TRENDS in Genetics Vol.18 No.2 February 200290 Review

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The realization that p53 [1], the archetypal tumorsuppressor in higher mammals, in fact belonged to afamily of related genes came in 1997, almost 20 yearsafter the discovery of p53 [2]. The report of the firsthomolog, p73 [3], and the fact it was located in a long-suspected tumor suppressor locus, was met with greatexcitement and anticipation. Was this another tumorsuppressor? And would decades of work on p53 enable usto understand this close relative readily? The situationwas quickly complicated by the appearance of yet

another homolog, p63 (also named KET, p51, p40, p73L)[4–8], and the myriad gene products it encoded. Far fromfitting into classic p53 roles in tumor suppression, thehomologs are claiming their own turf in stem cell biology,neurogenesis and a host of other physiological processes.The past four years of work on p63 and p73 have alsoadded layers of complexity to the p53 family as awhole. Here, we review the individual functions of thep53-related genes and explore evolutionary origins thatcould offer an intriguing perspective on the p53 family.

The burning question: tumor suppressors or not?

When they were first discovered, it seemed entirelyreasonable to imagine that p63 and p73 would follow in the footsteps of p53 and be involved in tumorsuppression and cell cycle control. The sequencesimilarity and conservation of functional domainsamong the p53 family members are indeed striking[3,4]. p63 and p73 both share the hallmark featuresthat identify p53 across all species – an acidic,

The discoveries of the p53 homologs, p63 and p73, have both fueled new insights

and exposed enigmas in our understanding of the iconic p53 tumor suppressor.

Although the pivotal role of p53 in cancer pathways remains unchallenged,

because p63 and p73 are now implicated in stem cell identity, neurogenesis,

natural immunity and homeostatic control. Despite their seemingly separate

tasks, there are hints that the p53 family members both collaborate and interfere

with one another. The question remains, therefore, as to whether these genes

evolved to function independently or whether their familial ties still bind them in

pathways of cell proliferation, death and tumorigenesis.

On the shoulders of giants:

p63, p73 and the rise of p53

Annie Yang, Mourad Kaghad, Daniel Caput and Frank McKeon

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N-terminal transactivation (TA) domain, a highlyconserved core DNA-binding domain, and a C-terminaloligomerization domain (Fig. 1). They can exert p53-likeactivities in various contexts, binding canonical p53sites and transactivating p53 target genes, includingp21, mdm2, BAX and GADD45 [3,9,10]. Moreover, p63and p73 can act as potent inducers of apoptosis [4,7,11],thus fulfilling another important p53 function.

Despite these encouraging findings, however, thefield has struggled to establish any role for the p63 andp73 genes in cancer. However, p73 is at least located in aregion on chromosome 1p36, which exhibits frequent lossof heterozygosity (LOH; Box 1) in human cancers [12].

Yet even the original cloning report noted that theremaining copy of the p73 gene in neuroblastoma cellswas wild type, and p73 protein levels were actuallyhigher in many tumor cell lines than normal tissues [3].Subsequent searches in many types of cancer haveyielded a similar lack of mutation in the remainingallele of p73 [3,13,14]. Considerable efforts have beendevoted to uncovering possible epigenetic means of genesilencing that might alter p73 expression. A consistentpattern has yet to emerge from these analyses, andthere remains no convincing evidence that p73 isinactivated in tumors [13,15]. These data would argueagainst p73 being a tumor suppressor, at least in aclassical sense such as that predicted by Knudson’s two-hit hypothesis [16]. Also, p73-null mice do not showincreased rates of spontaneous tumorigenesis [17].

Cancer aficionados have hardly fared better withp63. As with p73, few p63 mutations have been found intumors [7]. Furthermore, p63 is located in a region onchromosome 3q27-ter that is actually amplified, not lost,in various cancers [4,18], showing more the footprintof an oncogene rather than a tumor suppressor.

