Induction of Apoptotic Genes by a p73-Phosphatase andTensin Homolog (p73-PTEN) Protein Complex in Response toGenotoxic Stress*

Received for publication, January 3, 2011, and in revised form, August 25, 2011 Published, JBC Papers in Press, August 26, 2011, DOI 10.1074/jbc.M110.217620

Jason A. Lehman‡, David L. Waning‡, Christopher N. Batuello‡§, Rocky Cipriano¶, Madhavi P. Kadakia�,and Lindsey D. Mayo‡§**1

From the ‡Department of Pediatrics, Herman B. Wells Center for Pediatrics Research, and Departments of §Biochemistry andMolecular Biology and ¶Pathology, Case Western Reserve University, Cleveland, Ohio 44106, the �Department of Biochemistry andMolecular Biology, Boonshoft School of Medicine, Wright State University, Dayton, Ohio 45435, and the **Indiana UniversitySimon Cancer Center, Indiana University School of Medicine, Indianapolis, Indiana 46202

The p53 family member, p73, has been characterized as atumor suppressor and functions in a similar manner as p53 toinduce cellular death. The phosphatase and tensin homolog(PTEN) can function as a dual specificity lipid/protein phospha-tase. However, recent data have described multiple roles fornuclear PTEN independent of its lipid phosphatase activity.PTEN can directly or indirectly activate p53 to promote apopto-sis. We examined whether PTEN would interact and regulatep73 independent of p53. Co-localization in the nucleus andcomplex formation of p73/PTEN were observed after DNAdamage. Furthermore, we also demonstrate that p73�/PTENproteins directly bind one another. Both overexpressed andendogenous p73-PTEN interactions were determined to be thestrongest in the nuclear fraction after DNA damage, which sug-gested formation of a transcriptional complex. We employedchromatin immunoprecipitation (ChIP) and found that p73 andPTENwere associatedwith thePUMApromoter after genotoxicstress in TP53-null cells. We found that another p73 target,BAX, had an increased expression in the presence of p73 andPTEN. In addition, in virus-transduced cell lines stably express-ing p73, PTEN, or both p73/PTEN,we found that the p73/PTENcells were more sensitive to genotoxic stress and cellular deathas measured by increased poly(ADP-ribose) polymerase cleav-age and PUMA/Bax induction. Conversely, knockdown ofPTEN dramatically reduced Bax and PUMA levels. Thus, a p73-PTEN protein complex is engaged to induce apoptosis inde-pendent of p53 in response to DNA damage.

p73 and p63 belong to the p53 tumor suppressor family(1–4). Genetically, both p73 and p63 are components of a p53-dependent network to induce apoptosis (5). All p53 familymembers can have multiple isoforms as follows: �N (no trans-activation domain) or TA (containing transactivation domain),which result from alternative promoter usage (6). As forTAp73, it is capable of transactivating a number of genesincluding, but not limited to, p21 andGADD45 involved in cell

cycle arrest and BAX, NOXA, and 14-3-3 involved in apoptosis(7). In response to apoptotic stimuli, the PUMA gene (p53 up-regulated modulator of apoptosis) is induced by TAp73, whichtriggers Bax mitochondrial translocation and release of cyto-chrome c to activate the caspase cascade. The �Np73 isoformcan repress the caspase cascade by acting as a dominant nega-tive to both p73 and p53 (8). Recently, a ubiquitin ligase namedp73-induced ring protein 2 (PIR2) has been demonstrated to beinduced by TAp73, which leads to an increase in the ratio ofTAp73/�Np73 with preferential ubiquitin-mediated degrada-tion of �Np73 (9). The regulation by this ubiquitin ligase sup-ports the pro-apoptotic function of TAp73. Therefore, p73induces apoptosis in a similar fashion to p53, and isoform-spe-cific regulation of p73 dramatically affects the balance betweencell survival and programmed cell death.The PTEN2 tumor suppressor has been extensively investi-

gated with respect to somatic mutations associated with inher-ited human genetic diseases and post-translational modifica-tions, which have defined the role of PTEN in cell polarity,genomic maintenance, and regulating survival signaling (10–15). Thus, PTEN is a multifaceted protein involved in tumorsuppression networks (16). PTEN functions as a dual specificityphosphatasewhose activity has been shown to dephosphorylatephosphatidylinositol 3,4,5-triphosphate and some proteins(17–20). The loss of phosphatidylinositol 3,4,5-triphosphateopposes Akt function through inhibition of phosphatidylinosi-tol 3-kinase (PI3K) for regulation of cellular migration and cellcycle and proliferation and apoptotic events (21–24). PTENmay also undergo nuclear translocation, although its functionin the nucleus remains unclear, it seems to be involved ingenomic regulation.The PTEN gene is also a transcriptional target of p53 in

response to DNA damage (25), and at the biochemical levelPTEN can regulate the tumor suppressor p53 by a direct pro-tein-protein interaction or indirectly regulating the p53 antag-onist Mdm2 by blocking nuclear localization (26–28). PTENcan form a direct protein interaction with p53 and has beenmapped to the C2 domain amino acids 186–351 on PTEN andon the C-terminal negative regulatory region of p53 (29).

* This work was supported, in whole or in part, by National Institutes of HealthGrant CA109262 from NCI (to L. D. M.) and NRSA T32 CA 111198 (to J. A. L.).

1 To whom correspondence should be addressed: 980 West Walnut St.,R3-c518, Indianapolis, IN 46202. Fax: 317-274-8046; E-mail: ldmayo@iupui.edu.

