The p53 pathway

J. Pathol. 187: 112–126 (1999)

THE P53 PATHWAY

1 . 2*1Department of Biological Sciences, Columbia University, New York, 10027 U.S.A.

2Department of Cellular and Molecular Pathology, University of Dundee, Ninewells Hospital and Medical School,Dundee DD1 9SY, Scotland, U.K.

SUMMARY

Abnormalities of the p53 tumour suppressor gene are among the most frequent molecular events in human and animal neoplasia.Moreover, p53 is one of the most studied proteins in the whole of contemporary biology, with more than 12 500 papers so far written!In this review the choice has been deliberately made not to be fully comprehensive in the coverage of the huge p53 literature. Ratherattention is focused on a small number of recent developments which are reviewed in the context of modern models of p53 function.Progress in the analysis of signalling to p53 including phosphorylation cascades, and interactions with proteins such as mdm2 and ARFare highlighted. The plethora of protein–protein interactions is discussed, as are the strategies for defining downstream targets of p53.Finally, the emerging biology of p53 homologues is considered. The need for bridging the gap between reductionist, biochemical andbiophysical studies and biological and genetic analysis is emphasized. Only this will provide the needed framework for utilizing theinformation in clinical care. Copyright ? 1999 John Wiley & Sons, Ltd.

KEY WORDS—p53; homologue; phosphorylation; p19ARF; mdm2; protein–protein interaction; transcriptional regulation

*Correspondence to: P. A. Hall, Department of Cellular andMolecular Pathology, University of Dundee, Ninewells Hospitaland Medical School, Dundee DD1 9SY, Scotland, U.K. E-mail:

AN OVERVIEW OF p53 STRUCTURE ANDFUNCTION

A large body of evidence places the transcriptionfactor p53 at a critical nodal point of converging path-ways from diverse cellular insults which can elicit co-ordinated cellular responses that result in adaptation tothe insult.1 The mass of literature that has accumulatedover the past two decades has been well reviewed byothers2–4 and an invaluable historical perspective hasbeen provided by Harris.5 Many important data havecome from the analysis of clinical material and thisindicates that abnormalities of p53 are among the mostprevalent molecular abnormalities in human (and otheranimal) cancer,6,7 with missense point mutations in thesequence-specific DNA binding domain and allelic lossat the p53 locus on chromosome 17 being particularlyprevalent. In addition, p53 is a frequent target forinactivation by virally encoded proteins such as SV40large T antigen, the HPV E6 ORF, hepatitis B Xantigen, and adenovirus E1a. The potential clinicalutility of p53 has attracted considerable attention andhas been reviewed elsewhere.8–11 Hence, the plethoraof information concerning p53 and its role in neoplasiahas been well documented (see Table I) and it is notthe purpose of this review to attempt to reprise this.Consequently, we feel that it is more useful to providea concise overview of accepted ‘facts’ concerning thebiology of p53 and its role in neoplasia, emphasizing ourcurrent view of the physiological role of the p53 path-way. Onto this we will then build a series of shortperspectives on important recent advances in the field.Inevitably we cannot be comprehensive, but rather wehave sought to consider topical areas of interest.

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CCC 0022–3417/99/010112–15$17.50Copyright ? 1999 John Wiley & Sons, Ltd.

Our current view of the normal function of p53 is thatit is a transcription factor capable of regulating theexpression of a range of downstream genes (see below).As with most other transcription factors, p53 is amodular protein with several regions with distinct, butinter-dependent, functions.12,13 These functions arecoordinated such that p53 protein within a cell acts tointegrate signals emanating from a wide range of cellularstresses and allows the cell to respond to these insults byactivating a set of genes whose products facilitate adap-tive and protective activities, which include apoptosisand growth arrest.1–5 In order to function in such acomplex manner, p53 protein is subject to a diversearray of regulatory mechanisms that keep this poten-tially dangerous protein in check until needed.1–5 Thebiochemical properties that underpin the complex biol-ogy are intimately correlated with the structure of thep53 molecule (see Fig. 1).

The core of the p53 protein is a region which folds insuch a way as to form a domain which can interact withDNA in a sequence-specific manner.14,15 The majority ofmissense mutations seen in human (and animal) tumoursoccur in regions of the gene encoding this domain suchthat either critical residues involved in DNA contact arealtered or that the whole conformation of the coredomain of the protein is disrupted. The consequence ofeither of these events is the loss of the ability of p53 tospecifically bind DNA in a sequence-specific manner.The binding to DNA is optimal when the protein is ina tetrameric state as a consequence of interactions offour separate p53 molecules via the tetramerizationdomain.16 The C terminal region is composed of pre-dominantly basic residues and forms a region that haskey regulatory properties. As discussed below, modifica-tion of this region by acetylation, phosphorylation,O-glycosylation, and RNA binding has been reported butthe physiological significance of these post-translational
modifications remains uncertain. The DNA binding

113THE p53 PATHWAY

Table I—Some useful WWW links relating to p53

p53 in Dundee http://www.dundee.ac.uk/pathology/p53.htmProtein interactions with p53 http://www.dundee.ac.uk/pathology/p53inter.htm9th p53 Workshop http://athens.wistar.upenn.edu/2p53/p53 researchers http://athens.wistar.upenn.edu/2p53/dirfp.htmlp53 database at IARC http://www.iarc.fr/p53/homepage.htmp53 database at Institut Curie http://perso.curie.fr/Thierry.Soussi/index.htmlWeizmann Institute p53 site http://bioinformatics/weizmann.ac.il/hotmolecbase/entries/p53.htmp53 structure http://www.pds.med.umich.edu/users/frank/logo.htmlp53 database http://www.biomedcomp.com/4d.acgi$tsrchname?Name=p53Apoptosis sites http://www.access.digex.net/2regulate/apolist.htmlOMIM p53 site http://www3.sncbi.nlm.nih.gov/htbin-post/Omim/dispmim?191170OMIM mdm2 site http://www.ncbi.nlm.nih.gov/htbin-post.Omim/dispmim?164785mdm2 database http://www.infosci/coh.org/mdm2asp/default.asp

Fig. 1—A representation of the structure of p53 protein. In man, p53 is composed of 393 residues. Thereare five highly conserved boxes and five identifiable regions sub-serving different functions. However, itshould be recognized that the functions are inter-dependent and regulation in one domain can profoundlyinfluence other domains. Interactions with other macromolecules are of great significance, with binding toDNA in a sequence-specific manner being of critical importance. In addition, p53 interacts with a widerange of proteins, facilitating both the regulation of p53 activity and the control of its concentration by thecontrol of its degradation

domain is separated from the transcriptional activationdomain by a region containing a series of repeatedproline residues typical of a polypeptide that can inter-act with signal transduction molecules that contain aSH3 binding domain.17 Through this domain p53 isinfluenced by diverse signalling molecules including thec-abl oncogene.18 The acidic N-terminal transcriptionalactivation domain allows p53 protein, in the context ofits specific binding to a target DNA sequence, to recruitthe basal transcriptional machinery required for tran-scribing new mRNA and by so doing, activate theexpression of target genes. This region is also criticallyinvolved in regulating the stability and activity of p53protein via interactions with proteins such as mdm2,19,20

which allows targeting of p53 to the ubiquitin-mediatedproteolytic machinery. Mdm2 binding also blocks theability of p53 to interact with the transcriptional appa-ratus.21 Modification of p53 by phosphorylation mayalter many of these properties and, in particular, theinteraction of p53 with other proteins such as mdm2 (seebelow). By these means, much of the regulation of thep53 pathway occurs.

Given this biochemical background, what are thefunctions of p53? While much of the literature is focused

Copyright ? 1999 John Wiley & Sons, Ltd.

on the idea that p53 is activated by DNA damage toelicit alterations in cell cycle control and/or apoptoticcell death, a broader picture is now emerging as shownin Fig. 2. Furthermore, as we shall see, there are distinctactivation pathways that regulate p53. A diverse rangeof insults can activate p53 to elicit adaptive responsesthat include, but are by no means restricted to, growtharrest and apoptosis. These properties are profoundlyinfluenced by cell type and tissue-specific modifierswhich are of clear importance, although they remainpoorly understood in terms of mechanism.22–25 How-ever, we do have some general perspective of the mecha-nisms by which the pathway is controlled (Fig. 3). Forexample, the level of p53 is dependent on a balancebetween protein synthesis and degradation and it is clearthat the absolute level of p53 has potential for causingdifferent effects.26,27 However, additional levels ofcontrol exist, with protein–protein interactions and post-translational modifications being very important (seebelow). In addition, the regulation of sub-cellularlocalization of p53 is increasingly viewed as being sig-nificant.28,29 These mechanisms serve to regulate not justthe level and location of p53 protein, but its activity.Furthermore, different target genes are induced (or

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114 C. PRIVES AND P. A. HALL

Fig. 2—The p53 pathway is not a simple linear system. Rather, there are many inputs and many outputs.It is quite wrong to think of DNA damage only as the initiating stimulus to the pathway, and it is similarlyincorrect to view the resultant consequences as restricted to growth arrest and apoptosis. Recent evidence(see text) points to the existence of at least three genetically distinct activation pathways. Furthermore,there are many levels of control with inputs at all points from initiating insult to resultant adaptiveresponse. The activity of the system is profoundly altered by poorly understood cell type and tissue-specificeffects

Fig. 3—There are diverse levels of control of p53. p53 protein is tightly regulated by control of theproduction of protein (mRNA production, stability and efficiency of translation) and of its degradation byubiquitin-mediated proteolysis. Additional levels of control include diverse protein–protein interactions,post-translational modifications (including phosphorylation, RNA binding, and glycosylation) and also byregulation of sub-cellular localization. By this means, p53 protein can be switched from inactive to activeforms and as a result lead to transcriptional activation (or repression) of downstream target genes.Activities which do not relate to transcriptional control have also been suggested1

Copyright ? 1999 John Wiley & Sons, Ltd. J. Pathol. 187: 112–126 (1999)

115THE p53 PATHWAY

repressed) in different cell types and with differingkinetics and thresholds, leading to considerable subtletyin the p53 response. From this overview of the biochem-istry and biology of p53 a model of how p53 works hasemerged (Fig. 4) and how this is perturbed in neo-plasia (Fig. 5). Unfortunately, our understanding of p53biology is very much less sophisticated and in manyrespects the mechanistic basis by which p53 actuallyworks as a tumour suppressor remains uncertain.

