Physical Interaction with Human Tumor-derived p53 MutantsInhibits p63 Activities*

Received for publication, February 11, 2002Published, JBC Papers in Press, March 13, 2002, DOI 10.1074/jbc.M201405200

Sabrina Strano‡§, Giulia Fontemaggi‡¶, Antonio Costanzo�, Maria Giulia Rizzo‡, Olimpia Monti‡,Alessia Baccarini‡, Giannino Del Sal**, Massimo Levrero�, Ada Sacchi‡, Moshe Oren‡‡,and Giovanni Blandino‡§§

From the ‡Molecular Oncogenesis Laboratory, Regina Elena Cancer Institute, Rome 00158, Italy, �Laboratory of GeneExpression, Fondazione Andrea Cesalpino, University of Rome “La Sapienza” Rome 00161, Italy, **Laboratorio NazionaleCIB, AREA Science Park, Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole Trieste 34012, Italy,‡‡Molecular Cell Biology Department, The Weizmann Institute of Science, Rehovot 76100, Israel

The p53 tumor suppressor gene is the most frequenttarget for genetic alterations in human cancers,whereas the recently discovered homologues p73 andp63 are rarely mutated. We and others have previouslyreported that human tumor-derived p53 mutants canengage in a physical association with different isoformsof p73, inhibiting their transcriptional activity. Here, wereport that human tumor-derived p53 mutants can asso-ciate in vitro and in vivo with p63 through their respec-tive core domains. We show that the interaction withmutant p53 impairs in vitro and in vivo sequence-spe-cific DNA binding of p63 and consequently affects itstranscriptional activity. We also report that in cells car-rying endogenous mutant p53, such as T47D cells, p63 isunable to recruit some of its target gene promoters.Unlike wild-type p53, the binding to specific p53 mu-tants markedly counteracts p63-induced growth inhibi-tion. This effect is, at least partially, mediated by thecore domain of mutant p53. Thus, inactivation of p53family members may contribute to the biological prop-erties of specific p53 mutants in promoting tumorigen-esis and in conferring selective survival advantage tocancer cells.

Approximately half of human tumors bear p53 mutations (1).The most prevalent type of these mutations consists of mis-sense mutations that are frequently accompanied by loss of theremaining wild-type p53 (wt-p53)1 allele (2, 3). The major siteof the p53 mutations is the highly conserved DNA binding coredomain (4–6). Thus, mutant p53 proteins are unable to specif-ically bind DNA and to activate specific wt-p53 target genes.One certain outcome of p53 mutations is the loss of wild typeactivities such as growth arrest, apoptosis, and differentiation.

However, at variance with other tumor suppressor genes, cellswith p53 mutations maintain expression of full-length protein.This may suggest that, at least, certain mutant forms of p53can gain additional functions through which they contributeactively to cancer progression (6–9). Such evidence is providedby several in vitro and in vivo studies (10–17). We and othershave reported that conformational defective p53 mutants canincrease resistance of tumor cells to anticancer treatment andpromote genomic instability (18–20). The molecular mecha-nisms underlying such effects remain to be elucidated.

The recent identification of two p53-relatives, p63 and p73holds new perspectives in studying gain of function of mutantp53 (21–23). p63 and p73 share a significant homology witheach other and with p53. Indeed, they share the same modularorganization, comprising an N-terminal transactivation do-main, a central sequence-specific DNA binding domain, and aC-terminal oligomerization domain. Several p63 and p73 iso-forms are present in cells (21, 23–28). They result either fromthe use of a cryptic promoter that generates p63 isoforms (�Np63�, p63�, and p63�) lacking N-terminal transactivation do-main or by alternative splicing that generates p63 isoforms(p63�, p63�, and p63�) with different C-terminal sequences(30, 32). Exogenous expression of p63 or p73 causes growtharrest, apoptosis, and differentiation, recapitulating some ofthe most characterized p53 biological effects (9, 21, 23, 30, 31).These are mainly mediated by p73 and p63 through the acti-vation of specific p53 target genes such as Bax, IGF-BP3,p21waf1, and cyclin G (16, 21, 23, 24, 32–37). The respectivedeficient mice have provided further insights into the physio-logical role of p73 and p63. p73-deficient mice exhibit severedefects in the development of nervous and immune systems(38). p63 �/� mice are born alive but show striking defects indevelopment. Their skin does not progress from early stages ofdevelopment, lacking stratification as well as expression ofdifferentiation markers. The mammary glands, hair follicles,and teeth are absent in p63-deficient mice (39, 40). In agreementwith this phenotype, p63 was recently found mutated in patientsaffected by autosomal dominant disorder characterized by ectro-dactyly, ectodermal dyspalsia, and facial clefts (41).

The possibility that p53, p73, and p63 form hetero-oligomersin cells has been indicated by recent reports (8, 9). It wasoriginally shown that p73� but not p73� can interact with p53in two-hybrid screening (21). It was subsequently reported thathuman tumor-derived p53 mutants can engage in a physicalassociation in vitro and in vivo either with p73 or p63 (33, 34,42, 43). Recent findings indicate that the association betweenmutant p53 and p73 can be, at least partially, governed by acommon polymorphism at codon 72 of p53 that encodes Arg or

* This work was supported by European Community Grant QLG1–1999-00273, by the Italian Association for Cancer Research, and by theMinistero della Sanita’, Italy. The costs of publication of this articlewere defrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

§ Supported by European Community Grant QLG1-1999-00273.¶ Recipient of a fellowship from Fondazione Italiana per la Ricerca

sul Cancro.§§ To whom correspondence should be addressed: Molecular Onco-

genesis Laboratory, Regina Elena Cancer Institute, Via delle Messid’Oro, 156, Rome 00158, Italy. Tel.: 39-06-52662522; Fax: 39-06-4180526; E-mail: blandino@ifo.it.