But just when one is about to cast the p53 homologsoff as cheap imitations, they challenge us to reconsider.For instance, intriguing links have been made betweenp73 and DNA damage repair pathways; specifically,p73 binds to c-Abl and is stabilized upon gammairradiation [19–21]. By extension, p73 might functionin DNA damage signaling, to guard against geneticinstability and tumorigenesis. More recent studiesimplicate p73 in activation-induced cell death ofthymocytes, and show that this function is tied to theE2F-1 transcription factor [22–24]. Cytogeneticanalyses also suggest that loss of p73 could be involvedin radiation-induced murine T-cell lymphomas [25].

p63/p73: double-edged genes

A closer look at p63 and p73 reveals a probable basis forthe perplexing behavior of these genes. Unlike the p53gene, which encodes essentially one major transcript, thep63 and p73 genes each contain two separate promotersthat direct expression of two fundamentally differentclasses of protein [4,17]. One, denoted TAp63/p73, ismarked by an acidic N terminus with homology to thetransactivation domain of p53. A second promoter,located within an intron and over 30 kb downstream,gives rise to N-terminally truncated (∆N) products thatlack the TA domain. Alternative splicing generatesadditional complexity at the C terminus, rendering at least six major transcripts from each gene (Fig. 1).Numerous other sequences with minor variations atthe N and C termini have also been described [26,27].

A useful, if simplistic, way of classifying the variousp63/p73 isoforms is to determine whether they are ‘p53-like’or not (Table 1). Two activities are characteristic ofp53: transactivation and induction of apoptosis. Cellulartransfection assays have demonstrated that certainisoforms of p63 and p73 possessing transactivating(TA) domains are capable of transactivating p53target genes, and, when exogenously expressed,

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1 2 3 3′ 4 5 6 7 8 9 10 11 12 13 14 15

γ

β

TA/TA* ∆N

∆N

TA Oligo SAM

Oligo SAM PS

PS

Oligo

Oligo

TA Oligo

TA

TA

TA

TA

N

N

N

Oligo

TA DNA binding

DNA binding

DNA binding

DNA binding

DNA binding

DNA binding

DNA binding

Oligo

~25% ~65% ~35% % Identity

p53

1 2 3 3′ 4 5 6 7 8 9 10 11 12 13 14

δ

β

TA

γ

p63

p73

TA Oligo SAM

TA Oligo SAM

~40% % Identity ~85% ~50% ~60%

p63

p73

PS

PS

(a)

(b)

(c)

DNA binding

DNA binding

Fig. 1. p53 family isoforms and gene structure. (a) Comparison of the domain structure of p53 proteinand the six major isoforms encoded by p63 and p73. TA, transactivation domain; Oligo, oligomerizationdomain; SAM, sterile alpha motif domain implicated in protein–protein interactions; PS, post-SAMdomain implicated in transcriptional suppression; ∆N, truncated amino-terminal domain of isoformsderived from an intronic promoter in the p63 and p73 genes. (b) Exon–intron arrangement of the p63 andp73 genes in the human genome. Each gene is spread over ~100 kb, with introns ranging betweenseveral hundred base pairs and 40 kb. The proximal promoter gives rise to the TA/TA* isoforms (TA* is asubclass of TA isoform with an additional 39 amino acids at the N terminus), whereas the distal promoterdirects expression of the ∆N isoforms. The splicing patterns that give rise to the major C-terminalvariations in addition to the α isoform (the β, γand δ isoforms) are indicated. (c) Sequence identitiesbetween p63 and p73. Primary amino acid sequence comparisons between p63 and p73 reveal a highdegree of identity. These proteins are both more homologous to each other than to p53.

Loss of heterozygosity (LOH) commonly refers to the loss by mutation ordeletion of one of two heterogeneous alleles of a tumor suppressor gene incancer cells. For some but not all tumor suppressor genes, the remainingallele will also be inactivated or modified by mutation or deletion.