RECENT PROGRESS IN THE p53 field

Signalling to p53

It is clear that p53 functions within the framework ofa signal transduction pathway. Thus, signals arisingfrom an ever-increasing list of various forms of cellularstress, the most well studied of which is DNA damage,are transmitted by p53 to a similarly expanding compen-dium of genes and factors controlling various cellularresponses, including G1 and G2 arrest, differentiation,and apoptosis.1–4 Until the past year or so, the down-stream component of the p53 pathway was moreextensively understood and the upstream signalling com-ponent was a near complete mystery. Now it is clear thatthere are at least three genetically distinct pathways.Furthermore, the recent developments have, if nothingelse, begun to allow us to pose more defined questionsand design more informative experiments. Prior to theserecent advances it was known that after cellular stresses


such as DNA damage, hypoxia, nucleotide imbalance,or oxidative damage, on the one hand, and variousforms of oncogene imbalance, on the other, p53 levelsare greatly increased in cells. Additionally there is goodevidence that p53 itself is somehow activated to becomea potent sequence-specific transcriptional activator. Thisactivation is not fully understood, although there are anumber of studies which have shown that after varioustreatments, which will be discussed below, p53 is con-verted from a protein which binds very poorly to DNAto one which binds efficiently to its cognate sites.

In this section we will review three important sets ofrecent findings pertaining to the upstream regulation ofp53: first, data showing that the levels of p53 in cells areregulated by its interaction with Mdm2; second, theobservation that p53 can be modified at discrete andidentifiable sites after cellular stress, and that in at leastone case such stress-induced modification alters itsinteraction with Mdm2; and third, that as a result ofoncogene overexpression or imbalance, stabilization ofp53 occurs by a stress signal-independent pathway inwhich the product of the alternate reading frame of thep16INK4A locus (murine p19ARF or human p14ARF) isinduced and this leads to abrogation of Mdm2-targeteddestabilization of p53.

Fig. 4—The normal p53 pathway. A tonic loop exists in which the basal level of p53 activates a basal levelof downstream target genes including mdm2. Mdm2 protein binds p53, inactivating the ability of p53 tofunction as a transcription factor by blocking access to the basal transcription apparatus and alsotargeting p53 for ubiquitin-mediated degradation. A range of insults (see Fig. 2) alter a set of (as yet poorlydefined) signalling pathways such that there is alteration of the level and state of p53, perturbing the tonicloop. Activation of p53 occurs (see Fig. 3) with the transcriptional regulation of downstream target geneswhich elicit the required biological functions. It is likely that the kinetics and spectrum of downstreamtargets activated (or repressed) are modified by cell type-specific factors and, in particular, the profile ofother transcription factors and transcriptional co-activators present in a cell

Mdm2 targets p53 for destabilization—It is well estab-lished that p53 induction in cells after either DNAdamage or expression of viral oncogenes, such as SV40T antigen or adenovirus E1A, occurs largely through

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post-transcriptional mechanisms. Levels of the proteinunder both conditions rise in conjunction with itsmarkedly increased half-life. This, of course, poses thequestion as to what causes the rapid turnover of p53 innormal unstressed cells. The first hint came from workimplicating the ubiquitin-mediated proteasome in p53stability. The Oren and Vousden laboratories reportedthat overexpression of Mdm2 causes degradation of p53in transient assays, and that mutations of p53 or mdm2that prevented the interaction between the two lead togreatly stabilized p53.19,20 Furthermore, they showedthat Mdm2-targeted degradation of p53 is blocked byproteasome inhibitors. These observations were fol-lowed by a study from the Lane laboratory showing thatdisruption of the p53–mdm2 interaction in vivo by apeptide leads to increased quantities of p53.30 Sincethose observations were reported, a number of addi-tional studies have contributed to further understandingof this process. Honda et al.31 have provided evidencethat Mdm2 may serve as an E3 ubiquitin ligase. Rothet al.32 have published evidence that Mdm2 shuttles p53from the nucleus to the cytoplasm, where it is thendegraded, and Grossman et al.33 have provided anotherfacet to the story in their recent study showing thatMdm2 binds to p300 and this interaction is required forits ability to induce p53 breakdown. Given that it isbecoming increasingly clear that the major if not solecause of p53 turnover is mediated by its interaction withMdm2, it follows that disruption or prevention of thisinteraction is likely to be linked to p53 stabilization after


cellular stress or oncogene imbalance. It is now clearthat there are at least two separate and independentways that the p53 and Mdm2 interaction can be blocked,one through covalent modification of the proteins andthe other through the induction and action of anotherprotein, ARF (see below).

Fig. 5—p53 in neoplasia. Loss of function of p53 protein occurs as a consequence of missense mutation,allelic loss, or inactivation as a consequence of viral proteins such as HPV E6. As a consequence, thedownstream targets of p53 are not appropriately regulated and there is loss of their normal function; forexample, loss of p53-activated cell death, growth arrest, and/or control of genomic stability. Loss of theseimportant p53-dependent processes may occur early in the genesis of some tumours or, alternatively, maybe steps in tumour progression. One of the commonest features of mutant p53 is its accumulation withincells: a phenotype seen frequently in tumours and detectable by immunohistochemistry. While there aremultiple mechanisms for this clinically useful phenotype, one important mechanism is the loss ofactivation of mdm2 mRNA and hence mdm2 protein. Consequently, the mdm2-dependent degradationpathway of p53 does not operate and thus p53 protein accumulates to high levels, albeit the protein isinactive. Whether this model provides a complete explanation for p53 stability in neoplasia has recentlybeen questioned155

Regulation of p53 by stress-induced modification—p53is a nuclear phosphoprotein, and several protein kinaseshad been demonstrated to be able to phosphorylate p53sites believed to be of physiological relevance. However,until recently there has ben a dearth of evidence connect-ing stress or other signalling events to phosphorylationof p53. It is now clear that a number of phosphorylationsites on p53 are altered after DNA damage and, in somecases, new protein kinases have been shown to be ableto phosphorylate p53 at these sites. Furthermore, suchphosphorylation events have been shown to result inalterations in the protein which make it more stable andmore active. Phosphorylation, however, is not the onlyway in which p53 is regulated. Interestingly, anotherform of p53 modification, acetylation, has also beendemonstrated and shown to be DNA damage-inducible34 and the two may be temporally andmechanistically linked.35 Moreover, there is a class ofnon-covalent modifiers of p53 which are also likely to beinvolved in its ability to regulate cell cycle and cell death.To review these modifications, it is best to examine themodification of the relevant domains of p53, theirfunction, and regulation.

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117THE p53 PATHWAY

The N-terminus: DNA damage-induciblephosphorylation events and their impact on p53

The N-terminus consists of two transcription acti-vation sub-domains extending between residues 1 and63, and a region spanning residues 60–97, containingseveral copies of the sequence PXXP, which plays animportant role in the induction of apoptosis by p53.Several phosphorylation sites have been mapped withinthe N-terminus of human and/or murine p53 includingcasein kinase I (CK I) on murine p53: Ser 4, Ser 6, andSer 9; and DNA-activated protein kinase (DNA-PK):murine and human Ser 15 and human Ser 37.36 HumanSer 33 is phosphorylated in vivo and at least one proteinkinase, the TFIIH-associated trimeric cyclin-activatingkinase complex (CAK), consisting of cyclin H, CDK7,and p36/MAT1, has been shown to phosphorylate p53at that site in vitro.37 By contrast, murine p53 is phos-phorylated at Ser 34 in vivo and can be phosphorylatedby c-jun kinase (JNK) at that site.38 JNK can alsophosphorylate human p53 at its N-terminus, althoughthe exact site has not yet been reported.39 In addition,the murine p53 N-terminus can be phosphorylated invitro by mitogen-activated protein kinase (MAPK) atresidues 73 and 83.40 It should be noted that there are anumber of other potential threonine and serine phos-phate acceptors within the N-terminus of p53. Althoughtwo-dimensional phosphotryptic mapping has providedstrong support for the identification of the known sites,and perhaps can be tentatively used to rule out others, itremains possible that in some cells or conditions, novelsites may be utilized and such sites may indeed turn outto be very interesting. Although demonstration of theexistence of sites and relevant kinases is important,showing that a given site is phosphorylated in vivo inresponse to a given stimulus takes us considerablyfurther in understanding the biological relevance of suchmodifications.

A barrier to determining stress-induced changes inphosphorylation is that it is difficult to compare accu-rately the phosphorylation status of p53 from normalcells, where it is often present in nearly undetectableamounts, with that in stressed cells, where its levels haverisen dramatically, often by one or two orders of mag-nitude. One important technical advance has been theuse of phosphorylation site-specific antibodies. Thesereagents have made possible the rapid identification ofspecific phosphorylation events, either constitutive orinducible, and a number of groups have now reportedadvances with such antibodies. The first site to be shownto be inducibly phosphorylated after DNA damagewas Ser 15,41,42 as mentioned above, previously demon-strated to be a substrate for DNA-PK. Recently,another kinase, the product of the ATM gene, has alsobeen shown to be able to phosphorylate this site43,44 andit is likely that additional kinases will prove capable ofthis modification as well. Importantly the activity of theATM kinase is significantly increased when cells aresubjected to DNA damage. However, the observationsthat several protein kinases can phosphorylate a DNAdamage-responsive site leave critical questions to besolved. We still do not know which is the primary kinase


that actually phosphorylates p53 at this (or any) sitein vivo. The fact that phosphorylation of Ser 15 in p53 isdelayed in AT" cells is a provocative observation, butsince p53 does eventually become phosphorylated atthat residue in these cells,42 we are still left with thequestion as to whether ATM is a regulatory or aprimary kinase for p53. Indeed, a recent paper hasprovided compelling evidence that DNA-PK isupstream of p53.45 It is certainly possible that there aresituations (or cell types) where a given kinase predomi-nates and there is still much work to do in order todecipher the way that Ser 15 is regulated. To makematters even more complicated, it is now known that Ser15 is not the only DNA damage-inducible site on p53.It was recently shown that both Ser 33 and Ser 37 arealso DNA damage-inducible.46 Furthermore, the Meeklaboratory has identified protein kinases for whichp53 is a substrate whose activities are DNA damage-inducible.38,47 Moreover, Silicano et al.42 reported, inaddition to Ser 15, another site within the first 20 aminoacids of p53 which is increased in irradiated cells.