1 The abbreviations used are: wt-p53, wild type p53; FCS, fetal calfserum; mAb, monoclonal antibody; GST, glutathione S-transferase;ChIP, chromatin immunoprecipitation.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 21, Issue of May 24, pp. 18817–18826, 2002© 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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Pro (34). The biological outcome of these interactions results infunctional inactivation of p73 transcriptional activity as well asinduction of apoptosis (33, 34, 42, 43).

Therefore, we have further investigated the physical associ-ation between human tumor-derived p53 mutants and p63. Wefound that this association occurs under physiological condi-tions as shown by reciprocal co-precipitation experiments per-formed either in T47D breast cancer cells and HaCat immor-talized keratinocytes. The core domain of mutant p53 and theDNA binding domain of p63 mediate this association. We alsoreport that binding of mutant p53 strongly impairs in vitro andin vivo the binding of p63 to its target gene promoters andconsequently affects its transcriptional activity. Indeed, wefound that in cells carrying endogenous mutant p53, such asT47D cells, p63 is unable to recruit some of its target genepromoters. Similar to wt-p53, overexpression of p63 stronglyinhibits colony formation of SAOS2 cells. The concomitant ex-pression of specific p53 mutants, such as p53His175 andp53His273, but not p53Gly281, counteracts the p63-mediatedgrowth suppression. Such an effect requires the binding of p63to mutant p53 and is not exerted by the coexpression of wt-p53.Of note, the core domain of mutant p53 can also exert such animpairment. Thus, protein-protein interactions between mem-bers of the p53 family might impact on growth and drug resist-ance of cancer cells.

EXPERIMENTAL PROCEDURES

Cell Culture—The H1299 cell line is derived from a human large celllung carcinoma. H1299 cells were maintained in RPMI medium, sup-plemented with 10% fetal calf serum (FCS) (Invitrogen) (19). TheH1299-p53His175#41 cell line was generated as previously reported(43). SAOS2 osteosarcoma cells (a gift from M. Fanciulli) and HaCatimmortalized keratinocytes were maintained in Dulbecco’s modifiedEagle’s medium supplemented with 10% FCS. T47D breast cancer cellswere maintained in RPMI 10% FCS (43).

Plasmids and Transfections—Overexpression of p63 was achieved bytransfection of pcDNA3-myc-p63� (kindly provided by F. McKeon) andpcDNA3-HA-p63�. pcDNA3-HA-p63�-(141–321) was obtained by PCRfollowed by subcloning into pcDNA3-HA vector. Sequences of the oli-gonucleotides and primers are available on request. Overexpression ofmutant p53 was achieved by transfection of the following plasmids:pcDNA3-p53His175, pcDNA3-p53His273, pcDNA3-p53Trp248,pcDNA3-p53His175-(22–23), pcDNA3-p53His175-�proline, pcDNA3-p53His175-(1–338), and pcDNA3-p53His175-(1–355). Overexpressionof the core domain of mutant p53 was achieved by transfection of pEG-FP-p53His175-(74–298). The double mutants of p53His175 and its corewere prepared as previously described (43).

Transient transfections were done in Dulbecco’s modified Eagle’smedium plus 10% FCS by the calcium phosphate method in the pres-ence of BES (N,N-bis(2-hydroxyethyl-2-aminoethanesulfonic acid, so-dium salt) (Sigma). The precipitates were left for 12 h, after which themedium was changed again to RPMI plus 10% FCS. The cells wereharvested at 36 or 48 h, respectively (43).

Immunoprecipitation and Western Blot Analysis—H1299 cells weretransfected in 100-mm plates with 8 �g of DNA and harvested at 36 hafter transfection. Cells were lysed in 900 �l of lysis buffer (50 mM

Tris-HCl (pH 8), 100 mM NaCl, 10% glycerol, 1% Triton X-100, 1 mM

EDTA, 100 mM NaF, 1 mM MgCl2, 2 mM phenylmethylsulfonyl fluoride,protease, and phosphatase inhibitors), and the extracts were sonicatedfor 10 s and centrifuged at 14,000 rpm for 10 min to remove cell debris.Protein concentrations were determined by a colorimetric assay (Bio-Rad). After preclearing for 1 h at 4 °C with Protein G, immunoprecipi-tations were performed by incubating 1.5 mg of whole-cell extract with1.5 �g/sample of anti-p63 polyclonal antibody (H-129 that maps at thecarboxyl terminus of human p63�) (Santa Cruz Biotechnology, Inc.,Santa Cruz, CA) or with anti-hemagglutinin (anti-HA) antibody or witha mixture of anti-p53 mAbs DO1 and 1801 or with anti-IgG polyclonalantibody (Cappel). Immunocomplexes were precipitated with proteinG-agarose beads (KPL, Guilford, CA). The immunoprecipitates werewashed three times with 1 ml of wash NET-gel buffer (50 mM Tris-HCl(pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.25% gelatin, 0.1% Nonidet P-40).The excess liquid was aspirated, and 40 �l of 5� sample buffer wasadded. Immunoprecipitates as well as 1% of each extract were resolvedby SDS-10% PAGE. Protein gels were transferred to nitrocellulose

membranes (Sartorius). For p53 detection, a mixture of p53 mAbs DO1and 1801 (1:40 dilution) or anti-p53 FL393 (1 �g/ml) (Santa CruzBiotechnology) were used respectively; for c-Myc detection, we usedanti-c-Myc (9E10) (Pharmingen) at 2 �g/ml; for maltose-binding protein(MBP)-p63� detection, we used anti-MBP monoclonal antibody (CLON-TECH) at 2 �g/ml; for p21waf1 detection, we used anti-p21 polyclonalantibody (Santa Cruz Biotechnology) at 1:20,000 dilution; forp53His175-(74–298) (GFP-tagged) we used an anti-GFP polyclonal an-tibody (Invitrogen) at 1:5000.