2 The abbreviations used are: PTEN, phosphatase and tensin homolog; PARP,poly(ADP-ribose) polymerase; ARF, ADP ribosylation factor; CDDP, cispla-tin; DDA, dominant negative.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 42, pp. 36631–36640, October 21, 2011© 2011 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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Although PTEN traditionally functions as a lipid phosphatasein the cytoplasmic fraction of the cell, it has been reported toenter the nucleus. Interestingly, PTEN lacks classical nuclearlocalization signals and nuclear export signals, yet containsmotifs that appear to promote its nuclear entry (30).Here, we demonstrate in response to genotoxic stress that

human p73 and PTEN integrate into a common pathway toactivate apoptotic genes. In response to DNA damage, p73 andPTENprotein levels are increased and both proteins co-localizeto the nucleus. We found that the TAp73� isoform had thehighest affinity for binding to PTEN. Co-immunoprecipitationexperiments using both endogenous and overexpressed p73and PTENwere found to have increased interaction post-DNAdamage in nuclear fractions. This complex was found associ-ated with the PUMA promoter after genotoxic stress. The sub-sequent increase in apoptotic mediators, PUMA and Bax, cor-responded with increased PARP cleavage. Knockdown ofPTENdramatically reduced levels of Bax and PUMA.Ourworkdemonstrates that independent of p53, a p73-PTEN complexcan induce apoptosis.

EXPERIMENTAL PROCEDURES

Cell Culture and Transfection—The p53-null human non-small cell lung carcinoma cell line H1299, human kidney epi-thelial cell line 293T, and human foreskin fibroblast immortal-ized with hTert BJ-ERT and p53-null derivative Shp53 BJ-ERTdescribed previously (31) were all cultured at 37 °C in a humid-ified incubator with 5% CO2. The H1299 cell line was virallytransduced with PTEN (H1299 (PTEN)) and p73� (H1299(p73�)), and p73� and PTEN (H1299 (p73�/PTEN)) were con-structed from insertion of full-length human p73� or PTENinto a pLNCX2 vector. Knockdown of PTEN (ShPTEN) or con-trol (ShGFP) was made in H1299 cells in a pLVTHM vectorusing previously described sequences (31). Virus was producedas described previously (32). All cell lines were maintained inDulbecco’s modified Eagle’s medium with high glucose (Invit-rogen) supplementedwith 10% fetal bovine serum (Atlanta Bio-logical), 50 units/ml penicillin, and 50 �g/ml streptomycin sul-fate (Invitrogen). For transfection of 293T cells, cells wereplated overnight and transfected at �75% confluency in 6-wellplates with 1–2 �g of DNA of pcDNA3.1 HA-p73�, HA-p73�,myc-p73�, myc-p73� (a generous gift from Dr. MakotoHijikata), or pCMV5-FLAG PTEN using 3 �l each of Lipo-fectamine and Plus Reagent (Invitrogen) in 1 ml of serum-and antibiotic-free Iscove’s modified Dulbecco’s mediumovernight.Antibodies—The antibodies used for detection were as fol-

lows: anti-p73 and PTENpolyclonal antibodies (Bethyl Labora-tories); N-terminal PTEN monoclonal antibody (Abgent); p73mouse monoclonal antibody (BD Biosciences); mouse mono-clonal PTEN (A2B1), anti-Bax (2D2), anti-ARF, and anti-GST(1E5) antibodies (Santa Cruz Biotechnology); anti-FLAG (M2)monoclonal antibody and anti-PUMA C-terminal polyclonalantibody (Sigma); anti-hemagglutinin (HA) 12CA5 clone(Roche Diagnostics), and mouse monoclonal anti-His tag (EZBioLabs Inc.).Protein Expression and Purification—The PTEN(186–351)

construct was a generous gift from Dr. C. Eng and was sub-

cloned into pGEX4T3 vector. p73� was subcloned from apcDNA3.1 expression plasmid into a pRSET vector to generateHis-tagged p73�. Wild-type PTEN and mutant C124S weresubcloned fromapCMV5expression plasmid into pGEX4T3 togenerate GST-tagged PTEN and C124S versions. All recombi-nant proteins were expressed and induced with isopropyl1-thio-�-D-galactopyranoside in Escherichia coli. His-taggedproteins were purified over a 2-ml nickel-nitrilotriacetic acidcolumn or over glutathione for GST-tagged proteins.Immunoprecipitation, Cytoplasmic/Nuclear Fractionation,

and Western Blotting—Cells for both whole cell lysates andimmunoprecipitates (unless indicated) were solubilized in lysisbuffer: 25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NonidetP-40, 1 mM EDTA, 1 mM EGTA, 1% (octylphenoxy)poly-ethoxyethanol (IGEPAL), 1 mM phenylmethylsulfonyl fluoride(PMSF), 10 �g/ml aprotinin, 10 �g/ml leupeptin, 1 mM sodiumorthovanadate (Na3VO4), and 10 mM sodium fluoride (NaF).0.25–1 mg of protein (as indicated in the figure legends) wasimmunoprecipitated with either 1 �g of �-PTEN (A2B1)monoclonal antibody or epidermal growth factor (Santa CruzBiotechnology), �-p73 monoclonal antibody (BD Biosciences),or normal mouse IgG (Calbiochem). Immunocomplexes wereabsorbed onto 20 �l of A/G-Sepharose beads (Santa Cruz Bio-technology) for 5 h at 4 °C. The immunoprecipitates werewashed three times with 1 ml of lysis buffer and then boiled in30 �l of 2� Laemmli buffer prior to Western blotting analysis.Cytoplasmic and nuclear extracts were made as described pre-viously with immunoprecipitation from these extracts asdescribed above (33).Confocal Microscopy—H1299, BJ-ERT, and Shp53 BJ-ERT