What is the significance of phosphorylation ofN-terminal sites? First, it is often the case that modifi-cation of a given site leads to conformational changesthat are propagated to other regions of a protein.Second, p53 makes specific contact with a number ofproteins (see below) and with DNA, and phosphoryla-tion can affect these interactions. The N-terminus of p53associates with both components of the transcriptionalmachinery, with TAFs, and with its negative regulator,Mdm2.2,48 Shieh et al.41 demonstrated that phosphory-lation of p53 with DNA-PK in vitro both markedlyreduces the ability of p53 to bind to Mdm2 and causes asignificant change in conformation of the N-terminus.Mdm2 both destabilizes p53 and represses transactiva-tion by p53, and so this result provides a plausibleexplanation for how p53 is induced after DNA damage.It is likely that additional N-terminal DNA damage-inducible modifications will also be shown to affect theinteractions of p53 with Mdm2, and it was recentlyreported that phosphorylation of human p53 by JNKinhibits its interaction with Mdm2.49 It is also possiblethat phosphorylation at N-terminal sites will be shownto affect its interaction with transcription factors, as itwas reported that phosphorylation at Ser 15 and Ser 37decreases the ability of p53 to bind to TFIID.50

Regulation of p53 through its C-terminus works throughboth covalent and non-covalent modifiers

The C-terminus of p53 has been described both as aregion whose function is to control sequence-specificDNA binding and as a DNA damage recognitiondomain. It possesses an autonomous DNA binding andstrand reassociation ability but to date, there has beenno evidence of sequence specificity in these interactions.2The p53 protein exists in solution predominantly astetramers and tetramers are the most efficient bindingform of the protein.51 The structure of the oligomeriza-tion domain of human p53 reveals an á-sheet-turn–â-helix motif which forms a pair of dimers.52 Mutation ofthe p53 tetramerization region substituting residues with

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alanine53 or hydrophobic amino acids54 leads to alteredoligomerization properties. Substitution of this regionwith a heterologous tetramerization domain, however,produces a protein with many of the activities of wild-type p53.55 It is interesting that disruption or loss ofoligomerization function is associated with loss of cellcycle arrest but not growth suppression as measured bycolony formation assays.56,57 Recently mutations havebeen identified in this domain.58

The effects of covalent modification of the C-terminuson p53 DNA binding continue to spark much interest.Hupp et al.59 were the first to discover that phosphoryla-tion, antibody interaction or deletion of the C-terminusstimulates DNA binding by inert bacterially expressedp53, and this pivotal observation has led to a plethora ofrelated findings. It is now known that CKII sites (Ser392) or PK-C sites (Ser 371, 376, and 378) within thisregion result in stimulation of binding. The mechanismby which the C-terminus regulates DNA binding is notyet fully understood. Two models have been proposed:in the first, it has been hypothesized that the C-terminusallosterically regulates conversion of p53 from aninert ‘latent’ form to one which is active for DNAbinding.60,61 Evidence for this model is based on theobservation that p53 can be isolated from baculovirus-infected insect cells or synthesized in vitro in a form thatbinds DNA only weakly but which, upon treatments asoutlined above, becomes activated. The second modelpostulates that binding by the central domain is hin-dered by the interactions of the C-terminus with longerDNA molecules.62,63 It is not clear at this point which ofthese two models (or both) is correct. Validation of theallosteric model will require physical evidence for aconformational alteration in p53 protein upon one ormore of the modifications noted previously. Indeed, it isentirely possible that both explanations contribute to theway in which p53 is regulated by its C-terminus.

Effects of phosphorylation of the C-terminus

There have been a number of current developmentsin understanding how phosphorylation regulates p53DNA binding. We must preface this optimistic state-ment by noting that attempts to discern phenotypes ofphosphorylation site mutants in p53 have been confus-ing at best and disappointing at worst. Mutation ofmurine phosphorylation sites at N- and C-termini havefrequently yielded p53 proteins which are roughlyequivalent to wild-type protein in standard transienttransfection assays of transcriptional activation of p53-responsive reporters.2 Hao et al.,64 however, reportedthat wild-type p53 in G1-arrested murine cells is inac-tive, while mutation of the murine CKII site to acharged residue (Glu) rescued p53 function. Addition-ally, mutation of the murine CKII site to alanine hadlittle effect on transactivation of p21 and RGC report-ers, while it markedly inhibited the ability of p53 torepress the SV40 promoter.1 Although it is not yet wellunderstood, we might speculate that an interaction ofthe C-terminus with a protein component of the generaltranscription machinery such as TBP or P300, both ofwhich have been implicated in the repression function of


p53, either might require p53 phosphorylated at that siteor might be affected by mutating Ser 392. Furtherinsight into the mechanism by which p53 is stimulatedby CKII is derived from the observation that p53 isbound stably by the beta subunit of CKII,65 and afterphosphorylation, p53 can no longer reanneal DNA.66

This interaction, as well as phosphorylation, may con-tribute to the effect of the protein kinase on p53 DNAbinding. Inhibition of p53 annealing of DNA by CKIIphosphorylation supports the proposal that the C-terminus, when bound to DNA, interferes with specificbinding by the central region. Note, however, thatSakaguchi et al.46 have demonstrated that peptidesspanning the C-terminal protein (residues 303–393)display a ten-fold greater association constant forreversible tetramer formation after phosphorylation byCKII. Whether this striking observation is also true forfull length p53 awaits experimentation. Importantly,Kapoor and Lozano67 and Lu et al.,68 using antibodiesspecific for p53 phosphorylated at the CKII site, showedthat UV irradiation, but not gamma irradiation (IR),induces phosphorylation at this site.

The finding that PK-C phosphorylates human p53within the C-terminal basic regulatory region is tantaliz-ing because of the possibility that this is the end-point ofa signal transduction pathway leading to activation ofthe protein. Murine p53 is also phosphorylated oncomparable sites within the C-terminal 30 amino acids,69

and PK-C was shown to interact with murine p53residues 320–343.70 Milne et al.69 however, reported thatp53 may not be inducibly phosphorylated at PK-C sitesafter TPA treatment in SV3T3 cells. To gain furtherinsight into this protein kinase, one approach hasbeen to exploit the use of PK-C inhibitors and Chernovet al.7 found that H7 or bisindolemaleimide I treatmentleads to accumulation of p53 protein and, recipro-cally, that phorbol ester inhibits the accumulation ofp53 after DNA damage. Another group found thatalthough staurosporine inhibits p53 accumulation, otherPK-C inhibitors did not affect p53 in their system.72

Recently, another exciting observation was reported byHalazonetis and co-workers, who found that, after IR,p53 loses phosphate at residue 376, one of the PK-Csites.73 Moreover, such a DNA damage-induced altera-tion is correlated with the ability of p53 to bind 14-3-3proteins and become transcriptionally activated.73

Another region of the C-terminus which is regulatoryin in vitro studies spans the CDK site within the linkerregion and it was observed that phosphorylation of Ser315 selectively stimulates p53 DNA binding to a subsetof p53 sites.74 Whether Ser 315 phosphorylation oper-ates mechanistically in the same fashion as modificationof residues within the C-terminal basic region is not yetestablished. However, a monoclonal antibody (PAb 241)which binds in the vicinity of the site can stimulate DNAbinding analogous to the effects of PAb 421 at theextreme C-terminus.75 A binding site for CDK onhuman p53 has been identified within residues 330–339.76 Interestingly, substitution of Ser 392, but not Ser315, with the acidic residue Asp led to reduced intera-ction of CDK with p53.46 Reciprocally, whereas phos-phorylation of Ser 392 increases tetramerization, this

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119THE p53 PATHWAY

effect is reversed when p53 is phosphorylated at Ser 315.Taken together, these data suggest an inter-dependentrelationship between the CKII and CDK sites on p53.Finally, although the site has not been mapped, Levine’slaboratory reported that the CAK trimeric complex canphosphorylate site(s) within the C-terminus.77 WhetherCAK is the only kinase that can phosphorylate bothN- and C-terminal sites is not yet known.

Phosphorylation is not the only modification of p53.p53 has also been shown to be O-glycosylated within thePAb 421 epitope region in a cell type-specific manner.78

Such glycosylation renders p53 incapable of recognitionby PAb 421. Gu and Roeder34 discovered that p53 canbe acetylated by p300/CBP predominantly at lysines 373and 382, and that such modification leads to increasedDNA binding. Sakaguchi et al.46 have confirmed andextended these observations in their finding that whilep300 acetylates residues within the C-terminal 30 aminoacids, PCAF, another histone acetylase, acetylates lys320. Moreover, they have made the exciting discoverythat at least one of these acetylation sites (lys 382) isactually modified in response to DNA damage.

Non-covalent modifiers

While covalent modifications are clearly a mode bywhich cells can signal to and regulate p53, non-covalentregulators of p53 are also likely to become increasinglyimportant. In this respect, it is interesting to consider therelationship between p53 and DNA damage and repair.In addition to its possible role in regulation of thecentral core domain, the C-terminus of p53 has beenpostulated to be a damage recognition region, based onits ability to bind DNA ends and single strands,79

mismatches,80 Holiday junctions,81 and irradiatedDNA.82 Although the significance of these interactionsawaits further study, it is worth considering that theymay perform a regulatory function in a manner analo-gous to proteins that interact with the C-terminus.Moreover, a number of DNA repair proteins have beenidentified that interact with or regulate p53, presumablythrough its C-terminus. These include the XP-B andXP-D components of TFIIH74 and Rad51.83 The impactof these interactions on the sequence-specific transacti-vation function of p53 has not yet been fully explored.Moreover, the redox/repair protein Ref-1 was discov-ered to be a potent activator of p53 DNA binding andtransactivation.84 Thus, there are a surprisingly largenumber of disparate observations which connect p53to DNA repair processes both through signalling afterDNA damage and through its interactions with theDNA repair machinery. The physiological significanceof all of these modifications remains uncertain.