T47D and HaCat cells were lysed as previously described. Aliquots ofcell extracts containing 2.5 mg of total proteins were immunoprecipi-tated with anti-p63 polyclonal antibody, with anti-IgG polyclonal anti-body, or with a mixture of anti-p53 mAbs DO1 and 1801. For p53detection, a mixture of p53 mAbs DO1 and 1801 was used at 1:40dilution. For reciprocal co-precipitation experiments, T47D and HaCatcells were immunoprecipitated with anti-p53FL393 and with a mixtureof anti-p53 mAbs DO1 and 1801, respectively. For p63 detection, ananti-p63 monoclonal antibody (4A4 that was raised against amino acids1–205 of p63) (Santa Cruz Biotechnology) was used at 1:100 dilution.

Western blot analysis was performed with the aid of the enhancedchemiluminescence Supersignal West Pico Stable Peroxidase Solution(Pierce).

Recombinant Proteins and in Vivo Binding Assays—Recombinantproteins employed in the pull-down assay were produced as previouslydescribed (36, 43). Pull-down assays were performed using 20 �g ofimmobilized purified GST fusion proteins or wild type GST that wereincubated with 2 mg of total cellular proteins prepared from H1299 cellstransiently transfected with p53His273, p53Trp248, or p53His175-(74–298), from H-175#41 and T47D cells. The immunoblots were probedwith a mixture of anti-p53 mAbs DO1 and 1801 or with anti-p53 mAb240 or with anti-MBP antibody. Detection was performed with the aidof the enhanced chemiluminescence Supersignal West Pico Stable Per-oxidase Solution (Pierce).

Formaldehyde Cross-linking and Chromatin Immunoprecipitation—H1299–175#41 cells were treated with ponasterone A to induce theexpression of p53H175 for 24 h. DNA and proteins were cross-linked bythe addition of formaldehyde (1% final concentration) 20 min beforeharvesting. Formaldehyde cross-linking was stopped by the addition ofglycine, pH 2.5 (125 mM final concentration), for 5 min at room temper-ature. Cells were scraped off of the plates, resuspended in hypotonicbuffer, and passed through a 26-gauge needle. Nuclei were spun down,resuspended in 300 �l of SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM

Tris-HCl, pH 8, and a protease inhibitor mixture), and sonicated togenerate 500–2000-bp fragments. After centrifugation, the cleared su-pernatant was diluted 10-fold with immunoprecipitation buffer (50 mM

Tris-HCl, pH 8, 150 mM NaCl, 5 MM EDTA, 0.5% Nonidet P-40). Thecell lysate was precleared by incubation at 4 °C with 50 �l of Protein Abeads preadsorbed with sonicated single-stranded DNA and bovineserum albumin. The cleared lysates were incubated overnight with ananti-p63 polyclonal antibody (H-137, whose epitope corresponds toamino acids 15–151 of the DNA binding of �Np63�) (Santa Cruz Bio-technology) or with a control antibody. Immune complexes were precip-itated with protein A beads preadsorbed with sonicated single-strandedDNA and bovine serum albumin. After centrifugation, the beads werewashed and the antigen was eluted with 1% SDS, 100 mM sodiumcarbonate. DNA-protein cross-links were reversed by heating at 65 °Cfor 4–5 h, and DNA was phenol-extracted and ethanol-precipitated.Levels of Bax, p21waf1, 14-3-3�, and p53AIP1 promoter DNAs weredetermined by PCR using oligonucleotides spanning the p53 bindingsites. The following specific oligonucleotides were used: Bax (down,5�-CTG GGC AAC ATA GAG AGA CCT CAT; up, 5�-CCA GCC AGGACG TTA TAG ATG ACT); p21waf1 (down, 5�-CAT TGT TCC CAG CACTTC CTC TC; up, 5�-AGA AAG CCA ATC AGA GCC ACA G); 14-3-3�(down, 5�-CAT CAG AGTAAG ACC CTA TCT C; up, 5�-AAT GCT ACAGGG TTT CCA AGG); and p53AIP1 (down, 5�-TGG GTA GGA GGTGAT CTC ACC; up, 5�-GAG CAG CAC AAA TGG ACT GG). Oligonu-cleotides specific for glyceraldehyde-3-phosphate dehydrogenase pro-moter (down, 5�-AAA AGC GGG GAG AAA GTA GG; up, 5�-TCT CTTTGG GCC CTC CGA TC) were used as negative control.

For the chromatin immunoprecipitation (ChIP) performed on T47Dcells, the following oligonucleotides were used: 14-3-3� (down, 5�-CTGTAC TTC AGC CTG CAG ATC AGA G; up, 5�-CCG ACC TAA TAG TTGAGC CAG GAT); Bax (down, 5�-CTG GGC AAC ATA GAG AGA CCTCAT; up, 5�-CCA GCC AGG ACG TTA TAG ATG ACT); cyclin G (down,5�-GAT CTG ATA TCG TGG GGT GAG GT; up, 5�-CCC ACA CCA ACTAAA GAC AGG AAG); and Mdm2 (down, 5�-GCA GGT TGA CTC AGCTTT TCC TCT; up, 5�-GTG GTT ACA GCC CCA TCA GTA GGT A-3�).

Electrophoretic Mobility Shift Assay—The electrophoretic mobility

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shift assay was performed in a 20-�l DNA binding reaction, whichcontained 30 �g of whole cell extract, 4 fmol of labeled duplex oligonu-cleotide, binding buffer (50 mM Tris-HCl, pH 7.5, 40% glycerol, 100 mM

dithiothreitol, 2 �g/ml bovine serum albumin, and 0.2% Triton X-100)and 300 �g of salmon sperm. The reaction was carried out at roomtemperature for 10 min, and the protein-DNA complexes were subjectedto native electrophoresis on 5% acrylamide, 0.5� TBE gel. Anti-p73polyclonal antibodies C-17 and C-20 (Santa Cruz Biotechnology) andanti-p63 polyclonal antibody H-129 (Santa Cruz Biotechnology) wereadded to the labeled oligonucleotide and incubated on ice for 15 min.The following oligonucleotide was used: TCA CAA GTT AGA GAC AAGCCT GGG CGT GGG CAT TAT T.