Box 1. Loss of heterozygosity

Annie Yang

Frank McKeon*

Dept of Cell Biology,Harvard Medical School,240 Longwood Avenue,Boston, MA 02115, USA.*e-mail: fmckeon@hms.harvard.edu

Mourad Kaghad

Daniel Caput

Sanofi Biorecherche,Centre de Labege, CedexBP107, France.

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induce cell death [3,4,7,11]. Importantly, not allTA-p63/p73 proteins demonstrate p53-like properties tothe same extent. TA-p63/p73 α, for example, having thelargest C-terminal domains, appear less active in p53assays [4,27]. These findings hint at regulatory elementsin the α tail – already, a sterile α motif (SAM) has beenidentified here for both p63 and p73 [28,29]. Recentstudies also indicate that the extreme C terminus exertsan autoinhibitory effect of p63/p73 transactivation [30](V. Doetsch, pers. commun.) (Fig. 1).

As a whole, ∆Np63/p73 proteins not only lackp53-like functions including transactivation andpromotion of cell death, but might also act asdominant negatives against p53 activity [4,17,31,32].This latter phenomenon is probably attributable toseveral mechanisms of inhibition. First, ∆Np63/p73can readily compete with p53 (or transactivatingp63/p73 isoforms) for DNA target sites. A secondmode of inhibition might involve the formation oftransactivation-incompetent heterocomplexesbetween ∆N proteins and p53 or TA-p63/p73 [3,4,33](Fig. 2). A variation of this latter mode could bedefective interactions between DNA binding domainsof p53 and the ∆N variants of p63 and p73 [31,34,35].

At present, it is not known how these interactionsaffect p53 activity in a cell. In keratinocytes, for instance,∆Np63 is present at five times the concentration of p53(A. Michaelis and F. McKeon, unpublished), suggestingthe possibility that these cells are functionally p53-deficient under basal conditions (Fig. 3). However, afterexposure to ultraviolet light, this situation is rapidlyreversed through p53 induction coupled with p63degradation [32]. Similarly, ∆Np73 might also be actingto neutralize p53 apoptotic activity in sympatheticneurons, leading to their decreased survival in the p73-null mouse [31]. How such alterations are achieved inthe keratinocyte or any other cell is unknown, but theycertainly involve complex coordination of transcription,translation and protein stability of p53, p63 and p73. It is

also probable that the apparently contradictory TAand ∆N isotypes of the p63 and p73 genes are underindependent control. Specifically, the TA and∆N promoters are 30 to 40 kb apart and are known tohave unique regulatory sites (Fig. 4). For instance, E2F-1sites are present in the TA promoter of the p73 gene,but absent from the ∆N promoter. This arrangementis consistent with the ability of E2F-1 to induce a p73isoform that has apoptotic activities seen in activation-induced T-cell death [22,23,36]. Interestingly, p53 siteshave been characterized in the promoter region of∆Np73, suggesting the possibility that regulatory loopsinvolving ∆N and TA forms of p53, p63 and p73 existand are actively exploited in the cell (M. Kaghad andD.Caput, unpublished). Nevertheless, our understandingof the physiology underlying the networks of interactingp53 family members is rudimentary at best, and yetessential for understanding p53 in tumor suppressionas well as unique activities of the p53 homologs.

Twisted sister: p73 in signaling pathways

Despite the natural inclination to focus on the p53family as a whole, it is undeniable that the p53homologs deserve recognition in their own right(Box 2). The phenotype of the p73-knockout mouseassociates p73 with some of the more fundamentalsignaling systems in vertebrates, including those inpheromone-based social and reproductive interactions,infection control, intracranial pressure and importantaspects of neurogenesis [17]. Not surprisingly, all facetsof the p73-knockout phenotype can be linked to tissuesthat express high levels of p73. For instance, p73 ishighly expressed in airway epithelia, and newborn

Table 1. Differential activities of p63 and p73 isoforms

Isoform Transactivationa Apoptosisa Antagonize p53a Refs

TA*p63α (–) (–) NT [4]TA*p63β NT NT NTTA*p63γ (–) (–) NT [4]TAp63α –/+ (–) NT [4,7]TAp63β +++ +++ NT b