ARF mediates the p53 response to oncogenes—Despitethe well-studied anti-oncogene activities of p53, therehave been a number of reports documenting the fact thata number of viral (e.g. SV40 T antigen, adenovirus E1a)and cellular (c-myc and ras) oncogene products, whenoverexpressed in cells, can cause p53 to accumulate.While these observations run counter to the knownanti-proliferative effects of p53, it is also known that


in many cases cells and viruses can overcome the pro-apoptotic effects of p53. How viral and cellular onco-genes induce p53 was quite enigmatic until the recentdiscovery of the ARF product of the p16INK4a locus.85

It was observed by Kamijo et al.86 that primary cellsfrom ARF"/" mice become immortal and can betransformed by Ha-ras alone. Connecting these resultsto p53 was the fact that overexpression of ARF causesgrowth arrest,86 as well as repression of transformationby myc and ras87 in wild-type but not p53-null cells.These intriguing results were taken further in severalstudies showing that ARF can bind to Mdm2 andprevent it from inducing destruction of p53.87–90 Thus,ARF regulates p53 and is encoded by a gene overlappinga negative regulator of another tumour suppressor, pRb,namely p16INK4A. Further connections between p53and pRb have now been revealed. It has been shownvery recently that E2F1, whose negative regulation bypRb has been well studied, activates ARF transcription-ally91 and, entirely consistent with this important obser-vation, de Stanchina et al.92 have now shown that E1a,itself a negative regulator of pRb, can induce ARF.Importantly, the ability of E1a to stabilise p53 is absentin ARF"/" cells.92 Finally, it has been recently shownthat both myc93 and ras94 can also induce ARF. Thus,we can now construct a cellular pathway in whichoncogenes, through their induction of ARF with itsensuing inhibitory effects on Mdm2, cause p53 toaccumulate. Why would such a pathway be required?It can be speculated that when cells, either geneticallyor epigenetically, find themselves in inappropriatehyperproliferative states, cell arrest or even death is thebest response to such situations and p53 is clearly theappropriate conduit.

What is fascinating about these observations is thatthe DNA damage pathway (and perhaps the full set ofcellular stress responses) appears to be, at this point,genetically separable from the ARF pathway in regulat-ing p53. Kamijo et al.86 demonstrated that ARF"/"cells still have an intact DNA damage response, and deStanchina et al.92 found that, in contrast to DNA-damaged cells, ARF overexpression fails to induce phos-phorylation of Ser 15. We can certainly expect more newand exciting developments in the future further docu-menting the circuitry between p53, Mdm2, and ARF.What is clear, at present, is that just as p53 is regulatedby ARF and Mdm2, it too controls these products: p53transactivates its negative regulator, Mdm2, and, in amanner as yet unfathomed, also down-regulates itsactivator, ARF.89

These new data are very significant in two ways. First,they provide concrete molecular evidence for the ideathat there are distinct upstream inputs into the p53pathway. Second, they provide a new insight into therole of p53 oncogenesis. Specifically, these data lead tothe conclusion that the involvement of p53 in the genesisof tumours may have much more to do with loss of itsfunction as an inhibitor of inappropriate oncogenefunction than simply its role in DNA damage pathways.Therefore loss of p53 (or ARF) function gives a pro-found growth advantage as a result of loss of a criticalinhibitor of oncogene activity. Consequently, while it

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Table II—The reported protein interactions of p53 proteinwith a relevant reference (see also http://www.dundee.ac.uk/pathology/p53inter.htm)

ViralSV40 large T 95,96Adenovirus 5 and 12 E1b 55k 97Adenovirus E4orf6 98HPV E6 ORF 99EB virus EBNA-5 100Hepatitis B virus X protein 101Human CMV IE84 protein 102

Cellularc-abl 18p300/CBP 103TFIIH [CSB/XPD/ERCC3/XPB] 104HSP 70 105L5 ribosomal protein 106mdm2 107mdmX 108p53-BP1 109p53-BP2 109S100b 111Sp1 112TAFII31 113TATA box binding protein 114WT1 115cdc2 116RPA 117p19ARF 88RAD51 83p33ING1 118REF1 84HMG1 119Vimentin 12014-3-3 73E2F1 121BRCA1 122DP1 121JMY 123DNA-PK 124CK1 125JNK 38CKII 126PKC 127MAPK 128Cyclin H/CDK7/MAT1 43, 44

would be unwise to discount a role of genotoxic andother insults (e.g. hypoxia), the role of ARF-mediatedregulation of p53 is a central component of the mecha-nisms by which organisms avoid neoplasia by oncogeneactivation, and this itself now seems pivotal in thegenesis of cancer. Of course, a key question still remains:what is the exact set of downstream targets that p53activates which facilitate its role in tumour suppression?Nevertheless, of practical importance for the future, theconcentration of oncogenic events in the ARF-mdm2/p53 pathway suggests it being a key target in new cancertherapy.

The interaction of p53 with other macromolecules

Much of modern biology focuses on mechanisticissues and this inevitably requires knowledge of exactlyhow one molecule interacts with another in order toelicit some effect. Considerable progress has been madein structural biology, such that many biological pro-cesses are now understood in terms of specific physicalinteractions between macromolecules. One key area inthis is the definition of the physical interactions thatoccur between cellular proteins. Such interactions maybe long-lasting and very stable, as is the case for fila-ment formation by cytoskeletal proteins, or may be verytransient, as in the case of the contacts between a kinaseactive site and a target substrate. Understanding thephysical nature of interactions can provide considerableinsights into the mechanisms by which macromoleculesfunction and may indeed pave the way for rationaledesign drugs or at least modification of lead compoundsin order to enhance potential therapeutic effect. In thiscontext, it would seem that a large number of protein–protein interactions are observed to occur between p53and other cellular or virally encoded proteins (see TableII) and some have generated considerable interestbecause of their potential for being targets for thera-peutic benefit. It seems extraordinary, however, that apolypeptide of modest size should be the target for somany interactions, not to mention its binding to DNA,RNA, and modifications such as glycosylation, phos-phorylation, and acetylation. Central to interpretingthe plethora of interactions, however, are two keyquestions: first, what methods can be used for demon-strating such interactions; and second, what criteria canbe employed for defining the physiological relevance ofsuch interactions?

A wide range of methods can be employed to investi-gate protein–protein interactions and they includeco-localization studies, particularly by laser scanningconfocal microscopy and more recently by the use ofFRET (fluorescence energy transfer); immunochemicalstudies by co-immunoprecipitation (the method that wasoriginally employed to demonstrate the association ofT antigen and p53!95,96), in vitro association studies,whether using purified protein, in vitro transcribed andtranslated proteins, or recombinant protein; and the useof genetic approaches such as the application of theyeast two-hybrid method or a derivative thereof. All ofthese methods have intrinsic problems and ideally arange of methods must be used with particular caution

being placed on the interpretation of overexpressionstudies, and ideally some consideration of physiologicallevels and the stoichiometry of interactions. Geneticanalysis of mutants together with clear mechanisticevidence of physiological relevance is the gold standard,coupled with solid biochemical and co-localization datausing multiple methods. Having given this theoreticalbackground, in what interactions does p53 play a part?Table II lists the interactions reported so far with arelevant reference. As stated above, while the list isextensive, in many cases the physiological relevance ofthe interactions remains uncertain.110 A critical view isrecommended.

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121THE p53 PATHWAY

Downstream targets

The list of downstream targets of the p53 transcrip-tion factor continues to increase, as does our under-standing of the subtlety with which regulation occurs.To define a list of target genes would, at present, befoolish, as recent technical advances such as the use ofdifferential display, SAGE methods and CHIP-basedtechnology means that any list would rapidly be dated!More important are several theoretical points. First,while reports to the contrary exist, there is an over-whelming consensus that p53 functions primarily as atranscriptional regulator and not in non-transcriptionalprocesses. Second, the activity of the p53 transcriptionfactor is profoundly influenced by co-activator andco-repressor proteins that associate with p53. Otherexamples of such regulation abound in the transcrip-tional regulation field. The resultant complexity andsubtlety of regulation of downstream genes are thuslikely to be huge. Finally (and given the precedingdiscussion), there are considerable cell type and tissuetype differences in the spectrum of genes regulated byp53. Such complexity may be accentuated by a consid-eration of other p53-like transcription factors that canactivate similar (or the same) DNA sequences.

Fig. 6—Homologues of p53. See text for discussion. A recent paper156 has reported the existence of furtherisoforms as a consequence of alternate splicing and proposed yet further names. Interestingly several of theisoforms (ÄN p63á,â,ã) have lost the N-terminal transactivation domains and can thus act as dominantnegative forms

The discovery of p53 homologues

Whereas other critical cellular regulators such as pRband p16 are members of multi-gene families with over-lapping and often complementary functions, p53 was formany years not thought to be part of a family. In someretrospect, this view is perhaps surprising given, forexample, the fact that p53-null mice developed mostlynormally, even when lack of an expected phenotypeafter inactivation of a gene is often explained by func-


tional redundancy where related gene products fulfil therole of the missing gene. Recently, there has been rapidprogress in the definition of a series of p53 homologues.Three separate approaches have been taken. Friend’slaboratory reported some years ago the existence of amollusc cDNA with a considerable resemblance to p53,with conservation of the critical DNA binding resi-dues.12,129 Such sequences differed from the known p53cDNA in having a long C-terminal extension (Fig. 6). Atpresent, few functional data exist for this homologue butthey do suggest that the p53 transcription factor has along evolutionary history in some form.130

A second approach involved considerable serendipitysince the two mammalian p53 homologues cloned so far,p73 and p63 (KET, p51b), were identified by groups notworking in the p53 field and who were attempting toclone by polymerase chain reaction (PCR) genesinvolved in signal transduction. Kaghad et al.131 clonedp73, which shows very significant regions of sequenceidentity to p53 with the homology extensive in thetranscriptional activation domain, the DNA bindingdomain, and the oligomerization domain. The p73 geneencodes multiple polypeptides, the products of an alter-natively spliced mRNA transcript. The longest, p73á,consists of 636 residues; a shorter isoform, p73â, con-tains 499 residues, differing from p73á by only fiveresidues at the carboxy terminus. Of particular interest isthe fact that the p73 gene maps to the chromosomeregion 1p36, a region frequently deleted in neuro-blastoma and other human cancers. Neuroblastoma,unlike the majority of tumours, does not usually carryp53 mutations. Therefore it seems reasonable to specu-late that it is p73, rather than p53, that is performing acrucial tumour suppressor role in this type of tumouri-genesis. The original report suggested that p73 wasimprinted and that loss of this was associated with

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tumourigenesis. However, recent data suggest that p73may not be a classical tumour suppressor gene and maynot be the long sought after neuroblastoma susceptibil-ity gene at the 1p36 locus. In addition, analysis of p73 inprostate,132 lung,133,134 and colorectal cancer135 hasfailed to identify any somatic mutations, although anumber of polymorphisms have been reported. Further-more, in those series reported so far, while loss ofheterozygosity has been observed at the p73locus,132,134,135 it is not common. Clear data on this havebeen presented by Moll and co-workers,136 who foundbi-allelic expression of p73 in five of six neuroblastomas;mutations were not found (although polymorphisms arecommon). From such data it would seem that p73 isneither imprinted nor frequently mutated and is thus notlikely to be a true tumour suppressor.