Luciferase Assays—H1299 cells (3 � 105/60-mm plate) were trans-fected with reporter plasmid together with the indicated combinationsof plasmids. An equal number of pCMV-�-gal plasmids was added toeach transfection reaction mixture. 36 h later, cells were rinsed withcold phosphate-buffered saline, resuspended in cell lysis buffer (Pro-mega Corp., Madison, WI), and incubated for 10 min at room temper-ature. Insoluble material was spun down, and luciferase activity wasquantitated using a commercially available kit (Promega) with the aidof a TD-20E luminometer (Turner). The values were normalized for�-galactosidase and protein contents.

Indirect Immunofluorescence—Cells growing on glass coverslipswere fixed with cold methanol and incubated at �20 °C for 30 min.After rehydration with phosphate-buffered saline for 5 min, the cellswere stained for 1 h with a mixture of p53 mAbs DO1 and 1801.Staining with the secondary antibody and with 4�,6-diamidino-2-phe-nylindole was performed as described before (19), followed by visual-ization under a fluorescence microscope.

Colony Suppression Assays—SAOS2 cells were plated and tran-siently transfected as reported above. 24 h after the transfection, cellswere detached and replated in triplicate at 3 � 104/60-mm dish. Twodays later, puromycin (2 �g/ml) was added to each plate and main-tained for 72 h. Two weeks later, colonies were fixed in methanol andstained with Giemsa solution followed by washing with water.

RESULTS

The Association between Human Tumor-derived p53 Mutantsand p63 Occurs under Physiological Conditions—To determinethe biological relevance of the association between mutant p53and p63, we performed coprecipitation experiments usingT47D breast cancer cells and HaCat skin-derived immortalizedkeratinocytes carrying endogenous mutant p53Phe194 andmutant p53Y179/W282, respectively, as well as endogenousp63 (34, 44). Following a preclearing, equal portions of cellextract were taken for immunoprecipitation with control anti-

IgG polyclonal serum (Fig. 1, A–C, lane 3), with anti-p63 poly-clonal antibody (Fig. 1, A–C, lane 2), or with a mixture ofanti-p53 mAbs DO1 and 1801 (Fig. 1A, lane 4). Immunopre-cipitates were subjected to immunoblot with a mixture of anti-p53 mAbs DO1 and 1801. Aliquots of total cell lysate fromcontrol cells (Fig. 1, A and B, lane 1) were directly applied onthe gel. As shown in Fig. 1, A–C, p53 mutants were detectedonly in the immunoprecipitates with p53 or with p63 (lanes 2and 4) and not in the anti-IgG immunoprecipitates (lane 3).Reciprocal co-precipitation experiments of T47D and HaCatwere performed by immunoprecipitation with anti-p53FL393and a mixture of anti-p53 mAbs DO1 and 1801, respectively.Equal aliquots of lysates derived from the above mentionedcells were immunoprecipitated with anti-IgG serum or withanti-HA monoclonal antibody. Immunoblot was probed with amonoclonal anti-p63 antibody. As shown in Fig. 1, B–D, co-precipitated p63 was detected only in the p53-immunoprecipi-tates (lane 2). Of note, p63�, which is quite abundant in T47Dcells as verified by reverse transcription-PCR analysis (data notshown), is involved in the association with mutant p53 (Fig. 1B)

Thus, these results indicate that the association betweenmutant p53 and p63 occurs under physiological conditions.

The DNA Contact-defective p53 Mutants, p53His273 andp53Trp248, Engage in a Physical Association with Endogenousp63�—In an attempt to verify whether DNA contact-defectivep53 mutants, such as p53His273 and p53Trp248 can also as-sociate with endogenous p63�, we performed coprecipitationexperiments. To this end, H1299 cells were transiently trans-fected with a vector encoding p53His273 or p53Trp248 or withan empty vector. Cell lysates derived from these cells wereprocessed as previously reported (Fig. 1C). As seen in Fig. 2A,coprecipitated mutant p53His273 and p53Trp248 were broughtdown only in anti-p63 immunoprecipitates (lanes 8 and 9). Thespecificity of these interactions was further confirmed by apull-down assay in which identical cell lysates were incubated

FIG. 1. The association between mutant p53 and p63 occursunder physiological conditions. A–D, T47D human breast cancercells and HaCat skin-derived immortalized keratinocytes carryingp53Phe194 and p53Y179/W282, respectively, were extracted and sub-jected to immunoprecipitation (IP) as described under “ExperimentalProcedures,” followed by immunoblot (IB) using a mixture of anti-p53mAbs DO1 and 1801 (A and B) and anti-p63 monoclonal antibody (Cand D). Lanes 2–4 (A) and lanes 2 and 3 (B–D) represent immunopre-cipitates corresponding to 2.5 mg of total cell protein. Positions ofprotein molecular size markers are indicated on the left. FIG. 2. p63� engages in a physical interaction with p53His273

and p53Trp248. A, cell lysates derived from H1299 cells transientlytransfected with a plasmid encoding p53Trp248 (H-248) or p53His273(H-273) were subjected to immunoprecipitation as reported in Fig. 1A.B, cell extracts derived from H-273 and H-248 cells were incubated withGST-p63� and GST alone. Immunoblots relative to panels A and B wereprobed with a mixture of anti-p53 mAbs DO1 and 1801. Positions ofprotein molecular size markers are indicated on the left.

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with GST-p63� or GST alone. Specifically bound mutant p53was detected only when cell lysates (1.5 mg/lane) of H1299overexpressing p53 mutants were incubated with GST-p63�

(Fig. 2B, lanes 6 and 9). Aliquots of unprocessed lysates (100�g/lane) were processed and probed for each of the above re-ported experiments.

Taken together, these results indicate that other humantumor-derived p53 mutants distinct from those carried byT47D and HaCat cells can engage in a physical association withendogenous p63. Furthermore, they show that the two mostfrequent p53 mutations in human cancers, such as p53His273and p53Trp248, can interact with p63.

