A C-Terminal Inhibitory Domain Controls the Activity of p63 by an Intramolecular Mechanism

The human genome is far smaller than originally estimated, andone explanation is that alternative splicing creates greaterproteomic complexity than a simple count of open reading frameswould suggest. The p53 homologue p63, for example, is a tetramerictranscription factor implicated in epithelial development andexpressed as at least six isoforms with widely differing transactivationpotential. In particular, p63 ${alpha}$ isoforms contain a 27-kDa C-terminalregion that drastically reduces their activity and is of clearbiological importance, since patients with deletions in thisC terminus have phenotypes very similar to patients with mutationsin the DNA-binding domain. We have identified a novel domainwithin this C terminus that is necessary and sufficient fortranscriptional inhibition and which acts by binding to a regionin the N-terminal transactivation domain of p63 homologous tothe MDM2 binding site in p53. Based on this mechanism, we providea model that explains the transactivation potential of homo-and heterotetramers composed of different p63 isoforms and theireffect on p53.

INTRODUCTION

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Introduction

The recent discovery of two homologues of the archetypal tumorsuppressor protein p53, called p73 and p63 (p51/p40/p73L/ket)(1, 35, 39, 46, 49), has sparked speculations that surveillanceof cellular integrity might be achieved through a network ofthese p53-like tumor suppressors (10). These speculations werefurther fueled by the observation that the p73 gene is localizedto chromosome 1p36.3, a region that is frequently lost in neuroblastomasand other types of cancers (20, 43) while the p63 chromosomallocation, 3q27-9, is deleted in some bladder cancers and amplifiedin some cervical, ovarian, lung, and squamous cell carcinomas(13, 49). Moreover, both p73 and p63 can bind to p53 DNA-bindingsites in vitro and activate proteins under the control of ap53 promoter and induce apoptosis in vivo (18, 49).

Despite all this circumstantial evidence, however, only a veryfew mutations in p63 and p73 have been found in human tumors,and a direct link to carcinogenesis similar to that for p53has so far not been established (17, 22, 27, 32, 43). Furthermore,knockout mouse studies of both proteins have revealed that theyare involved in different biological processes. p73-knockoutmice are characterized by chronic infections and developmentaldefects, including hippocampal dysgenesis, hydrocephalus, andabnormalities in the pheromone sensory pathway (51). p63-knockoutmice show even more severe defects that include truncated limbsand a lack of all squamous epithelia (29, 50). Both phenotypesare very unlike that of the p53-knockout mice, which are withoutdevelopmental defects but have an elevated susceptibility totumorigenesis (9). These striking differences in biologicalfunction are surprising in light of the high homology amongall three proteins that reaches 65% identity in the DNA-bindingdomain, with all amino acids that contact DNA being conserved.Most likely then, these different biological functions are connectedto domains outside the DNA-binding domain unique to p73 andp63. Indeed, both proteins exist in multiple isoforms. In thecase of p63, at least six different isotypes with widely differingtransactivation potentials have been described elsewhere (49).Three of these isoforms are inactive due to the absence of theN-terminal transactivation (TA) domain ( ${Delta}$ N isoforms). The activityof the remaining isoforms (TA isoforms) is modulated by threeC-terminal sequences, ${alpha}$ , ß, and ${gamma}$ , not found in p53.The smaller p63ß and p63 ${gamma}$ isoforms are transcriptionallyactive while the presence of the 27-kDa ${alpha}$ C-terminal domain drasticallyreduces the activity of p63 (49). In addition, ${Delta}$ Np63 ${alpha}$ , which isthe major isoform found in cells, shows strong dominant-negativebehavior toward transactivating p63 isoforms and moderate dominant-negativebehavior toward p53. The overarching importance of the ${alpha}$ C-terminaldomain to the biological function of p63 is further emphasizedby clinical observations. A human patient with a mutation thatintroduces a premature stop codon in the ${alpha}$ C terminus shows skeletalreduction defects of the hands and feet, surprisingly similarto patients with a mutation in the DNA-binding domain (4). Thatthis mutation in the ${alpha}$ C terminus affects only the two ${alpha}$ isoforms(TAp63 ${alpha}$ and ${Delta}$ Np63 ${alpha}$ ) and yet produces very similar abnormalitiesas do mutations that eliminate DNA binding of all six p63 isoformssuggests that ${alpha}$ isoforms play a pivotal role in p63 processes.