However, the activities of p73 are remarkably similarto those of p53 in many respects. For example, Jostet al.137 reported that p73 can activate the p53-responsive elements in p21waf1 and can also induceapoptosis when overexpressed. More recent data indi-cate that p73alpha and beta can form heterodimers thatbind to canonical p53-responsive elements, but are nottargeted by viral oncoproteins such as SV40 large Tantigen (Kaelin, W., personal communication) and HPVE6.138 Using the two-hybrid system, Kaghad et al.131

demonstrated that p73â can form homotypic inter-actions and, perhaps of greater significance, heterotypicinteractions with p53, albeit weakly. The physiologicalsignificance of such interactions remains elusive, but it istempting to speculate that p73 may be capable ofregulating p53 function at least in some contexts.

At the same time as the p73 discovery, a groupsearching for signal transduction components that areexpressed specifically in rat taste receptor cells amplifiedanother p53 homologue which they called KET,139 nowcalled p63. Like p73, p63 shows close homology to squidp53 (Fig. 6), again suggesting that p63 and p73 mayrepresent primordial p53 ancestral genes. As with p73,isoforms of p63, as a consequence of alternate splicing,are seen (see Fig. 6).140,141 Considerable similarity existsbetween p73 and p63, including the long C-terminalextension. As with p73, a long form (denoted p63á) andshorter forms p63â and ã exist. Recent data indicate thatp51â (KET) is widely expressed in human and rodenttissues (Campbell and Hall, unpublished observations).It is noteworthy that both p73 (alpha and beta) and p63have remarkable conservation of critical contact resi-dues for mdm2 (equivalent to residues F19, D23, andL26 in p53) and the DNA binding domain (equivalent toresidues R175, G245, R248, R249, R273, and R282 inp53), as well as homology in the oligomerizationdomain. It might, then, be predicted that these proteinswill all interact with mdm2, have similar DNA bindingproperties, and potentially homo- and hetero-oligomerize. The long C-terminal extensions of p73á,p63á, and the squid homologue have a motif, called theSAM domain, seen in diverse proteins throughouteukaryotic phyla.142,143 Interestingly, this potentialprotein–protein interaction domain is specificallyexcluded from p73â, p63â and p63ã (see Fig. 6). Thebiological significance of this domain and the homo-


logues will await the generation of suitable reagents fortheir biochemical and genetic analysis.

A third approach to identifying p53 homologues isbased on functional assays and protein purification.Two reports indicate that this approach can be veryinformative, although the potential species have notbeen fully characterized as yet (p53CP;144 p53RE145).These approaches have been based on chromatographyand affinity purification of proteins capable of bindingto DNA-containing p53 binding sites. The exact natureof the proteins identified remains unclear, but based onimmunological criteria p53RE appears not to be p53 ora major p73 isoform.145 A final approach based onimmunological cross-reaction has not been veryinformative, and one recent claim should be consideredcritically, since the cross-reacting RA18A species146 isrestricted to an undefined cytoplasmic compartmentseen as scattered small dots (Hall, P. A., unpublishedobservations).

The discovery of p53 homologues means that p53 mayhave to share centre stage with its new cousins. Whilethe new homologues answer some old questions such asthe almost normal development of p53-null mice, theypose many more questions than they answer. Do thesehomologues activate the same, an overlapping or adifferent set of downstream genes? Do p73 and p53interact to yield novel interactions not displayed byeither molecule alone? Do the proteins serve distinctfunctions in a cell? What signals influence them? p73mRNA is not induced by DNA but protein levels havenot yet been examined. What is the functional signifi-cance of the carboxy-terminal extensions of p73 andp63? Presumably p73â and p63 will be regulated in adifferent manner from p53. Perhaps p73 and p63 substi-tute for developmental functions of p53. p73 and p63knockout mice are required to test this theory. More-over, are there more homologues to be identified andfurther isoforms of those currently known? The multi-faceted p53 protein provided us with a complex enoughstory in its own right. Now with the discovery of its newfamily members a re-evaluation of p53 function isnecessary. There is much work to be undertaken inelucidating the roles of the new homologues and we areonly at the beginning of the story.

FINAL PERSPECTIVES AND KEY QUESTIONSFOR THE FUTURE

The vignettes presented above are merely a snapshotof the huge p53 literature and even they fail to conveythe extent of our ignorance of the biology of p53. Forexample, there remains considerable uncertainty aboutthe role of specific p53 mutants in the biology ofneoplasia. Many authors have suggested that there are‘gain of function’ mutants that provide new or alteredproperties as well as the better understood loss of func-tion mutants. Recent work from several laboratorieshas, in part, clarified the issue with the demonstrationthat some mutants, such as R175H, do indeed providenew biological properties.147 Interestingly, Gualbertoet al.147 suggest that p53 mutations can contribute to the

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123THE p53 PATHWAY

progression of a cancer cell not only by absence of p53tumour suppressor activity, but also by the presence ofan activity that promotes genetic instability, and thatsuch mutants may act without the need for transcrip-tional activation. The possible clinical relevance of suchideas has been demonstrated by Blandino et al.,148 whohave demonstrated that different classes of p53 mutationhave differential effects on the resistance of tumour cellsto chemotherapy.

Another complexity in this area is the existence of anumber of polymorphisms in the p53 gene and recentdata suggest that these may have clinical significance byallowing differential sensitivity of cells bearing particularpolymorphisms to oncogenesis. Certain polymorphismshave been associated with an increased risk of sometumours.149,150 Recently, Storey et al.151 highlighted theproline/arginine polymorphism at position 72 andshowed that the arginine form of p53 was considerablymore susceptible to HPV E6-mediated proteolysis thanthe proline form. Provocatively, they also reported anoverrepresentation of homozygous R72 in HPV-associated tumours compared with the normal popula-tion. In a second study, however, individuals with R72were not found to be at increased risk of cervicalcancer.152 Clearly many further molecular epidemiologi-cal studies are required but the combination of bio-chemical and epidemiological studies reported by Storeyet al. would seem to provide a model for the future.

The complex effects of gene dosage in cancer shouldalso be noted. A well-established model of p53 functionin cancer follows the classical Knudsen two-hit para-digm, where mutation of one p53 allele is followed byloss of the second allele. While there are many situationswhere this has been observed, both experimentally andclinically, recent data suggest that alternative models arerelevant. Donehower and co-workers153 have recentlyreported a detailed analysis of tumours arising in p53heterozygous mice. Provocatively, they demonstrated inelegant experiments that the reduction of p53 genedosage to hemizygosity (with retention of a wild-typeallele) can be associated with neoplasia and that thereis not necessarily a need to lose the second allele. Thisprovides a striking example of the need to consider theabsolute level of p53 (and presumably other onco-proteins and tumour suppressor) protein in studies of itsrole in oncogenesis. Another level of complexity isillustrated by the striking observation that in theLi–Fraumeni cancer susceptibility syndrome, which isassociated in many cases with germline mutations in onep53 allele, phenotypically indistinguishable families existwithout p53 mutation.154 Moreover, linkage analysis insuch a family excludes not only p53 but also thep16/p19ARF locus, as well as BRCA1, BRCA2, MLH1,MSH2, and p57.154

Considering the information that we have reviewedand previous data in the field, where are we now? Well,it is clear that while we have learnt a great deal aboutp53, there is so much that we do not know: even somevery basic issues! For example, while it is clear that p53abnormalities occur frequently in cancer, what is the realfrequency of mutations in p53 in cells and tissues, andwhat proportion of these become selected as a conse-


quence of giving growth advantage. Moreover, which ofthe many properties of p53 are particularly important inoncogenesis? Is it the loss of apoptosis that is importantin oncogenesis, or of checkpoint function? Is it themitotic checkpoint or G1 checkpoint? To what extent isthe role of p53 in the DNA damage response of particu-lar relevance in tumour initiation or in promotion anddisease progression? Is it the loss of this activity (or setof activities) that is particularly relevant in humanneoplasia or is it (as seems possible) the role of p53 inopposing unrestrained oncogene activity via an ARF-dependent pathway? Posing questions such as thesehighlights our remarkable ignorance of p53 biology asopposed to our detailed (but still incomplete) knowledgeof p53 biochemistry. We have a long way to go!

ACKNOWLEDGEMENTS

We thank Emma Warbrick, David Meek, DavidLane, and the members of the Hall and Prives labora-tories for invaluable comment and discussion. We alsothank Bill Kaelin, Ute Moll, Moshe Oren, Gigi Lozano,and Larry Donehower for allowing us to see unpub-lished and in press data. We apologize to our manycolleagues whose work we cannot cite because of spaceconstraints. Work in the Hall laboratory is supported bythe Cancer Research Campaign, the Association forInternational Cancer Research, the Department ofHealth, the European Union, and the PathologicalSociety of Great Britain and Ireland. The Prives labora-tory is supported by the National Institute of Health.Carol Prives gave the Pathological Society Lecture at therecent 9th International p53 Workshop.

REFERENCES

1. Hall PA, Meek D, Lane DP. p53: integrating the complexity. J Pathol1996; 180: 1–5.

2. Ko LJ, Prives C. p53: puzzle and paradigm. Genes Dev 1996; 10:1054–1072.

3. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88: 323–331.

4. Agarwal ML, Taylor WR, Chernov MV, Chernova OB, Stark GR. Thep53 network. J Biol Chem 1998; 273: 1–4.

5. Harris CC. p53 tumor suppressor gene: from the basic research laboratoryto the clinic—an abridged historical perspective. Carcinogenesis 1996; 17:1187–1198.