The Core Domain of Mutant p53 Is Sufficient for the Associ-ation with p63�—We and others have previously shown thatthe core domain of mutant p53 is sufficient for the associationwith different isoforms of p73 (42, 43). To further characterizethe association between mutant p53 and p63, we aimed toidentify which p53 domain is involved in that association.

To this end, we transiently co-transfected H1299 cells with avector encoding p53His175 or the indicated double mutantstogether with a vector encoding a c-Myc-tagged version of p63�

(Fig. 3B). In agreement with previously reported data, wefound that p53His175 and p63� can associate in reciprocalcoprecipitation experiments (Fig. 3A, lane 4, and Fig. 3B, lane2). Furthermore, as shown in Fig. 3B (lanes 3–6), p53 doublemutants were present in anti-p63 immunoprecipitates. Wechecked the efficiency of the p63 immunoprecipitation by rep-robing the blot with anti-c-Myc monoclonal antibody (Fig. 3B,middle panel). Protein levels of p53His175, its double mutants,and c-Myc-tagged p63� reached in each transient co-transfec-tion are shown in Fig. 3B (lower panels). The reported resultsclearly suggest that the core domain of mutant p53 might besufficient for the association with p63�. To investigate thisissue, we employed an in vivo binding assay. Total cell lysatesof H1299 cells transiently transfected either withpEGFPp53His175-(74–298) vector or with its related emptyvector were incubated with GST-p63� or with GST alone. Spe-cifically bound mutant p53 was detected by probing the immu-noblot with anti-p53 mAb 240 (Fig. 4A, lane 4). To verifywhether the core of mutant p53 can also engage in a physicalinteraction with endogenous p63�, H1299 cells were tran-siently transfected with the above reported vectors. Cell lysatesderived from these cells were immunoprecipitated with anti-p63 and anti-IgG polyclonal sera and subjected to immunoblotwith anti-p53 mAb 240. As shown in Fig. 4B, p53His175 (74–298) was brought down only from extracts of cells transfectedwith pEGFPp53His175 (74–298) and immunoprecipitated withanti-p63 polyclonal serum (lane 3).

To further investigate whether the association between mu-tant p53 or its core domain and p63 occurs directly, we em-ployed an in vitro binding assay. Bacterially expressed andpurified MBP-p63� protein was incubated with GST-p53His175 or with GST-p53His175 (74–298) or with GSTalone. The immunoblot was probed with anti-MBP antibody.Specifically bound p63� was detected only when MBP-p63�

was incubated with mutant p53 or with its core domain (Fig.4C, lanes 3 and 4). GST fusion proteins employed in this ex-periment are shown in the lower panel.

Thus, the reported results demonstrate that the core domainof mutant p53 can engage in vitro and in vivo in a physicalinteraction with p63�. The association between human tumor-derived p53 mutants and p63� might occur directly.

The DNA Binding Domain of p63 Is Involved in the Associ-ation with Mutant p53—We have previously reported that theregion of p73� including the sequence-specific DNA bindingand the oligomerization domains is sufficient for the interac-

tion with mutant p53 (43). In an attempt to identify the mini-mal domain of p63 involved in that association, we analyzedwhether the DNA binding domain of p63� is sufficient for theassociation with mutant p53. To this end, we transiently co-

FIG. 3. In vivo association between p63 and mutant p53His175.A, H1299 cells overexpressing mutant p53His175 and a c-Myc taggedversion of p63� were lysed and subjected to immunoprecipitation witha mixture of anti-p53 MAbs DO1 and 1801. Immunoblot was probedwith anti-c-Myc monoclonal antibody (upper panel). The blot was rep-robed with a mixture of anti-p53 mAbs DO1 and 1801. B, H1299 cellswere transiently transfected with a plasmid encoding p63�/c-Myc incombination with p53His175, p53His175-(22–23), p53His175�proline,p53His175-(1–355), or p53His175-(1–338). Cell extracts were subjectedto immunoprecipitation (IP) with anti-p63 polyclonal antibody. Immu-noblot (IB) was probed with a mixture of anti-p53 mAbs DO1 and 1801(top panel). The blot was reprobed with anti-c-Myc monoclonal antibody(middle panel). Aliquots containing 100 mg of total cell protein weresubjected to immunoblot with a mixture of anti-p53 mAbs DO1 and 1801and with anti-c-Myc monoclonal antibody, respectively (lower panels).Positions of protein molecular size markers are indicated on the left.

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transfected H1299 cells with a vector encoding an HA-taggedversion of full-length p63� or its DNA binding domain togetherwith a plasmid encoding mutant p53His175. As seen in Fig. 5,p53His175 was detected in immunoprecipitates derived eitherfrom cells overexpressing full-length p63� or the p63 DNAbinding domain. Similar data were obtained employing an invivo binding assay in which GST-p63�, GST-p63�-(140–321),and GST alone were incubated with cell lysates of H1299 cellsoverexpressing p53His175 (data not shown).

Thus, the DNA binding domain of p63 is sufficient for theassociation with human tumor-derived p53 mutants.

Functional Inactivation of p63 by Mutant p53—We havepreviously reported that the association with mutant p53 in-terferes with transcriptional activity of p73�, -�, -�, and -� (43).To address the functional relevance of mutant p53 binding top63�, we performed DNA binding and transactivation assays.To this end, DNA binding reactions were employed using GST-p63�, radiolabeled oligonucleotide resembling the p53 bindingsite of Bax promoter with or without cell lysates derived fromthe indicated cell lines (Fig. 6A). As shown in Fig. 6A, thebinding of p63� to DNA was specific, because it was super-shifted by the addition of anti-p63 antibody but not by anti-p73antibody (Fig. 6A, lanes 7 and 8). Furthermore, a 200-foldmolar excess of unlabeled probe specifically inhibited this bind-ing (Fig. 6A, lane 5) but not a 1000-fold molar excess of anunrelated probe (Fig. 6A, lane 6). Mutant p53His175 stronglyreduced the binding of p63� to DNA (Fig. 6A, lane 4). Of note,

a similar effect was also seen in the presence of cell extractsderived from H1299 cells overexpressing the core domain ofmutant p53 (Fig. 6A, lane 12).