TAp63γ +++ +++ NT [4]∆Np63α (–) (–) +++ [4]∆Np63β (–) (–) +++ b

∆Np63γ (–) (–) +++ [4]

TAp73α ++ ++ NT [3,11]TAp73β ++/+++ ++ NT [3,11]TAp73γ + NT NT [26]TAp73δ ++ NT NT [26]∆Np73α (–) NT +++ [17,31]∆Np73β (–) NT +++ [17,31]∆Np73γ NT NT NT∆Np73δ NT NT NTaNT, not tested; slash, based on separate reports.bAnnie Yang and Frank McKeon, unpublished results.

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DBD DBD

TA

Target gene

DBD

Target gene

DBD

∆N

∆N

∆N

DBD

TA(a) Competition for DNA sites

Binding via DBD ??

X

X

(b) Transactivation incompetent

Hetero-oligomers

Fig. 2. Modes of transcriptional suppression by ∆N isoforms of p63 andp73. (a) Simple competition by non-transactivating ∆N isoforms forDNA target sites employed by TA isoforms (including p53), therebydecreasing expression of regulated genes. (b) Protein–proteininteractions between ∆N and TA isoforms through oligomerizationdomains or other, less well-defined associations, combined withcompetition for DNA sites, could also suppress TA isoform activity.

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p73−/− pups display airway inflammatory responses inthe absence of provocative bacterial infections.

p73-null mice also show various degrees ofhydrocephalus, the basis of which appears to beexcessive cerebral spinal fluid production by cells ofthe choroid plexus. As with the hyperinflammatoryresponses in these mice, it appears that the secretionof cerebral spinal fluid is left in some overdrive mode in the absence of p73. The reproductive andbehavioral defects in p73-null mice appear linked to a p73-dependent expression of pheromone receptorsin these animals. Finally, p73-null mice show veryunusual defects in the hippocampal formation of thebrain, primarily the result of a loss of a discrete set of neurons that act to guide the organization of thehippocampus. Given this complex array of defects in the p73-null mouse, any simplistic attempt tocategorize p73 function seems likely to fail. However,it is fair to say that p73 is central to control systemsthat monitor a wide range of contingency andhomeostatic mechanisms. In this regard, p73 fulfillsone expectation of p53-like genes: that they act assensors for intracellular and extracellular signals,and coordinate responses in the cell.

p63 and epithelial stem cells: use it or lose it

The discovery of p63 was significant because itestablished the concept of the contradictory TA and∆N isotypes that would prove to hold true for p73 aswell. Unlike the conditionally expressed p53, p63 isconstitutively present, especially in the stem cellcompartment of many epithelial tissues [4]. Notably,the high level of p63 in epithelial stem cells is made upalmost entirely of ∆Np63 isoforms from the internal p63promoter, highlighting a role for dominant–negative orrepressive versions of p63 in epithelial stem cell identity.

Only by examining the mouse knockout phenotypewas the real significance of the p63 gene to theepithelial stem cell revealed. p63-null mice are born

but lack limbs and a wide range of epithelial structuresincluding skin, prostate, breast and urothelia [37,38]. Aretrospective analysis of p63-deficient embryogenesisrevealed why these epithelia are lost: the tissues ineffect run out of stem cells that are critical for thedevelopment and maintenance of such multi-layered,regenerative epithelia [38]. The primitive, single-layered ectoderm lacking p63 apparently responds tothe mesodermal signals during late embryogenesis tostratify into a differentiating epidermis. However,unlike wild-type cells that undergo the asymmetricdivision definitive of stem cells, all p63-null basal cellsare triggered to undergo terminal differentiation.Consequently, no stem cells remain to sustain theepidermis and other epithelial tissues dependent onp63 (Box 2). Thus, p63 is the only gene known to beessential for the survival of epithelial stem cells. These findings were further complemented by theidentification of dominant mutations in p63 that yielda number of human syndromes involving limbdevelopment and ectodermal dysplasia [39,40].