6. Greenblatt MS, Bennett WP, Hollstein M, Harris CC. Mutations in thep53 tumor suppressor gene: clues to cancer etiology and molecularpathogenesis. Cancer Res 1994; 54: 4855–4878.

7. Hainaut P, Hernadez T, Robinson A, et al. IARC database of p53 genemutations in human tumors and cell lines: updated compilation, revisedformats and new visualisation tools. Nucleic Acids Res 1998; 26: 205–213.

8. Dowell SP, Hall PA. The p53 tumour suppressor gene and tumourprognosis: is there a relationship? J Pathol 1995; 177: 221–224.

9. Bosari S, Viale G. The clinical significance of p53 aberrations in humantumours. Virchows Arch 1995; 427: 229–241.

10. Bray SE, Schorl C, Hall PA. The challenge of p53: linking biology,biochemistry and patient management. Stem Cells 1998; 16: 248–260.

11. Nielsen LL, Maneval DC. p53 tumor suppressor gene therapy for cancer.Cancer Gene Ther 1998; 5: 52–63.

12. Soussi T, May P. Structural aspects of the p53 protein in relation to geneevolution: a second look. J Mol Biol 1996; 260: 623–637.

13. Arrowsmith CHP, Morin P. New insights into p53 function from struc-tural studies. Oncogene 1996; 12: 1379–1385.

14. El-Deiry WS, Kern SE, Pietenpol JA, Kinzler KW, Vogelstein B. Defini-tion of a consensus binding site for p53. Nature Genet 1992; 1: 45–49.

15. Bourdon JC, Deguin-Chambon V, Lelong JC, et al. Further characteris-ation of the p53 responsive element—identification of new candidate genesfor trans-activation by p53. Oncogene 1997; 14: 85–94.

J. Pathol. 187: 112–126 (1999)


16. Jeffrey PD, Gorina S, Pavletich NP. Crystal structure of the tetrameriza-tion domain of the p53 tumor suppressor at 1·7 angstroms. Science 1995;267: 1498–1502.

17. Gorina S, Pavletich NP. Structure of the p53 tumor suppressor bound tothe ankyrin and SH3 domains of 53BP2. Science 1996; 274: 1001–1005.

18. Yuan ZM, Huang Y, Whang Y, et al. Role for c-Ab1 tyrosine kinase ingrowth arrest response to DNA damage. Nature 1996; 382: 272–274.

19. Haupt Y, Maya R, Kazaz A, Oren M. Mdm2 promotes the rapiddegradation of p53. Nature 1997; 387: 296–299.

20. Kubbutat MHG, Jones SN, Vousden KH. Regulation of p53 stability byMdm2. Nature 1997; 387: 299–303.

21. Wu X, Bayle JH, Olson D, Levine AJ. The p53–mdm-2 autoregulatoryfeedback loop. Genes Dev 1993; 7: 1126–1132.

22. Midgley CA, Owens B, Briscoe CV, Thomas DB, Lane DP, Hall PA.Coupling between gamma irradiation, p53 induction and the apoptoticresponse depends upon cell type in vivo. Cell Sci 1995; 108: 1843–1841.

23. MacCallum DE, Hupp TR, Midgley CA, et al. The p53 response toionising radiation in adult and developing murine tissues. Oncogene 1996;13: 2575–2587.

24. Gottlieb E, Haffner R, King A, et al. Transgenic mouse model for studyingthe transcriptional activity of the p53 protein: age- and tissue-dependentchanges in radiation-induced activation during embryogenesis. EMBO J1997; 16: 1381–1390.

25. Komarova EA, Chernov MV, Franks R, et al. Transgenic mice withp53-responsive lacZ: p53 activity varies dramatically during normal devel-opment and determines radiation and drug sensitivity in vivo. EMBO J1997; 16: 1391–1400.

26. Chen X, Ko LJ, Jayaraman L, Prives C. p53 levels, functional domains,and DNA damage determine the extent of the apoptotic response of tumorcells. Genes Dev 1996; 10: 2438–2451.

27. Lassus P, Ferlin M, Piette J, Hibner U. Anti-apoptotic activity of lowlevels of wild-type p53. EMBO J 1996; 15: 4566–4573.

28. Moll UM, Ostermeyer AG, Haladay R, Winkfield B, Frazier M, ZambettiG. Cytoplasmic sequestration of wild-type p53 protein impairs the G1checkpoint after DNA damage. Mol Cell Biol 1996; 16: 1126–1137.

29. Ostermeyer AG, Runko E, Winkfield B, Ahn B, Moll UM. Cytoplasmi-cally sequestered wild-type p53 protein in neuroblastoma is relocated tothe nucleus by a C-terminal peptide. Proc Natl Acad Sci USA 1996; 93:15190–15194.

30. Bottger A, Bottger V, Sparks A, Liu WL, Howard SF, Lane DP. Design ofa synthetic Mdm2-binding mini protein that activates the p53 response invivo. Curr Biol 1997; 7: 860–869.

31. Honda R, Tanaka H, Yasuda H. Oncoprotein MDM2 is a ubiquitin ligaseE3 for tumor suppressor p53. FEBS Lett 1997; 420: 25–27.

32. Roth J, Dobbelstein M, Freedman DA, Shenk T, Levine AJ. Nucleo-cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of thep53 protein via a pathway used by the human immunodeficiency virus revprotein. EMBO J 1998; 17: 554–564.

33. Grossman SR, Perez M, Kung AL, et al. p300/MDM2 complexes partici-pate in MDM2-mediated p53 degradation. Mol Cell 1988; 2: 405–415.

34. Gu W, Roeder RG. Activation of p53 sequence-specific DNA binding byacetylation of the p53 C-terminal domain. Cell 1997; 90: 595–606.

35. Sakaguchi K, Herrera JE, Saito S, et al. DNA damage activates p53through a phosphorylation–acetylation cascade. Genes Dev 1998; 12:2831–2841.

36. Meek D. Post-translational modification of p53. Semin Cancer Biol 1994;5: 203–207.

37. Ko L, Chen X, Shieh S-, et al. p53 is phosphorylated by CDK7/cyclin H ina 36/MAT1 dependent manner. Mol Cell Biol 1997; 17: 7220–7229.

38. Milne DM, Campbell LE, Campbell DG, Meek DW. p53 is phos-phorylated in vitro and in vivo by an ultraviolet radiation induced proteinkinase characteristic of the c-Jun kinase, JNK1. J Biol Chem 1995; 270:5511–5518.

39. Adler V, Pincus MR, Minamoto T, et al. Conformation-dependentphosphorylation of p53. Proc Natl Acad Sci USA 1997; 94: 1686–1691.

40. Milne DM, Campbell DG, Caudwell FB, Meek DW. Phosphorylation ofthe tumor suppressor protein p53 by mitogen-activated protein kinases.J Biol Chem 1994; 269: 9253–9260.

41. Shieh S-Y, Ikeda M, Taya Y, Prives C. DNA damage-induced phos-phorylation of p53 alleviates inhibition by mdm2. Cell 1997; 91: 325–334.

42. Siliciano JD, Canman CE, Taya Y, Sakaguchi K, Appella E, Kastan MB.DNA damage induces phosphorylation of the amino terminus of p53.Genes Dev 1997; 11: 3471–3481.

43. Banin S, Moyal L, Khostravi R, et al. Enhanced phosphorylation of p53by ATM response to DNA damage. Science (in press).

44. Canman CE, LIM DS, Cimprich KA, et al. Activation of the ATM kinaseby ionizing radiation and phosphorylation of p53. Science (in press).

45. Woo RA, McLure KG, Lees-Miller SP, Rancourt D, Lee PWK. DNA-dependent protein kinase acts upstream of p53 in response to DNAdamage. Nature 1998; 394: 700–704.

46. Sakaguchi K, Herrera JE, Saito S, et al. DNA damage activates p53through a phosphorylation–acetylation cascade. Genes Dev 1998; 12:2831–2841.


47. Knippschild U, Milne DM, Campbell LE, et al. p53 is phosphorylated invitro and in vivo by the delta and epsilon isoforms of casein kinase 1 andenhances the level of casein kinase 1 delta in response to topoisomerase-directed drugs. Oncogene 1997; 15: 1727–1730.

48. Gottlieb MT, Oren M. p53 in growth control and neoplasia. BiochemBiophys Acta 1996; 1287: 77–102.

49. Fuchs SY, Adler V, Pincus M, Ronai Z. MEKK1/JNK signalling stabilizesand activates p53. Proc Natl Acad Sci USA 1998; 95: 10541–10546.

50. Pise-Masison CA, Radonovich M, Sakaguchi K, Appella E, Brady JN.Phosphorylation of p53: a novel pathway for p53 inactivation in humanT-cell lymphotropic virus type 1-transformed cells. J Virol 1998; 72:6348–6355.

51. Jeffrey PD, Gorina S, Pauletich NP. Crystal structure of the tetrameriza-tion domain of the p53 tumour suppressor at 1·7 angstroms. Science 1995;267: 1498–1502.

52. Waterman JL, Shenk JL, Halazonetis TD. The dihedral symmetry of thep53 tetramerization domain mandates a conformational switch uponDNA binding. EMBO J 1995; 14: 512–519.

53. Chene P, Mittl P, Grutter M. In vitro structure–function analysis of thebeta-strand 326–333 of human p53. J Mol Biol 1997; 273: 873–881.

54. McCoy M, Stavridi ES, Waterman JL, Wieczorek AM, Opella SJ,Halazonetis TD. Hydrophobic side-chain size is a determinant of thethree-dimensional structure of the p53 oligomerization domain.EMBO J1997; 16: 6230–6236.

55. Waterman MJ, Waterman JL, Halazonetis TD. An engineered four-stranded coiled coil substitutes for the tetramerization domain of wild-typep53 and alleviates transdominant inhibition by tumor-derived p53mutants. Cancer Res 1996; 56: 158–163.

56. Ishioka C, Englert C, Winge P, Yan YX, Engelstein M, Friend SH.Mutational analysis of the carboxy-terminal portion of p53 using bothyeast and mammalian cell assays in vivo. Oncogene 1995; 10: 1485–1492.