Transactivation assays were performed by cotransfection ofH1299 cells with p63� together with mutant p53His175 or withmutant p53His175 (74–298) and a luciferase reporter genedriven by the p53-responsive Bax promoter. In agreement withDNA binding results, mutant p53His175 as well as its coredomain markedly reduced the transcriptional activity of p63�(Fig. 6B).

To determine whether mutant p53 can also interfere withthe activation of endogenous target genes by p63�, we analyzedthe levels of p21waf1 protein in cells overexpressing p63� aloneor together with a plasmid encoding mutant p53His175 as wellas its core domain. As seen in Fig. 7, overexpression of p63�caused a clear accumulation of p21waf1 protein (lanes 2 and 6).This is markedly reduced when mutant p53 or its core domainis concomitantly overexpressed (lanes 3 and 8).

Taken together, these results demonstrate that human tu-mor-derived p53 can interact with p63 not only physically butalso functionally.

The Binding of Mutant p53 to p63 Interferes in Vivo with theRecruitment of Its Target Genes—To further investigatewhether the physical association with mutant p53 can interferein vivo with the binding of p63 to a specific DNA binding site,we performed chromatic immunoprecipitation (ChIP) and co-precipitation experiments in the same cell context such asH1299 cells whose mutant p53His175 expression is tightlyregulated by ponasterone A (H1299-p53His175#41) (43). Inorder to induce mutant p53 expression, the cell line was grownin the presence of ponasterone A (2.5 �M/ml) for 24 h.

To perform ChIP experiments, cells with or without ponas-terone A addition were treated with formaldehyde to cross-linkproteins to DNA. The cross-linked chromatin derived fromequivalent numbers of cells was immunoprecipitated by usingeither an anti-p63 or an unrelated anti-IgG antibody. Follow-ing immunoprecipitation, the cross-linking was reversed, and

FIG. 4. The core domain of mutant p53 binds in vitro and invivo to p63. A, cell extracts derived from H1299 cells transientlytransfected either with a plasmid encoding a GFP-tagged version ofp53His175-(74–298) or with the relative empty vector were incubatedwith GST-p63� or with GST alone. Immunoblot (IB) was probed withanti-p53 mAb 240. B, cell extract employed in A was subjected toimmunoprecipitation (IP) with anti-p63 or anti-IgG polyclonal sera,followed by immunoblotting with anti-p53 mAb 240. C, bacterial puri-fied MBP-p63� was incubated with GST-p53His175 or GST-p53His175-(74–298) or with GST alone and transferred to nitrocellulose mem-brane. The blot was probed with anti-MBP antibody (upper panel).Coomassie staining of replica gel shows the GST fusion proteins (lowerpanel).

FIG. 5. The specific DNA binding domain of p63 is sufficient forthe binding to mutant p53. H1299 cells were transiently transfectedwith a plasmid encoding a HA-tagged version of p63� or p63�-(140–321)together with a plasmid encoding p53His175. Cells were lysed andsubjected to immunoprecipitation (IP) with anti-HA monoclonal anti-body. Immunoblot was probed with a mixture of anti-p53 mAbs DO1and 1801 (upper panel). Aliquots containing 100 �g of total protein fromunprocessed lysates were subjected to immunoblot (IB) with a mixtureof anti-p53 mAbs DO1 and 1801 (middle panel) or with anti-HA mono-clonal antibody (lower panels).

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the amount of endogenous Bax, p21waf1, 14-3-3�, and p53AIP1promoters was monitored in each sample by PCR amplificationusing internal primers (33, 35, 45–47). We found that Bax,p21waf1, and 13-3-3� promoters were present in the chromatinimmunoprecipitates with anti-p63 antibody (Fig. 8A, upperpanels). Of note, these promoters were not present in identicalchromatin immunoprecipitates derived from cells overexpress-ing p53His175 upon the addition of ponasterone A (Fig. 8A,upper panels). As expected, anti-IgG immunoprecipitates de-rived from the above mentioned cells did not contain the abovementioned promoters. As shown in Fig. 8A, p63 does not bind tothe p53AIP1 promoter. This may be related to target specificityor to the lack of p63 modifications that make it able to recruitdirectly such a p53 target gene. To evaluate whether the bind-ing of p63 to the Bax, p21waf1, and 14-3-3� promoters wasspecific, we applied the chromatin immunoprecipitation assayto the GADPH promoter that does not contain the binding sitefor p63. Thus, the glyceraldehyde-3-phosphate dehydrogenase

promoter was present neither in anti-p63 nor anti-IgG immu-noprecipitates (Fig. 8A, lower panel).

To perform co-precipitation experiments, cell extracts de-rived from H1299-p53His175#41 with or without ponasteroneA stimulation were immunoprecipitated either with anti-p63and anti-IgG polyclonal sera and subjected to immunoblot witha mixture of anti-p53 mAbs DO1 and 1801. As shown in Fig.8B, mutant p53His175 was only present in immunoprecipitatesderived from H1299-p53His175#41 cells upon induction withponasterone A (lane 6). The predominant nuclear localization ofmutant p53His175 upon ponasterone A induction is shown inFig. 8C.

To further verify whether the binding of p63 to its targetgene promoters was impaired in cells carrying endogenousmutant p53, we performed ChIP experiments in T47D cells. Tothis end, cross-linked chromatin derived from T47D cells wasimmunoprecipitated and processed as reported in Fig. 8A. Wefound that the binding of p63 to Bax, 14-3-3�, and cyclin Gpromoters (upper panels) is impaired, whereas the binding toMdm2 promoter (lower panel) is still present (Fig. 9) (48).

Taken together, the results of the ChIP (Figs. 8A and 9) andthe co-precipitation experiments (Figs. 1 (A and B) and 8B)clearly indicate that, only upon binding to mutant p53, endog-enous p63 is unable to recruit specific target genes.