We have identified an inhibitory domain within the ${alpha}$ C terminusthat is both necessary and sufficient for transcriptional inhibition.In this paper we demonstrate that this domain uses a new modeof inhibition by binding to a region in the N-terminal TA domainof p63 that is homologous to the MDM2 binding site in p53, maskingresidues that are important for transactivation. Based on thisintramolecular inhibition, we provide a model that makes senseof many past experiments and explains how alternative splicingmodulates the transactivation potential of homo- and heterotetramerscomposed of different p63 isoforms and how they regulate p53.Given the high homology between the ${alpha}$ C termini of p63 and p73,the results presented here are also very likely relevant forp73.

MATERIALS AND METHODS

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Materials and Methods

Cell culture and transactivation assays. SAOS-2 cells were obtained from the American Type Culture Collection.Cells were maintained in Dulbecco modified Eagle medium with10% fetal bovine serum at 37°C in a 5% CO₂ atmosphere. Transactivationassays were carried out with the dual-luciferase reporter assaysystem (Promega) in SAOS-2 cells with 13 tandem repeats of anoptimized p53-binding sequence (from Bert Vogelstein) subclonedinto a firefly luciferase reporter vector (49). All experimentalresults were normalized for transfection efficiency and samplepreparation variations with a pRL-CMV control Renilla luciferaseplasmid (Promega). Experiments were performed in triplicate.

Expression vectors. For the transactivation cell culture assays all p63 and p53constructs and mutants were cloned into a pcDNA3 vector (Invitrogen)as XhoI-XbaI fragments and containing an N-terminal myc tag.Point mutants were made with a QuikChange (Stratagene) mutagenesiskit. Glutathione S-transferase (GST) fusion vectors were constructedby inserting BamHI-EcoRI fragments into a pGEX-6P-2 vector (PharmaciaBiotech). For the expression of the TID ${Delta}$ 25 peptide, the constructwas cloned into a pET-31b(+) vector (Novagen) as a single AlwNIfragment according to the kit instructions.

Immunoblotting. Transfected cell lysates were resolved on a sodium dodecyl sulfate(SDS)-4 to 12% polyacrylamide gel electrophoresis (PAGE) gradientgel (NuPAGE Bis-Tris; Invitrogen) and transferred to a polyvinylidenedifluoride (PVDF) membrane (Immobilon-P; Millipore). p63 isoforms,mutants, and chimeras were detected with the 4A4 monoclonalp63 antibody and/or the 9E10 c-Myc antibody. Secondary anti-mouseimmunoglobulin G antibodies, conjugated to horseradish peroxidase(Santa Cruz Biotechnology), were applied, followed by treatmentwith enhanced chemiluminescence (ECL) reagent (Amersham Biosciences)and exposure to Kodak Biomax ML film.

Immunofluorescence. Transfection and immunofluorescence experiments were carriedout with BHK cells attached to 18-mm-diameter round glass coverslipsas described earlier (49). The 9E10 c-Myc-specific antibodywas used as the primary antibody against the N-terminally myc-taggedTAp63 ${alpha}$ , followed by staining with a Cy3-conjugated goat anti-mousesecondary antibody. DNA was stained with the fluorochrome Hoechst33278 (Sigma). myc-tagged TAp63 ${alpha}$ was stained with the c-Myc-specificantibody 9E10.