57. Pellegata NS, Cajot JF, Stanbridge EJ. The basic carboxy-terminaldomain of human p53 is dispensable for both transcriptional regulationand inhibition of tumor cell growth. Oncogene 1995; 11: 337–349.

58. Lomax ME, Barnes DM, Gilchrist R, Picksley SM, Varley JM,Camplejohn RS. Two functional assays employed to detect an unusualmutation in the oligomerisation domain of p53 in a Li–Fraumeni likefamily. Oncogene 1997; 14: 1869–1874.

59. Hupp TR, Meek DW, Midgley CA, Lane DP. Regulation of the specificDNA binding function of p53. Cell 1992; 71: 875–886.

60. Hupp TR, Lane DP. Allosteric activation of latent p53 tetramers. CurrBiol 1994; 4: 865–875.

61. Halazonetis TD, Davis LJ, Kandil AN. Wild-type p53 adopts a ‘mutant’-like conformation when bound to DNA. EMBO J 1993; 12: 1021–1028.

62. Bayle JH, Elenbaas B, Levine AJ. The carboxyl-terminal domain of thep53 protein regulates sequence-specific DNA binding through its non-specific nucleic acid binding activity. Proc Natl Acad Sci USA 1995; 92:5729–5733.

63. Anderson ME, Woelker B, Reed M, Wang P, Tegtmeyer P. Reciprocalinterference between the sequence-specific core and nonspecific C-terminalDNA binding domains of p53: implications for regulation. Mol Cell Biol1997; 17: 6255–6264.

64. Hao M, Lowy AM, Kapoor M, Deffie A, Liu G. Lozano G. Mutation ofphospherine 389 affects p53 function in vivo. J Biol Chem 1996; 271:29380–29385.

65. Appel K, Wagner P, Boldyreff B, Issinger OG, Montenarh M. Mapping ofthe interaction sites of the growth suppressor protein p53 with theregulatory beta-subunit of protein kinase CK2. Oncogene 1995; 11: 1971–1978.

66. Filhol O, Boudier J, Chambaz EM, Cochet C. Casein kinase 2 inhibits therenaturation of complementary DNA strands mediated by p53 protein.Biochem J 1996; 316: 331–335.

67. Kapoor M, Lozano G. Functional activation of p53 via phosphorylationfollowing DNA damage by UV but not gamma radiation. Proc Natl AcadSci USA 1998; 95: 2834–2837.

68. Lu H, Taya Y, Ikeda M, Levine AJ. Ultraviolet radiation, but not gammaradiation or etoposide-induced DNA damage, results in the phosphoryla-tion of the murine p53 protein at serine-389. Proc Natl Acad Sci USA 1998;95: 6399–6402.

69. Milne D, McKendrick L, Jardine LJ, Deacon E, Lord JM, Meek DW.Murine p53 is phosphorylated within the PAb421 epitope by proteinkinase C in vitro, but not in vivo, even after stimulation with the phorbolester o-tetradecanoylphorbol 13-acetate. Oncogene 1996; 13: 205–211.

70. Delphin C, Huang KP, Scotto C, et al. The in vitro phosphorylation of p53by calcium-dependent protein kinase C: characterization of a protein-kinase-C-binding site on p53. Eur J Biochem 1997; 245: 684–692.

71. Chernov MV, Ramma CV, Adler VV, Stark GR. Stabilization andactivation of p53 are regulated independently by different phosphorylationevents. Proc Natl Acad Sci USA 1998; 95: 2284–2289.

72. Pitkanen K, Haapajarvi T, Laiho M. U.V.C.-induction of p53 activationand accumulation is dependent on cell cycle and pathways involvingprotein synthesis and phosphorylation. Oncogene 1998; 16: 459–469.

73. Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD. ATM-dependent activation of p53 involves dephosphorylation and associationwith 14-3-3 proteins. Nature Genet 1998; 19: 175–178.

J. Pathol. 187: 112–126 (1999)

125THE p53 PATHWAY

74. Wang Y, Prives C. Increased and altered DNA binding of human p53 byS and G2/M but not G1 cyclin-dependent kinases. Nature 1995; 376:88–91.

75. Hansen S, Midgley CA, Lane DP, et al. Modification of two distinctCOOH-terminal domains is required for murine p53 activation by bacte-rial Hsp70. J Biol Chem 1996; 271: 30922–30928.

76. Wagner P, Fichs A, Gotz C, Nastainczyk W, Montenarh M. Fine mappingand regulation of the association of p53 with p34cdc2. Oncogene 1998; 16:105–111.

77. Lu H, Fisher RP, Bailey P, Levine AJ. The CDK7–cycH–p36 complex oftranscription factor IIH phosphorylates p53, enhancing its sequence-specific DNA binding activity in vitro. Mol Cell Biol 1997; 17: 5923–5934.

78. Shaw P, Freeman J, Bovey R, Iggo R. Regulation of specific DNA bindingby p53: evidence for a role for O-glycosylation and charged residues at thecarboxy-terminus. Oncogene 1996; 12: 20797–20802.

79. Balkalkin G, Selivanova G, Yakovleva T, et al. p53 binds single-strandedDNA ends through the C-terminal domain and internal DNA segmentsvia the middle domain. Nucleic Acids Res 1995; 23: 362–369.

80. Lee S, Elenbaas B, Levine A, Griffith J. p53 and its 14 kDa C-terminaldomain recognize primary DNA damage in the form of insertion/deletionmismatches. Cell 1995; 81: 1013–1020.

81. Lee S, Cavallo L, Griffith J. Human p53 binds Holliday junctions stronglyand facilitates their cleavage. J Biol Chem 1997; 272: 7532–7539.

82. Reed M, Woelker B, Wang P, Wang Y, Anderson ME, Tegtmeyer P. TheC-terminal domain of p53 recognizes DNA damaged by ionizing radia-tion. Proc Natl Acad Sci USA 1995; 92: 9455–9459.

83. Buchhop S, Gibson MK, Wang XW, Wagner P, Sturzbecher HW, HarrisCC. Interaction of p53 with the human Rad51 protein. Nucleic Acids Res1997; 25: 3868–3874.

84. Jayaraman L, Murthy KGK, Curran T, Xanthoudakis S, Rives C.Identification of redox/repair protein Ref-1 as an activator orf p53. GenesDev 1997; 11: 558–570.

85. Quelle DE, Zindy F, Ashmun RA, Sherr CJ. Alternative reading frames ofthe INK4A locus tumor suppressor gene encode two unrelated proteinscapable of inducing cell cycle arrest. Cell 1995; 83: 993–1000.

86. Kamijo T, Zindy F, Roussel MF, et al. Tumor suppression at the mouseINK4a locus mediated by the alternative reading frame product p19ARF.Cell 1997; 91: 649–659.

87. Pomerantz J, Schreiber-Agus N, Liegeois NJ, et al. The Ink4a tumorsuppressor gene product, p19Arf, interacts with MDM2 and neutralizesMDM2’s inhibition of p53. Cell 1998; 92: 713–723.

88. Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ.Functional and physical interactions of the ARF tumor suppressor withp53 and Mdm2. Proc Natl Acad Sci USA 1998; 95: 8292–8297.

89. Stott F, Bates SA, James M, et al. The alternative product from the humanCDKN2A locus, p14ARF, participates in a regulatory feedback loop withp53 and MDM2. EMBO J 1998; 17: 5001–5014.

90. Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradationand stabilizes p53: ARF–INK4a locus deletion impairs both the Rb andp53 tumor suppression pathways. Cell 1998; 92: 725–734.

91. Bates S, Phillips A, Clarke P, et al. E2F-1 regulation of p14ARF links pRband p53. Nature (in press).

92. de Stanchina E, McCurrach ME, Zindy F, et al. E1a signalling to p53involves the p19ARF tumor suppressor. Genes Dev 1998; 12: 2434–2443.

93. Zindy F, et al. MYC-induced immortalization and apoptosis targets theARF–p53 pathway. Genes Dev 1998; 12: 2424–2433.

94. Palermo I, Pantoja C, Serrano M. p19ARF links the tumour suppressor p53to RAS. Nature 1998; 395: 125–126.

95. Lane DP, Crawford L. T antigen is bound to a host protein in SV40transformed cells. Nature 1979; 278: 261–263.

96. Linzer DI, Levine AJ. Characterization of a 54K dalton cellular SV40tumor antigen present in SV40-transformed cells and uninfected embryo-nal carcinoma cells. Cell 1979; 17: 43–52.

97. Kao CC, Yew PR, Berk AJ. Domains required for in vitro associationbetween the cellular p53 and the adenovirus 2 E1B 5KK proteins. Virology1990; 179: 806–814.

98. Dobner T, Horikoshi N, Rubenwolf S, Shenk T. Blockage by adenovirusE4orf6 of transcriptional activation by the p53 tumor suppressor. Science1996; 272: 1470–1473.

99. Scheffner M, Takahashi T, Huibregtse JM, Minna JD, Howley PM.Interaction of the human papillomavirus type 16 E6 oncoprotein withwild-type and mutant human p53 proteins. J Virol 1992; 66: 5100–5105.

100. Szekely L, Selivanova G, Magnusson KP, Klein G, Wiman KG. EBNA-5,an Epstein–Barr virus-encoded nuclear antigen, binds to the retinoblas-toma and p53 proteins. Proc Natl Acad Sci USA 1993; 90: 5455–5459.

101. Feitelson MA, Zhu M, Duan LX, London WT. Hepatitis B#antigen andp53 are associated in vitro and in liver tissues from patients with primaryhepatocellular carcinoma. Oncogene 1993; 8: 1109–1117.

102. Speir E, Modali R, Huang ES, et al. Potential role of human cytomega-lovirus and p53 interaction in coronary restenosis. Science 1994; 265:391–394.

103. Lill NL, Grossman SR, Ginsberg D, DeCaprio J, Livingstone DM.Binding and modulation of p53 by p300/CBP coactivators. Nature 1997;387: 823–827.


104.105. Pinhasi-Kimhi O, Michaalovitz D, Ben-Zeev A, Oren M. Specific inter-action between the p53 cellular tumour antigen and major heat shockproteins. Nature 1986; 320: 182–184.