The functions of p63 and p53, deduced from theirrespective mouse knockout phenotypes, seemdiametrically opposed. Whereas p63 is most closely tiedto the ‘immortal’properties of epithelial stem cells, p53 isindelibly linked to cell cycle arrest, senescence and cell

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Time post-UV

Pro

tein

leve

ls

∆N p63 p53

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1 2 3 3′ 4

TA ∆NE2F-1

> 30 kb

p53

p63 p73

E2F-1

Fig. 3. Antagonistic interactions between ∆N isoforms and p53. Relativeprotein levels of ∆Np63 (red) and p53 (green) in human keratinocytesupon ultraviolet treatment. In ‘resting cells’ ∆Np63 is predominant, thuspotentially blocking p53 function. Following ultraviolet radiation ofkeratinocytes, ∆Np63 is lost by degradation and p53 levels enhanced bystabilization in response to DNA damage. A similar situation might existin sympathetic neurons where ∆Np73 isoforms are expressed and couldact to suppress the activity of p53 [31].

Fig. 4. Differential control of TA and ∆N promoters in p73 gene. The TAand ∆N promoters of p73 gene are shown, together with regulatorytranscription factors that could differentially alter relative promoterstrength. E2F-binding sites are present in both promoters, whereas p53-binding sites are present only in the ∆N promoter, suggesting thepossibility that any of the p53 family members could contribute toregulation of ∆Np73 expression. Analogous systems of controlprobably exist for the two promoters of p63.

p63

• Involved in epithelial cell maintenance• Essential for development of skin, limbs and

appendages, breast, urothelium, limb formation,prostate

• Possibly involved in the p53 response toultraviolet light.

p73

• Gamma irradiation response• T-cell apoptosis• Sympathetic neuron survival• Hippocampal neurogenesis• Cerebral spinal fluid homeostasis• Inflammatory response• Pheromone detection

Box 2. The separate roles of p73 and p63

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death. It is this stark contrast in functions that againraises the issue of p63’s ‘anti-p53’, possibly oncogenic,activities. Does the high level of ∆Np63 expression inepithelial stem cells indeed render them functionallyp53-null, and is p63 expression significant to thecarcinomas that show 3q26–28 amplification [18,41,42]?The answers to these questions will determine the extentof the links between p63, the limitless proliferativepotential of stem cells, and whether counteracting of p53in these cells by ∆Np63 could contribute to oncogenesis[32,43,44]. Finally, the apparent role of p63 inmediating proliferation versus cellular differentiationcould provide intriguing therapeutic strategies forcancer and other hyperplastic disorders.

Evolution of the p53 family

Although it is probable that the p53 family membersinteract in mammalian cells, it is still striking how the knockout phenotypes indicate their functionalcompartmentalization. Did these genes evolve from aprecursor whose job was primarily to suppress tumors,or rather from one engaged in stem cell control ordetection of invading organisms? Alternatively, thesefunctions might be related somehow such that a singleprecursor molecule could have monitored all three.

The Drosophila and Caenorhabditis eleganssequencing projects now offer a glimpse into theorigins of the family, as each of these organisms has asingle p53-like gene. The fly ‘p53’, highly divergent

from the mammalian p53 isotypes, has beenimplicated in certain genoprotective mechanisms[45–47]. In particular, expression of engineereddominant–negative versions of Dm-p53 appears toprevent the induction of the reaper gene in responseto DNA damage, thus short-circuiting an importantpathway of cell death in flies. In a similar fashion, ‘p53’-deficient worms are developmentally normal but showgerm cells that are resistant to radiation-induced celldeath [50]. Strikingly, Rothman and colleagues notedthat the p53-null animals show chromosome instability,another function attributed to p53 in mammals.