cells were fixed in 3% paraformaldehyde in PBS for 15 min,washedwith PBS, and permeabilized in 1%TritonX-100 in PBSfor 15 min. Slides were blocked with 5% fetal bovine serum inPBS/Tween and incubated with a rabbit polyclonal antibody top73 (Bethyl Laboratories), followed by an anti-rabbit Cy3 sec-ondary antibody (The Jackson Laboratory). Additionally, slideswere blocked and then incubated with a rabbit polyclonal anti-body to PTEN (Bethyl Laboratories) followed by an anti-rabbitCy5 secondary antibody (The Jackson Laboratory). Slides werewashed with PBS-T following primary antibody and secondaryantibody incubation. Nuclei were stained with DAPI, and slideswere mounted with nonfading gel-mount prior to visualizationon a Zeiss multiphoton microscope.ReporterAssay/(ChIP) Assay—The PTEN-luc reporter has

been previously described, and reporter assay methods havebeen previously described (28). The ChIP assay to detect p73and PTENprotein-DNA interactions in vivowere performed asdescribed previously (34). Immunoprecipitations were per-formed overnight with specific antibodies �-p73 (Bethyl Labo-ratories) or a mixture of antibodies against PTEN as follows:�-PTEN (A2B1) (Santa Cruz Biotechnology), �-PTEN (BethylLaboratories), and �-PTEN (N-terminal) (Abgent) or with sec-ondary �-mouse or �-rabbit antibodies (Santa Cruz Biotech-nology) as negative controls. Complexes were then incubatedwith protein G-Sepharose beads (Pierce). Immunoprecipitateswere washed with TE and eluted by incubation at 65 °C over-night in 1% SDS (in 0.1 M NaHCO3 at 65 °C for 4 h). DNA waspurified using a PCR purification kit (Roche Diagnostics)

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according to the manufacturer’s instructions and eluted withTE; templates were used in PCRs to detect PUMA promoterregions bound by specific proteins. PCR primer sequences usedwere as follows: PUMA, forward 5�-CTGTGGCCTTGTGTC-TGTGAGTAC-3� and reverse 5�-CCTAGCCCAAGGCAAG-GAGGAC-3�. PCR products were resolved on a 2% agarose geland visualized by ethidium bromide staining.

RESULTS

Increased Levels of p73 and PTEN in Response to DNADamage—To determine whether there was an integrated path-way that connected p73 and PTEN, which was independent ofp53, we transfected p73 and human PTEN promoter fused to aluciferase construct into p53-null H1299 cells. The �Np73�isoform has been reported to bind the PTEN promoter andnegatively repress its activity in thyroid cancer cells (35). Weobserved that p73� robustly stimulated the human PTEN pro-moter (Fig. 1A). The dominant negative p73 DDA abrogatedthe ability of p73� to stimulate the PTEN promoter (Fig. 1A).Thus, PTEN can be induced by the TAp73� isoform. The factthat TAp73 activated the PTEN promoter and the �Np73 iso-form repressed this activity is in agreement with these two iso-forms differentially regulating promoters (35).To examine if p73 and PTEN protein levels change in

response to DNA-damaging agents, a time course in responseto doxorubicin was conducted in multiple cell lines. Normalhuman diploid BJ fibroblasts immortalized with E1a, H-Ras-V12, and hTERT (BJ-ERT cells) and BJ-ERT cells transducedwith control ShRNA or ShRNA to p53 (Shp53 BJ-ERT) were

used (31). We treated both Shp53 and ShControl BJ-ERT celllines with 2 �M doxorubicin. The protein levels of p53 wereanalyzed to ensure the cells were responsive to genotoxic stress.An increase in p53 levels was observed in BJ-ERT at 4 h withlevels maintained through 16 h (Fig. 1B, top panel) but not inShp53 BJ-ERT (Fig. 1C, top panel). p73 and PTENprotein levelswere increased in response to DNA damage in BJ-ERT cells(Fig. 1B, lower panel). Shp53 BJ-ERT cells had a rapid inductionof p73 protein levels following doxorubicin treatment begin-ning at 2 h, although PTEN induction occurred at 8–16 h (Fig.1C, lower panel). A similar response for p73 and PTEN wasdetected in H1299 cells, a p53 null cell line (Fig. 1D). Thus, inboth immortalized fibroblasts devoid of p53 and a p53-null can-cer cell line, we observed that DNA damage results in anincrease in p73 and PTEN protein levels.Because the levels of p73 and PTEN were increased, we

next examined the cellular localization of p73 and PTEN inresponse to DNA damage. A time course of ShControl BJ-ERT cells untreated or with 2 �M doxorubicin over a 16-htime course and was used to detect p73 and PTEN by confo-cal microscopy. In response to DNA damage, both p73 andPTEN localized to the nucleus (Fig. 2A). Merged imagesrevealed that p73 and PTEN co-localized at 4 h. To show thatthe p73/PTEN co-localization was not dependent on p53,Shp53 BJ-ERT cells were subjected to DNA damage followedby confocal imaging. Once again in response to DNA dam-age, p73 and PTEN were found in the nucleus, which wasindependent of p53 (Fig. 2B).