106. Marechal V, Elenbaas B, Piette J, Nicolas JC, Levine AJ. The ribosomalL5 protein is associated with mdm-2 and mdm-2–p53 complexes. Mol CellBiol 1994; 14: 7414–7420.

107. Momand J, Zambetti GP, Olson DC, George D, Levine AJ. The edm-2oncogene product forms a complex with the p53 protein and inhibitsp53-mediated transactivation. Cell 1992; 69: 1237–1245.

108. Shvarts A, Steegenga WT, Riteco N, et al. MDMX: a novel p53-bindingprotein with some functional properties of MDM2. EMBO J 1996; 15:5349–5357.

109. Iwabuchi K, Bartel PL, Li B, Marraccino R, Fields S. Two cellularproteins that bind to wild-type but not mutant p53. Proc Natl Acad SciUSA 1994; 91: 6098–6102.

110. Warbrick E. Two’s company, three’s a crowd: the yeast two hybrid systemfor mapping molecular interactions. Structure 1997; 5: 13–17.

111. Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. Charac-terization of the tumor suppressor protein p53 as a protein kinase Csubstrate and a S100b-binding protein. Proc Natl Acad Sci USA 1992; 89:11627–11631.

112. Gualberto A, Baldwin AS Jr. p53 and Sp1 interact and cooperate in thetumor necrosis factor-induced transcriptional activation of the HIV-1 longterminal repeat. J Biol Chem 1995; 270: 19680–19683.

113. Lu H, Levine AJ. Human TAFII31 protein is a transcriptional coactivatorof the p53 protein. Proc Natl Acad Sci USA 1995; 92: 5154–5158.

114. Seto E, Usheva A, Zambetti GP, et al. Wild-type p53 binds to theTATA-binding protein and represses transcription. Proc Natl Acad SciUSA 1992; 89: 12028–12032.

115. Maheswaran S, Park S, Bernard A, et al. Physical and functional inter-action between WT1 and p53 proteins. Proc Natl Acad Sci USA 1993; 90:5100–5104.

116. Milner J, Cook A, Mason J. p53 is associated with p34cdc2 in transformedcells. EMBO J 1990; 9: 2885–2889.

117. Dutta A, Ruppert JM, Aster JC, Winchester E. Inhibition of DNAreplication factor RPA by p53. Nature 1993; 365: 79–82.

118. Garkavtsev I, Grigorian IA, Ossovskaya VS, Chernov MV, ChumakovPM, Gudkov AV. The candidate tumour suppressor p33ING1 cooperateswith p53 in cell growth control. Nature 1998; 391: 295–298.

119. Jayaraman L, Moorthy NC, Murthy KG, Manley JL, Bustin M, Prives C.High mobility group protein-1 (HMG-1) is a unique activator of p53.Genes Dev 1998; 12: 462–472.

120. Klotzsche O, Eisman T, Hohenburg H, Bohn W, Deppert W. Cytoplasmicretention of mutant tsp53 is dependent on an intermediate filament protein(vimentin) scaffold. Oncogene 1998; 16: 3423–3434.

121. O’Connor DJ, Lam EW, Griffin S, et al. Physical and functional inter-actions between p53 and cell cycle co-operating transcription factors, E2F1and DP1. EMBO J 1995; 14: 6184–6192.

122. Zhang H, Somasundaram K, Peng Y, et al. BRCA1 physically associateswith p53 and stimulates its transcriptional activity. Oncogene 1998; 16:1713–1721.

123. LaThangue, et al., personel communication.124. Lees-Miller SP, Chen YR, Anderson CW. Human cells contain a DNA-

activated protein kinase that phosphorylates simian virus 40 T antigen,mouse p53, and the human Ku autoantigen. Mol Cell Biol 1990; 10:6472–6481.

125. Milne DM, Palmer RH, Campbell DG, Meek DW. Phosphorylation of thep53 tumour-suppressor protein at three N-terminal sites by a novel caseinkinase I-like enzyme. Oncogene 1992; 7: 1361–1369.

126. Meek DW, Simon S, Kikkawa U, Eckhart W. The p53 tumour suppressorprotein is phosphorylated at serine 389 by casein kinase II. EMBO J 1990;9: 3253–3260.

127. Baudier J, Delphin C, Grunwald D, Khochbin S, Lawrence JJ. Charac-terization of the tumor suppressor protein p53 as a protein kinase Csubstrate and a S100b-binding protein. Proc Natl Acad Sci USA 1992; 89:11627–11631.

128. Milne DM, Campbell DG, Caudwell FB, Meek DW. Phosphorylation ofthe tumor suppressor protein p53 by mitogen-activated protein kinases.J Biol Chem 1994; 269: 9253–9260.

129. Ishioka C, Englert C, Winge P, Yan YX, Engelstein M, Friend SH.Mutational analysis of the carboxy-terminal portion of p53 using bothyeast and mammalian cell assays in vivo. Oncogene 1995; 10: 1485–1492.

130. Hall PA, Lane DP. p53—a developing role? Curr Biol 1997; 7: 144–147.131. Kaghad M, Bonnet H, Yang A et al. Monoallelically expressed gene

related to p53 at 1p36, a region frequently deleted in neuroblastoma andother human cancers. Cell 1997; 90: 809–819.

132. Takahashi H, Ichimiya S, Nimura Y, et al. Mutation, allelotyping, andtranscription analyses of the p73 gene in prostatic carcinoma. Cancer Res1998; 58: 2076–2077.

133. Mai M, Yokomizo A, Qian C, et al. Activation of p73 silent allele in lungcancer. Cancer Res 1998; 58: 2347–2349.

134. Nomoto S, Haruki N, Kondo M, et al. Search for mutations andexamination of allelic expression imbalance of the p73 gene at 1p366.33 inhuman lung cancers. Cancer Res 1998; 58: 1380–1383.

J. Pathol. 187: 112–126 (1999)


135. Sunahara M, Ichimiya S, Nimura Y, et al. Mutational analysis of the p73gene localized at chromosome 1p36.3 in colorectal carcinomas. Int J Oncol1998; 13: 319–323.

136. Kovalev S, Marchenko N, Swendeman S, laQuaglia M, Moll UM.Expression level, allelic origin and mutation analysis of the p73 gene inneuroblastoma tumours and cell lines. Cell Growth Different (in press).

137. Jost CA, Marin MC, Kaelin WG Jr. p73 is a human p53-related proteinthat can induce apoptosis. Nature 1997; 389: 191–194.

138. Prabhu NS, Somasundaram K, Satyamoorthy K, Herlyn M, El-DeiryWS.p73, unlike p53, suppresses growth and induces apoptosis of humanpapillomavirus E6-expressing cancer cells. Int J Oncol 1998; 13: 5–9.

139. Schmale S, Bamberger C. A novel protein with strong homology to thetumour suppressor p53. Oncogene 1997; 15: 1363–1367.

140. Osada M, Ohba M, Kawahara C, et al. Cloning and functional analysis ofhuman p51, which structurally and functionally resembles p53. NatureMed 1998; 4: 839–843.

141. Senoo M, Seki N, Ohira M, et al. A second p53 related protein, p73L, withhomology to p73 . Biochem Biophys Res Commun 1998; 248: 603–607.

142. Bork P, Koonin EV. Predicting functions from protein sequences—whereare the bottlenecks? Nature Genet 1998; 18: 313–318.

143. Schultz J, Ponting CP, Hofmann K, Bork P. SAM as a protein interactiondomain involved in developmental regulation. Protein Sci 1997; 6: 249–253.

144. Bian J, Sun Y. p53CP, a putative p53 competing protein that specificallybinds to the consensus p53 DNA binding sites: a third member of the p53family? Proc Natl Acad Sci USA 1997; 94: 14753–14758.

145. Zeng X, Levine AJ, Lu H. Non-p53 sp53RE binding protein, a humantranscription factor functionally analogous to P53. Proc Natl Acad SciUSA 1998; 95: 6681–6686.

146. Drane P, Barel M, Balbo M, Frade R. Identification of RB19A, a 205 kDanew p53 regulatory protein which shares antigenic and functional proper-ties with p53. Oncogene 1997; 15: 3013–3024.


147. Gualberto A, Aldape K, Kozakiewicz K, Tlsty TD. An oncogenic form ofp53 confers a dominant, gain-of-function phenotype that disrupts spindlecheckpoint control. Proc Natl Acad Sci USA 1998; 95: 5166–5171.

148. Blandino G, Levine AJ, Oren M. Mutant p53 gain of function: differentialeffects of different p53 mutants on resistance of cultured cells to chemo-therapy. Oncogene (in press).

149. Wang-Gohrke S, Rebbeck TR, Besenfelder W, Kreienberg R, RunnebaumIB. p53 germline polymorphisms are associated with an increased risk forbreast cancer in German women. Anticancer Res 1998; 18: 2095–2099.

150. Buller RE, Sood A, Fullenkamp C, Sorosky J, Powills K, Anderson B. Theinfluence of the p53 codon 72 polymorphism on ovarian carcinogenesisand prognosis. Cancer Gene Ther 1997; 4: 239–245.

151. Storey A, Thomas M, Kalita A, et al. Role of a p53 polymorphism in thedevelopment of human papillomavirus-associated cancer. Nature 1998;393: 229–234.

152. Rosenthal AN, Ryan A, Al-Jehani RM, Storey A, Harwood CA, JacobsIJ. p53 codon 72 polymorphism and risk of cervical cancer in the UK.Lancet 1998; 352: 871–872.

153. Venkatachalam S, Shi Y-P, Jones SN, et al. Retention of wild-type p53tumors from p53 heterozygous mice: reduction of p53 dosage can promotecancer formation. EMBO J 1998; 17: 4657–4667.

154. Evans SC, Mims B, McMasters KM, et al. Exclusion of a p53 germlinemutation in a classic Li–Fraumeni syndrome family. Hum Genet (in press).

155. O’Neill M, Campbell SJ, Save V, Thompson A, Hall PA. An immuno-chemical analysis of mdm2 expression in human breast cancer and theidentification of a growth regulated cross reacting species, p170. J Pathol1998; 186: 254–261.

156. Yang A, Kaghad M, Wang Y, et al. P63, a p53 homolog at 3q27-29,encodes multiple products with transactivating, death inducing and domi-nant negative activities. Molecular Cell 1998; 2: 305–316.

J. Pathol. 187: 112–126 (1999)

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The p53 pathway