Overexpression of Specific Human Tumor-derived p53 Mu-tants Markedly Reduces p63-mediated Growth Suppres-sion—To further investigate the biological relevance of theassociation between mutant p53 and p63, we investigatedwhether exogenous expression of mutant p53 interferes withp63�-mediated growth suppression. To this end, SAOS2 cellswere cotransfected with p63� together with a plasmid encodingp53His175, or p53His273, or p53His175-(74–298) and as acontrol with an empty vector. Overexpression of p63� sup-pressed colony formation of SAOS2 cells as compared with thatof cells transfected with empty vector (Fig. 10A). Conversely,SAOS2 cells regained colony formation when mutant p53 wasoverexpressed (Fig. 10A). Interestingly, the core domain ofmutant p53 could also promote such an effect (Fig. 10A). Inagreement with the above reported observations, we found thattransient overexpression of p63� strongly inhibited cell growth

FIG. 6. Mutant p53 markedly reduces p63 transcriptional ac-tivity. A, gel shift assays were performed by the incubation of GST-p63� with H1299 transiently transfected with the indicated plasmids(lanes 3–12) and with a 32P-radiolabeled p53 DNA-binding site of hu-man Bax. Specific and nonspecific competitions are shown in lanes 5and 6. Supershift with anti-p63 polyclonal antibody is shown in lane 7.B, H1299 cells were transiently transfected with the indicated combi-nations of plasmids encoding p63� (25 ng/60-mm dish), p53His175 (100ng/dish), or p53His175-(74–298) (100 ng/dish) or vector control togetherwith a Bax luciferase reporter plasmid (50 ng/dish). The total amount oftransfected DNA in each dish was kept constant by the addition ofempty vector wherever necessary. Cell extracts were prepared 36 hlater and subjected to determination of luciferase activity. Results arerepresented as repression of p63 transcriptional activity. Histogramsshow the mean of a typical experiment of three performed in triplicate;bars indicate S.D.

FIG. 7. Overexpression of mutant p53His175 or of its core do-main markedly reduces the amount of p63-inducible p21waf1

protein. H1299 cells were transiently transfected with the indicatedplasmid combinations. The total amount of transfected was maintainedconstant by the addition of a control vector. Cell extracts (50 �g/lane)were prepared 36 h later, subjected to SDS-PAGE, and immunoblottedwith anti-p21waf1 polyclonal serum, with a mixture of anti-p53 mAbDO1/1801, with anti-c-Myc monoclonal antibody, with anti-GFP serum,or with anti-�-Hsp70 antibody for equal loading.

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of SAOS2 cells, whereas coexpression of p53His175 restoredsuch property to a similar extent as control cells (Fig. 10B).Unlike mutant p53, wt-p53 is per se able to inhibit colony forma-tion of SAOS2 cells, but when co-expressed with p63� it does notinterfere with p63-mediated growth suppression (Fig. 10A).

In an attempt to verify whether each p53 mutant can bindand counteract p63 activities, we assessed the effects inducedby mutant p53Gly281 overexpression on the p63-mediatedgrowth suppression. As shown in Fig. 11A, mutant p53Gly281does not counteract p63-mediated suppression of SAOS2 colonyformation. The requirement of the physical association of mu-tant p53 to p63, in order to impair the activities of the latest, isclearly indicated by the finding that mutant p53Gly281 doesnot interact with p63� (Fig. 11B), whereas, under the sameexperimental conditions, p53His175 binds to p63� (Fig. 11C).

To explore whether the counteracting effect of mutant p53 onp63-mediated growth suppression occurs through the reductionof p63 protein levels, we looked at the protein levels of p63�upon overexpression of p53His175 or p53Gly281 in SAOS2cells. We found that such overexpression does not impact on theprotein levels of p63� (Fig. 11D).

Taken together, these results contribute to further define afunctional role for the association between specific human tu-mor-derived p53 mutants and p63.

DISCUSSION

Recent reports have clearly indicated the existence of a net-work of protein-protein interactions between the members ofthe p53 family in cancer cells (33, 34, 42, 43). Here we providefurther evidence on the physical association between humantumor-derived p53 mutants and p63. In agreement with thedata reported by Gaiddon et al. (42), we show that the interac-tion between mutant p53 and p63 occurs under physiologicalconditions. The simplest way to interpret such in vivo interac-tion could be that in tumors bearing mutant p53 growth inhi-bition, apoptosis or differentiation induced by p63 is impaired.In support of this, we show that overexpression of mutant p53markedly reduces transcriptional activity of p63 as well as

FIG. 8. Mutant p53 interferes in vivo with the binding of p63 to target gene promoters. A, cross-linked chromatin from H175#41 withor without ponasterone A treatment was immunoprecipitated with antibodies to p63 and IgG and analyzed by PCR with primers specific for theindicated promoters (see “Experimental Procedures”). Input corresponds to nonimmunoprecipitated cross-linked chromatin. B, H1299-induciblecell line was generated as reported under “Experimental Procedures.” Cell extracts were prepared from H1299-p53His175#41 24h after theaddition of 2.5 �M of ponasterone A (lanes 1–3). Identical cell extracts were prepared from untreated cells. Aliquots of 1.5 mg of protein weresubjected to immunoprecipitation with anti-p63 or with anti-IgG polyclonal sera. C, subcellular localization of mutant p53His175 is shown. Cellswere stained with 4�,6-diamidino-2-phenylindole to visualize nuclei and a mixture of anti-p53 mAbs DO1 and 1801 to visualize mutant p53.

FIG. 9. The binding in vivo of p63 to some of its target genepromoters is impaired in T47D cells. Cross-linked chromatin fromT47D cells was immunoprecipitated with antibodies to p63 and IgG andanalyzed by PCR with primers specific for the indicated promoters (see“Experimental Procedures”). Input corresponds to nonimmunoprecipi-tated cross-linked chromatin.