FIGURE 1. p73 and PTEN induction in response to DNA damage. A, H1299 were transfected with human PTEN promoter coupled to the luciferase geneRSV-�-gal (control), p73�, or p73�-DDA. Each sample was done in triplicate and expressed as relative luciferase units (RLU). B, BJ-ERT cells were treated with 2�M doxorubicin for the indicated times, and whole cell lysates were prepared for Western blotting. Blots were probed separately with polyclonal antibodies top73 and PTEN and monoclonal to GAPDH for protein loading (bottom panels) or p53 (top panel). C, BJ-ERT (shp53) cells were treated and probed with antibodiesas in B. D, H1299 cells were treated and probed with antibodies as in C.

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Complex Formation of PTEN and p73—Because we observedboth p73 and PTEN nuclear co-localization with similar kinet-ics, we tested if there was a direct protein-protein interaction.Purified His-p73� and GST-PTEN and a lipid phosphatasemutant, GST-C124S, were all produced from bacteria fromwhich GST pulldown assays were performed. TAp73� waspulled down with both GST tagged PTEN and C124S but notGST (Fig. 3A). This indicates that both wild-type and lipidphosphatase mutant forms of PTEN directly bind TAp73�protein.We next tested if the interaction was TAp73 isoform-spe-

cific. An alignment of TAp73 isoforms �, �, �, and � aredepicted (Fig. 3C). p73 shares extensive homology to p53 intheir transactivation domain, DNA binding domain, and oligo-merization domain. To test which p73 isoforms would interactwith PTEN, we overexpressed multiple TAp73 isoforms andPTEN in 293T cells and examined binding interactions byimmunoprecipitation. HA-tagged p73� and PTEN interactionwas evident, and the PTEN-p73� was not (Fig. 3B, left panel).We also tested the ability of p73� and p73� isoforms and foundamarginal interaction with PTEN (Fig. 3B, right panel). A sum-mary of the various isoforms of TAp73 and binding to PTEN isdepicted in Fig. 3C which illustrates that the C-terminaldomain is important for binding to PTEN.

Previous reports have demonstrated that p53 and PTENform a direct protein-protein interaction (29). To test wherep73 binds PTEN, recombinant GST-PTEN andGST(186–351)were expressed and purified from E. coli and then mixed withHis-p73� with a GST pulldown performed. As depicted in Fig.3D, PTEN C-terminal domain bound to p73�. This is in agree-ment with full-length PTEN and the 186–351 fragmentincreasing p53 transactivation (36).Based on the observation that p73 and PTEN bind directly

and DNA damage appears to enhance cellular compartmental-ization at a similar time course, we determined if DNA damageenhanced this interaction. To test if p73 and PTEN complexformation would increase with DNA damage, whole cell lysatesfrom H1299 cells that were transduced with both TAp73� andPTEN and treated with cisplatin (CDDP) were prepared. Anal-ysis of p73 protein levels increased at 8 and 16 h, althoughPTEN levels remained unchanged (Fig. 4A, bottom panel).When cells were treated with CDDP and p73 was immunopre-cipitated, an increase in PTEN binding was observed at 8 and16 h (Fig. 4A, top panel). Thus, the interaction of p73 and PTENwas increased upon platinum treatment because levels of bothproteins were elevated after DNA damage.Next, we examined cellular fractionation on p73 and PTEN

protein levels after platinum-induced DNA damage. H1299(p73/PTEN) cells were treated with CDDP over a 16-h timecourse. Cytoplasmic and nuclear fractions were prepared, and aWestern blotwas used to analyze p73 andPTEN localization. Inresponse to CDDP, we observed an increase in nuclear p73levels. In addition, the overall levels of p73 levels were increasedin response to DNA damage (Fig. 4B). PTEN levels did notappear to change after damage in the cytoplasmic fraction;however, a robust increase in nuclear PTEN occurred at 16 h(Fig. 4B). The data from H1299 cells is in agreement with theconfocal data results in Fig. 2 from Shp53 BJ-ERT cells.Based on our immunoprecipitation results where the inter-

action of p73 and PTEN was more abundant after DNA dam-age, we wanted to see if this occurred in the nuclear fraction.H1299 cells were used to immunoprecipitate endogenous p73from cytoplasmic and nuclear fractions after CDDP treatment.Mock immunoprecipitationwith IgG antibodies did not co-pu-rify p73. However, PTEN was detected at 16 h after genotoxicstress, as denoted by the arrow in Fig. 4C. Nuclear p73 versuscytoplasmic p73 increased with DNA damage (Fig. 4C). PTENwas detected with p73 in all nuclear fractions compared withthe cytoplasmic fractions at 16 h (Fig. 4C). To examine if endog-enous p73 would co-precipitate with PTEN, whole cell extractswere prepared from Shp53 BJ-ERT cells after 16 h of CDDPtreatment. Immunoprecipitation with p73 antibody led to anincreased amount of PTEN co-purified with p73 after CDDPtreatment (Fig. 5A, top panel). In addition, immunoprecipita-tion of PTEN detected endogenous p73 under DNA damageconditions with little detection of p73 with no treatment (Fig.5A,middle panel). Counter-blotting revealed an enhanced levelof PTEN protein immunoprecipitated with CDDP treatmentversus control, which is in agreement with levels observed inlysates (Fig. 5A, bottom panel). These results demonstrate thatan endogenous PTEN-p73 complex forms after DNA damage,which likely stems from higher levels of both proteins being

FIGURE 2. p73 and PTEN co-localize in response to DNA damage. A, DNAdamage in transformed fibroblast cell lines leads to co-localization of p73 andPTEN. DNA damage was induced with 2 �M doxorubicin for the indicatedtimes in BJ-ERT fibroblasts that have wild-type p53, which were then pre-pared for confocal microscopy. Merged images show co-localization of p73and PTEN in the nucleus. Nuclei are stained with DAPI. B, Shp53 BJ-ERT fibro-blasts devoid of p53 were treated and prepared as described above.