Based on the genoprotective activities of theDrosophila p53-like gene, as well as the absence of theC-terminal SAM domains seen in the mammalianp63 and p73 genes, it has been argued that p53 wasthe ancestor gene and, by extension, that p63 and p73evolved to serve other purposes [45–47]. Counteringthese arguments is the observation that BLASTsearches with Dm-p53 and Ce-p53 reveal a gradient ofsimilarities starting with ‘p53’of squid and clams, thenthe core regions of mammalian p63, p73, and finally p53.The single p53-like gene of mollusks in fact resemblesmammalian p63 very closely, and contains an extendedC-terminus with a SAM domain. A phylogeneticanalysis of the invertebrate and vertebrate p53 genefamilies reveal SAM domains in both protostomes(Mollusca, Arthropoda, Annelidia) and deuterostomes(Echinodermata and Chordata), indicating that theancestral p53 molecule indeed possessed a SAMdomain similar to those of mammalian p63 and p73[48,49] (Fig. 5). This observation, in turn, supportsthe notion that p53 is actually a recent product ofevolution, and not the ancestor gene.

p53: devolution for tumor suppression?

The importance of the p53 family’s origins extendsbeyond an academic interest – understanding the

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ProtostomesTAp63 (+ SAM)

DeuterostomesTAp63 (+ SAM)

Mollusks

Arthropoda

Chordata

Vertebratetransition

Squid, clamTAp63 (+ SAM)

Drosophila (fast-evolving)TAp63 (no SAM)

Xenopus, mouse, human

TAp63 (+ SAM)

Gain ofintronicpromoter

Loss of SAM

Geneduplication

Geneduplication

Loss ofSAM

TAp63 (+ SAM)∆Np63 (+ SAM)

TAp73 (+ SAM)∆Np73 (+ SAM)

p53

AdditionalC-terminalsplicingvariants

Fig. 5. Evolution of the p53 family members. Major branches of metazoanevolution are shown together with known p53 family members. That theprimordial p53 family member is most similar to a TA-p63 protein bearinga C-terminal SAM domain is suggested by the presence of TA-p63(SAM)molecules on both side of the split between deuterostomes andprotostomes. On the protostome side (represented by mollusks andarthropods such as Drosophila), TA-p63 with or without SAM domains isthe only p53-like molecule reported. In contrast, the deuterostome branch,and in particular the vertebrates, show TA-p63, ∆Np63, TA-p73, ∆N73 andp53, suggesting a co-evolution of ∆N isoforms with p73 and p53.

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evolutionary relationships among p53 familymembers could elucidate the present-day functions of these genes, as well as the extent of theirphysiological interactions.

In this regard, a key event is the invertebrate-to-vertebrate transition, where we see the geneduplication events that gave rise to two additionalhomologs of the ancestral p53 (perhaps moreappropriately, p63). This transition is also marked by an acquisition of the dual promoter structure that yields TA and ∆N isoforms of p63 and its close relative, p73. With this added feature, p63 and p73 were now equipped with an array ofpotential signaling molecules that could in turnregulate a multitude of cellular and homeostaticprocesses.

p53 itself, however, seems to have got the ‘shortend’of the evolutionary stick. The single gene producthere is remarkably simple – a TA isoform without anyof the C-terminal elements seen in p53’s relatives(Fig. 1). Yet what p53 lacks in size and complexity, itcertainly makes up for in finesse and potency. Could itbe then, that p53 evolved to embody the most salientfeatures of its predecessors – the exquisite sensorycapabilities of p73, and the cell-fate determination ofp63 – for the essential tasks of genoprotection andtumor suppression in more-complex and longer-livingmammals? And how much do p63 and p73 continue toinfluence p53’s activity in the cell? Deciphering thecooperative and internecine interactions within thep53 family is certain to offer intriguing insights intotumorigenesis and cancer treatment.

Acknowledgements

We thank Volker Doetsch,Joel Rothman, GrahamWalker, Pasqual Ferraraand Mohini Lutchman forhelpful discussions. Thiswork was supported bygrants from the NIH.

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