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counteracting growth suppression by p63. The tumor suppres-sor function of p63 and p73 has been challenged by severalobservations (49). To date, both p63 and p73 have been foundrarely mutated in human tumors. Indeed, several studies havereported that overexpression of p73 or �Np63 is present inneuroblastoma, colorectal cancer, bladder cancer, nasopharin-geal carcinomas, squamous-cell carcinoma of head and neck,and hepatocellular carcinoma (4, 50–57). Thus, it is possiblethat overexpression of p63 and p73 in tumors is tolerated bythe presence of mutant p53. p73 and possibly p63 can be acti-vated in response to DNA damage. Indeed, it has been reportedthat p73 can be stabilized and tyrosine-phosphorylated in re-sponse to cis-platin and �-irradiation, respectively (58–60).These post-translational modifications of p73 require a compe-tent kinase-active c-Abl (58–60). A more recent work has re-

ported that, in response to DNA damage, p73 can be acetylatedand consequently driven to specific target genes (61). We haverecently shown that the co-activator YAP depicts specificity inbinding and enhancing transcriptional activity of p53 familymembers (36, 62, 63). YAP binds to p63 and p73 but not p53and promotes their ability to activate proapoptotic genes suchas Bax (36).2 Taken together, these results indicate that inresponse to DNA damage or diverse types of stress, p63 and/orp73 can promote apoptosis, being involved in alternative path-ways to those recruiting p53. The identification of specific p63or p73 target genes should highlight the downstream events ofsuch pathways. Gain of function of mutant p53 could result in

2 S. Strano and G. Blandino, unpublished observations.

FIG. 10. Mutant p53 overexpressionrescues p63-induced growth suppres-sion. A, SAOS2 cells were grown in60-mm dishes and transfected with theindicated plasmids together with a select-able marker plasmid. The ratio betweenp63 expression plasmid (2 �g/transfec-tion) and the ones encoding for p53 mu-tants (6 �g/transfection) and its core do-main was 1:3. An equal amount of pBabe-puro (0.5 �g/transfection) was added toeach transfection. Cells were replated andselected with puromycin as reported un-der “Experimental Procedures.” The datashown represent the average of number ofcolonies formed relative to cells trans-fected with the marker alone. Error barsindicate S.D. of a representative experi-ment out of three performed in triplicate.B, SAOS2 cells were transiently trans-fected with the indicated plasmid combi-nations. The total amount of transfectedDNA was maintained constant by the ad-dition of an empty control vector. 48 hlater, cells were fixed in paraformalde-hyde for 10 min at room temperature. Af-ter rehydration with phosphate-bufferedsaline for 5 min, cells were visualized andphotographed with the aid of phase-con-trast microscopy.

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binding and sequestering p63 from pathways, culminating ineither growth arrest or apoptosis upon cancer treatment. Asshown by chromatin immunoprecipitation experiments (Figs. 8and 9), the binding of endogenous p63 to the Bax, p21waf1,14-3-3�, cyclin G, and p53AIP1 promoters is impaired in cellscarrying either exogenous or endogenous mutant p53. Thismight represent a molecular basis to the impaired apoptoticactivity of p63 in tumors bearing mutant p53.

We originally reported that the core domain of mutant p53and a large region of p73, including the DNA binding and theoligomerization domains, mediate this association in vitro andin vivo (43). A more recent work has reported that the coredomain of mutant p53 can either bind to p73 or to p63 (42). Bydeletion studies, we now report that the interaction betweenmutant p53 and p63 occurs through the respective DNA bind-ing domains. The identification of the minimal stretch of resi-dues of p63 and mutant p53 directly involved in this interactionmay allow the design of small peptides aimed to disassemblethe protein-protein complex and to make available p63 foranti-tumor effects. The core domain of mutant p53 has beenregarded as inactive, since it cannot bind and activate wt-p53target genes. Taken together, these observations with the pre-viously reported data define the core domain of mutant p53 asa protein-protein interaction module that might contribute togain of function of mutant p53 by sequestering and inactivatingproteins required for anti-tumor functions. Further support forthe potential role of the core domain in gain of function activityof mutant p53 might be provided by its ability to markedlyreduce p63 transcriptional activity and to counteract p63-in-duced growth inhibition as found in transactivation and colonysuppression assays. These findings do not exclude the possibil-ity that the N terminus and/or C terminus (or termini) ofmutant p53 can play a role in oncogenic activity of mutant p53(64–67). Indeed, mutant p53Gly281 that has been reported to

exert, in vitro and in vivo, gain of function activity through itsN-terminal transactivation domain does not associate with andcounteract p63-mediated growth suppression (Fig. 11, A andB). These findings might allow the definition of two classes ofgain of function p53 mutants. The first one may account formutants (p53His175 and p53His273) whose gain of functionactivity relies on protein-protein interactions with p63 and p73,whereas the second one includes p53 mutants (p53Gly281) thatexert such activity independently from the physical associationwith the p53 family members. Thus, gain of function of mutantp53 might result from a combination of specific protein-proteininteractions as well as from the activation or repression ofspecific target genes. In this case, a model based on the sequen-tial role for the domains of mutant p53 can be considered. Arather speculative hypothesis might suggest that the core do-main of mutant p53 plays the major role in sequestering andinactivating proteins required for biological activities of onco-suppressor proteins, such as p63 and p73, whereas both N-andC-terminal regions can mainly control activation or repressionof specific target genes.

Acknowledgments—We thank F. McKeon, A. Levine, B. Vogelstein,and G. Cesareni for expression plasmids; D. Lane for DO1 antibody; andM. Fanciulli for SAOS2 cells. We are grateful to M. Sudol for helpfulsuggestions and revision of the manuscript.

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Mutant p53 Interacts with p6318826

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Giovanni BlandinoLevrero, Ada Sacchi, Moshe Oren and

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Sabrina Strano, Giulia Fontemaggi, Antonio  ActivitiesTumor-derived p53 Mutants Inhibits p63 Physical Interaction with HumanDEVELOPMENTAL BIOLOGY:MOLECULAR BASIS OF CELL AND

doi: 10.1074/jbc.M201405200 originally published online March 13, 20022002, 277:18817-18826.J. Biol. Chem. 

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