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present. To further define the endogenous localization of thesetumor suppressors, we made cytoplasmic/nuclear extractsfromH1299 cells after treatment with DNA damage. After 16 hof CDDP treatment, levels of nuclear PTEN and p73 increasedcompared with cytoplasmic fractions (Fig. 5B), which is inagreement with confocal microscopy and lysates from cellsoverexpressing p73 and PTEN in Figs. 2, A and B, and 4B,respectively. Both H1299 cytoplasmic and nuclear extractswere immunoprecipitated with PTEN or ARF (control) anti-bodies and then probed for reactivity to p73 (Fig. 5C). Resultsshow a clear increase in the amount of endogenous p73 thatco-precipitates with PTEN after 16 h of CDDP treatment innuclear extracts. This immunoprecipitation with PTEN anti-bodies used in other experiments mirrors the results observedin Fig. 4C (immunoprecipitated p73 in overexpressed cells),where in nuclear extracts post-CDDP treatment increased thep73-PTEN complex. Thus, DNA damage levels clearly increase

the levels of nuclear p73 and PTEN proteins, which forms ap73-PTEN complex.Induction of Apoptotic Genes by p73/PTEN—p73 will induce

BAX and PUMA gene expression independent of p53 (8). Totest if a p73-PTEN complex would influence BAX expression,the human BAX promoter upstream of luciferase was used.Control or H1299 cells stably overexpressing PTEN (H1299-PTEN)) were transiently transfected with RSV-�gal, BAXreporter, and either control TAp73� or p73��DDA con-structs. In Fig. 6A, overexpression of TAp73� increases BAXluciferase activity compared with the control plasmid. Thisactivity was increased 2-fold in H1299 (PTEN) cells versus con-trol H1299. This effect was abrogated by p73� DDA (dominantnegative) for the induction of BAX in either cell line (Fig. 6A).Next we examined if Bax protein was induced in response toDNA damage. A time course with 2 �M doxorubicin treatmentrevealed an increase in Bax levels at 8–16 h (Fig. 6B). Also,

FIGURE 3. p73 and PTEN form an interaction in vivo and in vitro. A, p73 and PTEN form a direct interaction in vitro. Recombinant GST-PTEN, GST-C124S (PTENlipid phosphatase mutant), and His-p73� were expressed and purified from E. coli and used in GST-pulldown assays. Interactions were detected with anti-His,and pulldown efficiency was counter-blotted with anti-GST. B, p73 and PTEN interact in vivo. FLAG-PTEN and HA-tagged p73� or p73� were overexpressed in293T cells (left panel), and immunoprecipitation (I.P.) with PTEN antibodies was performed. Blots were probed for HA and then stripped and re-probed withPTEN. FLAG-PTEN and Myc-tagged p73� or p73� were overexpressed (right panel) and immunoprecipitated with PTEN antibodies as described for the previouspanel and then stripped and re-probed with Myc antibodies. C, schematic diagram of p73 isoforms. Specific protein domains are labeled throughout thediagram as follows: transactivation domain, DNA binding domain, oligomerization, and the C terminus. Positive results from PTEN binding to various p73isoforms is denoted by plus symbols. D, PTEN domain mapping with p73. An in vitro GST pulldown assay was performed with GST-PTEN and GST(186 –351) afteraddition of His-p73�. A Western blot was probed for anti-His and counter-blotted for anti-GST, and positive results of p73 interaction with PTEN are displayedas plus symbols.

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another p73 target, PUMA, was also elevated at 8–16 h (Fig.6B). The increase of Bax and PUMA protein levels coincideswith the co-localization of p73/PTEN in the nucleus (Fig. 2, Aand B) and protein complex formation (Figs. 4C and 5C).Because Bax and PUMA levels are increased with the com-

plex formation of nuclear p73 and PTEN,we next determined ifp73 and PTEN were associated with the PUMA genomic pro-moter using a ChIP assay. p73 will bind to apoptotic promotersin response to genotoxic stress (37). However, PTEN has neverbeen shown to be in a complex on a promoter element. Weanalyzed in Shp53 BJ-ERT and H1299 cancer cells. These cellswere treated with 5 �M doxorubicin for 24 h followed by cova-lent cross-linking of proteins to DNA and immunoprecipita-tion of either p73 or PTEN. We observed an increase in p73binding to the PUMA promoter after 24 h in response to DNAdamage in Shp53 BJ-ERT cells (Fig. 6C, left graph). A represent-ative ChIP assay from Shp53 BJ-ERT cells depicting bothimmunoprecipitations with p73 and PTEN antibodies is shownbelow the graphs in Fig. 6C. PTEN was also found associatedwith the PUMA promoter at the 24-h time point (Fig. 6C, leftgraph). These experimentswere replicated inH1299 cells, and asimilar trend was observed (Fig. 6C, right graph) with each cellline data coming from at least two independent experiments.In response to genotoxic stress, the elevation of p73/PTEN

