phosphorylation ofMdm2 results in reduction of its stability due to its increasedself-ubiquitination, rendering less Mdm2 available for targetingthe degradation of p53. Phosphorylation by ATM causes p53stability due to interference with Mdm2 association (27,28),whereas ATM phosphorylation of Mdm2 affects its nuclearexport (103). AKT phosphorylation of Mdm2 was demon-strated to be required for its nuclear import, an essential stepthat allows Mdm2 access to the cellular compartment in whichit affects p53 activity and stability (97).

In addition to regulation by phosphorylation, various Mdm2forms are products of alternate splicing or caspase cleavage(107,108). Each of these forms renders Mdm2 inactive, eitherdue to loss of the amino terminal domain that is required forassociation with p53, or due to deletion of the RING domain,

Fig. 5. The interplay. p53 in its transcriptionally active form, which occursin response to stress and DNA damage, is capable of activating distincttarget genes that contribute to either apoptosis or growth arrest, asillustrated in each of the two blocks, respectively. Among the targets forp53 is Mdm2, which in turn serve to limit the amount of p53 via itsefficient targeting of p53 degradation under non-stressed and normal growthconditions. p53 could activate PTEN which in turn inactivates AKT therebyinhibiting anti-apoptotic signals that are otherwise elicited from AKT. PKBphosphorylation of Mdm2 is required for Mdm2 entry to the nucleus, whereit can exercise the regulation of p53 stability and activity, thereby resultingin p53 degradation. E2F1, normally regulated by Rb, is capable ofincreasing p14ARF expression levels, similar to the effects of β-catenin.p14ARF in turn inhibits Mdm2 E3 ligase activities, via accelerateddegradation of Mdm2, which require phosphorylation of Mdm2 by p38.Consequently, activation of p14ARF results in the stabilization of p53. Otherfactors that affect Mdm2 stability and activity include Raf/Ras signalingwhich could induce Mdm2 transcription, or p14ARF expression, therebyeliciting opposing signals towards Mdm2 and p53. Arrows colored in bluereflect transcriptional activation of downstream effector; red arrows reflectphosphorylation that acquires stability or activity; orange arrows representdegradation pathways.


which is required for its activity as an E3 ligase. Truncatedforms of Mdm2 efficiently inhibit the activity of full-lengthMdm2, thereby serving to inhibit Mdm2 targeted ubiquitinationand degradation of p53, resulting in elevated levels of p53(109,110). Truncated forms of Mdm2 found in human tumors(111) further support the notion that Mdm2 may play a rolein tumorigenesis, independent of p53. Along these lines, onewould expect that Mdm2 would regulate proteins independentof p53. Indeed, recent studies highlight proteins that areaffected by Mdm2, including Numb (112), MTBP (113) andβ2-adrenergic receptor and its regulatory protein β-arrestin(114). A novel pathway for Mdm2 targeted ubiquitination wasrecently demonstrated through its effects on glucocorticoidreceptors (GR), where the ability of Mdm2 to alter GR stabilitywas found to be p53-dependent (115). One would expectadditional proteins to be targets for Mdm2 via p53, a pathwaythat offers a novel layer in the regulation of p53-associatedproteins and the function of p53 as well.

The interplay between p53 and Mdm2

A growing number of proteins are implicated in the regulationof both p53 and Mdm2 (Figure 4). These include p300, E2F1,and at least 3 kinases, p38, CK1 and ATM (116–119). Ofinterest is the reciprocal effect of ATM and p38 on Mdm2versus p53. While phosphorylation of MDM2 inhibits itsactivities due to increased self-ubiquitination and altered cellu-lar localization, both kinases increase p53 stability and limitthe association of p53 with Mdm2. Figure 4 outlines the genesthat are common as well as those that are distinct p53 andMdm2 effectors. Under normal growth conditions, Mdm2effectively targets, in concert with JNK and COP9, the degrada-tion of p53. PKB signaling appears to increase Mdm2 targetingof p53 via alteration of Mdm2 localization and E3 ligaseactivities (97). Important components that affect this auto-regulatory feedback loop include the tumor suppressor protein,p14ARF (p19ARF in mice) (120,121). The p14ARF protein has atumor suppressor function that relies on wild type p53 function.The p14ARF protein binds MDM2 protein, and inhibits the E3activity of MDM2 (122), in addition to sequestering MDM2into the nucleolus (123,124). Consequently, p14ARF disruptsthe negative feedback inhibition of p53 by the binding toMDM2 (125,126). Somewhat similar to the effect of p14 isthat of MDMx (Mdm4), a homologue of Mdm2 that associ-ates with Mdm2 and attenuates its E3 ligase activities(Z.Ronai, unpublished data), and is capable of attenuating p53transcriptional activities in vitro (127–130). The importanceof Mdm4 to cellular growth functions and possible tumorigenic-ity is best illustrated in the finding that Mdm4 knock out micefail to develop, and can be rescued by crossing with p53 knockout mice, thereby resembling some of the major charac-teristics of Mdm2 (131).

β-catenin was also shown to affect the Mdm2 p53 regulatorycascade in its ability to increase the amount of transcriptionallyactive p53 (132). This effect appears to be mediated atleast in part through the ability of β-catenin to increase thetranscription of p19ARF which inactivates Mdm2 E3 ligaseactivity, thereby stabilizing the amount of p53 available tomediate its transcriptional output. In as much, the availabilityof wt p53 may serve to attenuate the oncogenic effects ofβ-catenin. Furthermore, the β-catenin–p19-Mdm2-p53 cascadeappears to cooperate with Ras mediated cellular transformation(133). Of interest is that while Ras is capable of inducing

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p19ARF expression (which would support the above pathway),it could also increase the levels of Mdm2, through increasedMdm2 transcription, thereby limiting availability of p53 (134).The mechanism that underlies the balance between the twopathways requires further investigation. Somewhat similar tothe effect of β-catenin is that of E2F1 which is also capableof increasing p19ARF expression, thereby limiting Mdm2 E3ligase activities and enabling the accumulation of p53 (135).Mutations in Rb, or in β-catenin, which are a commonoccurrence in human tumors, would therefore serve to furtherelevate the levels of p19ARF with consequent effects on Mdm2and p53. Similarly, mutant forms of the Ras oncogene or overexpression of c-myc would result in elevated E2F1 expression,thereby enabling an independent pathway for the up-regulationof p19ARF (135). Figure 5 outlines the components known toaffect the complex interplay between p53 and Mdm2.

The affair that never ends—epilogue

The best evidence for the vital need for the regulation of therelationship between p53 and Mdm2 for cell development andproper protection from DNA damage and stress comes fromgenetic studies where the corresponding partners were deleted.Biochemical analysis adds more layers to the complexity ofthis affair, which includes almost every pathway involved inthe control of cell cycle and growth. Charge and conformationappear to be the critical requirements for the regulation ofthe affair between p53 and Mdm2. The p53–Mdm2 relationshipis vital not only for essential functions of the cell, but it alsoappears to be an integrated part of the complex cellularnetwork involving other signaling cascades, including Wnt(via β-catenin), Ras, Rb, myc and more. The greater complexitysupports the importance of this affair, which is a hallmark forits co-existence. This affair is also among the most intensetargets for design and development of therapeutic strategiesfor cancer treatment (136,137).

Acknowledgements

We apologize to the many authors of papers, which were not cited in thiscommentary due to space limitations. We thank members of the Ronai Labfor discussions and Curt Harris and Jim Manfredi for comments. Support byNCI grant CA78419 is gratefully acknowledged.

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Received November 21, 2001; revised and accepted November 21, 2001

p53-mdm2--the affair that never ends

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