leading to induction of Bax and PUMA would be predictive of

an activated caspase cascade and consequently the initiation ofapoptosis. One of the downstream targets of caspase activationis the cleavage of PARP. To test if PARP cleavage is dependenton the levels of p73 and PTEN, H1299, H1299 (p73�), H1299(PTEN), and H1299 (p73�/PTEN) were treated with doxorubi-cin. AWestern blot was made from whole cell lysates from thevarious cells lines to show overexpression of PTEN and p73(Fig. 7A). Next, H1299 cells were treated in a dose response tofind the optimal dose for PARP cleavage. A Western blot forcleaved PARP revealed that 10 �M doxorubicin was the maxi-mal dose after 20 h (Fig. 7B). Moreover, PARP cleavage wasinitiated at a lower dose of 2.5 �M doxorubicin in cells overex-pressing both p73/PTEN. A comparison of all cell lines wasperformed after treatment with 10 �M doxorubicin at both 8and 16 h. At 8 h the p73/PTEN-overexpressing cell line showedmaximal PARP cleavage that was sustained through 16 h. Allcell lines showed some level of PARP cleavage at 16 h. The levelsof PARP cleavage were not as robust in other cell lines (PTEN,p73, or control), which is not surprising as H1299 cells are veryresistant to genotoxic stress (Fig. 7C). To test if overexpressionof p73/PTEN led to an induction in Bax protein levels, cellextracts were made after 16 h of CDDP treatment or no treat-ment. p73 protein levels in cells overexpressing p73 alone andcells overexpressing both p73/PTEN were increased inresponse to CDDP (Fig. 7D). Bax levels are already elevated incells overexpressing both p73/PTEN but not each individualtumor suppressor protein (Fig. 7D). However, in response to

FIGURE 4. Interaction between overexpressed p73 and PTEN is enhancedwith DNA damage. A, Western blot of immunoprecipitated p73 and PTENfrom overexpressing cell lines. H1299 (p73/PTEN) cells were treated with 10�M cisplatin for the indicated times. Cell extracts were immunoprecipitated(IP) with polyclonal p73 antibodies (top panel), and the blot was probed forPTEN (A2B1) and stripped and re-probed with anti-p73. Whole cell extracts(bottom panel) were probed with monoclonal p73 and PTEN (A2B1) antibod-ies and GAPDH for loading controls. B, cytoplasmic (C)/nuclear (N) fraction-ation of p73 and PTEN in response to DNA damage. H1299 (p73/PTEN) cellswere treated as in A, and cytoplasmic and nuclear fractions were made fromwhole cell lysates. Blot was probed with monoclonal antibodies against p73and PTEN (A2B1). GAPDH and PARP were probed for cytoplasmic and nuclearfractionation controls. C, immunoprecipitations were performed from cyto-plasmic and nuclear extracts from B with anti-p73 (monoclonal) or normalmouse IgG as control. The Western blot was probed with polyclonal antibod-ies to PTEN (denoted by arrow) to detect co-precipitation and then strippedand re-probed with monoclonal p73 antibodies.

FIGURE 5. Endogenous p73-PTEN interactions are enhanced in thenuclear compartment in response to DNA damage. A, Western blot ofimmunoprecipitated (IP) endogenous p73 and PTEN in response to DNAdamage. Shp53 BJ-ERT cells were treated with 20 �M cisplatin for the indi-cated times. Whole cell extracts were immunoprecipitated with polyclonalp73 antibodies or ARF as a control (top panel), and the blot was probed forPTEN (monoclonal) and stripped and re-probed with anti-p73. Whole cellextracts were immunoprecipitated with monoclonal PTEN antibodies or ARFfor control (middle panel), and the blot was probed for p73 (polyclonal) andstripped and re-probed with anti-PTEN. Lysates (bottom panel) were probedwith polyclonal p73, PTEN (A2B1) antibodies, and GAPDH. B, cytoplasmic (C)/nuclear (N) fractionation of endogenous p73 and PTEN in response to DNAdamage. H1299 cells were treated as in A, and cytoplasmic and nuclear frac-tions were made from whole cell lysates. Blot was probed with monoclonalantibodies against p73 and PTEN (A2B1), PARP, and GAPDH. C, immunopre-cipitations were performed from cytoplasmic and nuclear extracts from Bwith anti-PTEN (A2B1) or ARF as a control. The Western blot was probed withp73 monoclonal antibodies to detect co-precipitation and then stripped andre-probed with PTEN polyclonal antibodies for counter blot.

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CDDP, the levels of Bax are increased in cells overexpressingPTEN and to the highest level in the cell line overexpressingboth p73/PTEN (Fig. 7D). Thus, overexpression of both p73/PTEN leads to maximal activation of the pro-apoptotic proteinBax in cells independent of p53. To validate the role of PTEN instimulation of apoptosis, we silenced PTEN levels in H1299cells with ShRNA to PTEN or GFP (control). Levels of PTENwere decreased�90%, and levels do not increase in response togenotoxic stress. ShGFP control cells showed a predictableincrease in Bax (Fig. 7E), whereas ShPTEN cells showed adecrease in Bax induction. Puma levels were not detectable inShPTEN cells and were not induced following genotoxic stress(Fig. 7E). Thus, the presence of both p73 and PTEN tumorsuppressors facilitates optimal induction of apoptotic genesafter DNA damage. Our model shows a p73-PTEN interactionenhanced in the nuclear compartment (Fig. 7F). These tumorsuppressors are capable of binding to and activating the BAXand PUMA promoters independent of p53.

DISCUSSION

The responsiveness of cancer cells to chemotherapeuticagents depends on intact tumor suppressor pathways. Thedegree that p53 regulates apoptosis without contribution fromp63 and p73 remains understudied (5, 38). Although there areclear similarities and possible overlapping functions and regu-lation of p53 and p73, there are distinct differences. For

instance, Mdm2 represents the predominant E3 ubiquitinligase for proteasomal degradation of p53, although p73 is not atarget of Mdm2 (39). The NEDD4-like E3 ligase Itch has beendemonstrated to interact with p73 and mediate its degradationthrough ubiquitination, yet this enzyme does not influence p53(40). Additionally, other proteins that are known to bind p53such as human papillomavirus E6, adenovirus E1B 55K, andsimian virus 40Tdo not associatewith p73� and p73� isoforms(41). Although there are distinct differences in p73 and p53protein-binding partners and how these proteins are degraded,there are functional overlaps in transcriptional gene targets.p73� and p73� have both been shown to be competent in tran-scription of p53-responsive genes such as BAX, GADD45, andothers, but they differ in the degree of transcription betweenp73 isoforms and with respect to p53 (7). Thus, there are cleardelineations of p73 and p53 in the manner in which theyrespond to DNA damage and protein binding partners.A mouse phenotype where complete p73 isoform knock-out

(Trp73�/�), including both TA and �N, has been performedreveals a variety of neurological and inflammatory defects.However, spontaneous tumorswere not observed, which servesas a hallmark for identification of a tumor-suppressor protein(42). Recently, to delineate the differences between TAp73 and�Np73, separate mouse knockouts were performed. TheTAp73 null mouse (TAp73�/�) was found to have a higher

FIGURE 6. p73/PTEN enhances BAX apoptotic promoter activity and PUMA promoter binding. A, H1299 or cells overexpressing PTEN, H1299 (PTEN), weretransfected with human BAX promoter coupled to the luciferase gene, RSV-�-gal (control), p73�, or p73�-DDA. Each sample was done in triplicate andexpressed as relative luciferase units (RLU). B, H1299 cells were treated with 5 �M doxorubicin for the indicated times, and Western blots were probed withanti-Bax or PUMA antibodies and GAPDH for loading controls. C, PTEN and p73 bind the human PUMA promoter. Shp53 BJ-ERT or H1299 cells were treated with5 �M doxorubicin for the indicated times, and then chromatin immunoprecipitation (ChIP) was performed with antibodies against p73 or PTEN as describedunder “Experimental Procedures.” Input was nonimmunoprecipitated (I.P.) chromosomal DNA. Bar graphs are the average of two independent experimentswith no DNA control subtracted and represented as the percent change in PUMA promoter binding. A representative ChIP experiment from Shp53 BJ-ERT cellsis depicted below the graphs.

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incidence of carcinogen-induced and spontaneous tumorsalong with infertility, genomic instability, and aneuploidy (43).A knock-inmousemodel of the�Np73 isoforms (�Np73�/�) issensitive to chemotherapeutic agents and has increased p53-dependent apoptosis (44). Thus, TAp73 appears to be a tumorsuppressor like p53 capable of transactivating genes involved inapoptosis, whereas the�Np73 opposes this effect by interferingwith DNA damage signaling pathways.PTEN represents a very important armof cellular tumor sup-

pression by negatively regulating PI3K/Akt activity. The PTEN

gene is frequently lost or mutated in human cancers affirmingits role as a primary tumor suppressor (14). We observed arobust induction of PTEN protein levels in response to DNAdamage in the absence of p53 (Figs. 1, C and D, and 3A). PTENlevels followed a similar time course profile for induction ofprotein levels to p73 with (Fig. 1, C and D). This is consistentwith p73 activation in response to DNA damage and its abilityto impair chemoresistance in cancer cells (45).Our data shows that PTEN can bind both in vitro and in vivo

to the p73� isoform (Fig. 3, A and B). p73� is the only isoform

FIGURE 7. Cooperative induction of apoptosis through p73 and PTEN. A, H1299 and cell line derivatives overexpressing p73, PTEN, or both were lysed, andwhole cell extracts were probed with p73, PTEN, and GAPDH antibodies. B, H1299, H1299 (p73�), H1299 (PTEN), and H1299 (p73�/PTEN) cell lines were treatedwith 0 –10 �M doxorubicin (dox) as indicated for 20 h. Cells were harvested, and whole cell lysates were probed for cleaved PARP and GAPDH. C, H1299, H1299(p73�), H1299 (PTEN), and H1299 (p73�/PTEN) cell lines were treated with 10 �M doxorubicin for 8 and 16 h, and Western blots were probed as in B. Lane C,control. D, H1299 cell line derivatives (described in C) were treated with 10 �M cisplatin for 16 h, and lysates were probed with the indicated antibodies. E, H1299stable cell lines for ShGFP or ShPTEN were treated with 10 �M cisplatin for 16 h, and lysates were probed with the indicated antibodies. F, model for p73-PTENinteractions stimulating p53-independent apoptosis. PTEN and p73 are present in the cytoplasmic fraction and have limited binding with each other. DNAdamage signals nuclear p73/PTEN levels, which lead to an enhancement in p73-PTEN complex formation. The end result is recruitment of PTEN binding to thePUMA promoter by transcriptional activity of p73, which in turn enhances BAX activity.

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that contains a complete C-terminal domain that includes thesterile �-motif (Fig. 3C). The sterile �-motif domain in p73� isdispensable for membrane binding to phospholipids yet func-tions as an autoinhibitory domain for its transcriptional activ-ity, which prevents the interaction with p300/CBP (46). PTENbinding to this region on p73� could compete for p73 bindingto membranes and make it more transcriptionally competentby relieving this inhibition. We also observed that in cellsexpressing p73 and PTEN, the overall levels of p73 were ele-vated (Fig. 7A). This may implicate a role in PTEN in alteringthe stability of p73 by protecting it from ubiquitin-mediatedproteolysis. Further investigation to understand this mecha-nism is currently underway. In addition, PTEN is known tofacilitate p53 acetylation by p300 (47). Interestingly, p73 tran-scriptional activity can also be inhibited bymutant p53, becausethe mutant rather than wild-type p53 can bind to p73 (7). Theselective pressure of cancer cells to favor mutant p53 presentsan interesting problem for determining how to activate p73.Therefore, theremust be othermechanisms present to alleviatethe negative repression on p73 from mutant p53.Previous reports have hinted that a p73-PTEN interaction

may exist. 1) A PTEN-regulated pathway, mammalian target ofrapamycin in the PI3K signaling pathway, is an upstream regu-lator of p73 (48). 2) �Np73 has shown to inhibit PTEN expres-sion in thyroid cancer cells (35). We have demonstrated thatTAp73� can stimulate thePTENpromoter, which can be inhib-ited with the addition of the p73 DDA construct (Fig. 1A),which is in agreement with Ref. 35. An additional luciferasereporter assaywas performedwhich showedPTENoverexpres-sion with p73� enhanced BAX transcription (Fig. 6A). Ournovel finding of PTEN (direct or indirect DNA binding) on anapoptotic promoter further validates its role as a tumor sup-pressor, especially in cells devoid of p53 (Fig. 6C).Opposing the tumor suppressor functions of p73 and p53 is

the murine double minute (Mdm2) oncoprotein. Additionally,there is further interplaywithMdm2 in conjunctionwith PTENand p53 which is reviewed in Ref. 16. A strong link betweensignaling in the PI3K pathway and Mdm2 has been demon-strated. PTEN impedes Akt activity to promote Mdm2 nuclearentry through phosphorylation of serine 166 and 186 onMdm2(26). This allows for cytoplasmic retention and destabilizationof Mdm2, thereby keeping p53 active. Furthermore, a func-tional connectivity between PTEN and p53 has been estab-lished, whereby cancer cells are more sensitive to DNA-dam-aging agents when both tumor suppressors are present (27).Perhaps this is the same case for p73 and PTEN in the absenceof p53. In agreement is our observation that DNA damageincreases the levels of both p73 and PTEN protein. From ourco-precipitation experiments utilizing both overexpressedand endogenous p73 and PTEN, we detected enhancedinteractions under conditions of DNA damage (Figs. 4,A andC, and 5, A, C, andD). The p73-PTEN interaction is substan-tially increased in endogenous nuclear fractions after DNAdamage (Fig. 5C). However, our experiments are not able todiscern if the enhanced p73-PTEN nuclear protein interac-tion is directly stimulated by DNA damage or if this is merelya secondary effect due to the induction of both proteins afterdamage. Some data links PTEN nucleoplasmic shuttling to

the major vault protein through Ca�2 signaling (49). Pre-cisely, how PTEN nuclear levels would increase after DNAdamage and required upstream signaling events clearly war-rants further investigation.In support of our observation that a p73-PTEN protein com-

plex acts as a co-activator of apoptosis, is the observation thatcells overexpressing both tumor suppressors induce an overallhigher level of PARP cleavage in cells (Fig. 7B). Furthermore,overexpression of both p73/PTEN leads to the greatest level ofinduction of Bax (Fig. 7D), whereas silencing PTEN completelyreduces levels of PUMA and substantially decreases Bax in thebackground of p73 activation by DNA damage (Fig. 7E). Theseresults provide interesting evidence to the degree of the impor-tance of PTEN in activating downstream apoptotic targets inresponse to p73 activation after DNA damage. These experi-ments substantiate our observations that the p73 and PTENtumor suppressors interact together to enhance apoptosis.Mdm2 expression is also regulated by PTEN independent ofp53. Cells null for PTEN expressed higher levels of the Mdm2P1 promoter, and consequently PTEN through its lipid phos-phatase activity negatively regulates expression of the p90Mdm2 (50). Interestingly, a p53-PTEN-Mdm2 complex couldnot be co-precipitated from cells (29). Therefore, a fine-tuningmechanism between PTEN, p53, p73, and Mdm2 is in place inhuman cells to mediate cellular apoptosis in response to DNAdamage. To this end, Mdm2 represents a viable target for drugdesign and chemotherapeutic intervention (51–53).Thus, our data reveal an important role for nuclear PTEN in

facilitating p73 binding to apoptotic promoters and inducingtheir expression. This is the first demonstration that PTEN canbe foundon an apoptotic promoter, which further substantiatesits role as a tumor suppressor. Given the high incidence oftumors with mutant or null p53, stimulation of the p73/PTENaxis to enhance apoptosis is an evolving area for furtherinvestigation.

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PTEN/p73 Induces Apoptotic Genes

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P. Kadakia and Lindsey D. MayoJason A. Lehman, David L. Waning, Christopher N. Batuello, Rocky Cipriano, Madhavi

(p73-PTEN) Protein Complex in Response to Genotoxic StressInduction of Apoptotic Genes by a p73-Phosphatase and Tensin Homolog

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