Peptide expression and purification. The TID ${Delta}$ 25 (residues 568 to 616) peptide was expressed as a fusionto ketosteroid isomerase and a His tag, separated from eachby a methionine. The fusion was purified from inclusion bodiesin a denaturing buffer (6 M guanidine-HCl, 0.5 M NaCl, 5 mMimidazole, and 20 mM Tris-HCl, pH 7.9) by Ni-affinity chromatography.The purified protein precipitated upon dialysis into water andwas collected by centrifugation. The protein was then dissolvedin 80% formic acid in a round-bottomed flask and treated withCNBr in the dark, in a fume hood, and under argon for 24 h.The solution was evaporated to dryness at 28°C and resuspendedin 20 mM KPO₄ (pH 7.4) and 100 mM NaCl overnight. The resultingsupernatant was passed over a high-pressure liquid chromatographunder a CH₃CN-H₂O gradient, and the eluted fractions were assessedby mass spectrometry. The fraction containing the TID ${Delta}$ 25, a 49-merpeptide, contained two species: one corresponded to the expectedmolecular mass of the peptide and the other was of the expectedmolecular mass plus six histidines. A subsequent Ni-affinityassay also indicated that the larger of the two peptide speciesstill contained a C-terminal His tag.

GST pull-down assays. The transactivation-inhibitory (TI) domain (residues 566 to641 or 569 to 616) and the TA domain (residues 1 to 26, 1 to34, or 1 to 141) of p63 were each expressed as a fusion proteinwith an N-terminal GST and a C-terminal His tag in Escherichiacoli and purified by Ni-affinity and glutathione-affinity chromatography.The GST fusion protein bound to glutathione beads was incubatedfor an hour with different isoforms and mutants of p63 thatwere expressed with an in vitro transcription-translation system(Promega) and labeled with [³⁵S]methionine or with a purifiedpeptide (TID ${Delta}$ 25). Unbound protein was removed by centrifugationthrough a 0.65-µm-pore-size filter unit (Amicon). Thebeads on the filter were washed four times with 200 µlof binding buffer (100 mM NaCl, 10% glycerol, 50 mM Tris-HCl,pH 8.0) by centrifugation. Finally, the proteins were elutedfrom the beads by incubation with hot SDS sample buffer andremoved from the beads by centrifugation. After PAGE (4 to 12%NuPAGE Bis-Tris), the gels were vacuum dried at 80°C for2 h and the amount of each protein bound to the beads was quantifiedwith a PhosphorImager (Storm 840; Molecular Dynamics). Analysisof the band intensities was conducted with ImageQuant 1.2. Valuesreported are the fractions of protein bound compared to theamount of labeled protein loaded on the beads. As a negativecontrol, the experiments were repeated with only GST bound tothe beads. All experiments were performed in triplicate.

RESULTS

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Results

The last 71 residues of the ${alpha}$ C terminus of p63 contain an inhibitory domain. To identify the inhibitory domain in the large ${alpha}$ C terminus andto investigate its mechanism of inhibition, we created C-terminaldeletion mutants of TAp63 ${alpha}$ and monitored their transactivationwith a luciferase assay in SAOS-2 cells. Wild-type TAp63 ${alpha}$ , thelargest p63 isoform containing both the TA domain and the ${alpha}$ Cterminus, shows only very little transcriptional activity inthese assays. Deleting the last 71 residues, however, restoredthe activity of the protein (TAp63 ${alpha}$ ${Delta}$ TID in Fig. 1A) to a levelapproaching that of a p53 control. This TI domain shows no homologyto any known sequence, aside from the equivalent region of p73 ${alpha}$ ,and is directly C-terminal to a SAM (sterile ${alpha}$ motif) domainthat was recently found in the ${alpha}$ C terminus of both p63 and p73(5, 28). Our finding agrees with an earlier study demonstratingtransactivation activity for a mutant p63 identified in a humanectrodactyly-ectodermal dysplasia-clefting syndrome patientwith a frameshift mutation toward the beginning of the SAM domain,creating an illegitimate 13-amino-acid peptide sequence followedby a premature stop codon after tyrosine-525 (4). Similar resultshave been obtained in a study with the p63-homologous proteinp73 (36).

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FIG. 1. The last 71 residues of the ${alpha}$ C terminus of p63 contain an inhibitory domain. (A) The relative transactivation levels of different p63 isoforms and deletion mutants were compared with those of the wild type and a DNA-binding-incompetent mutant of p53, as described in Materials and Methods. All values are scaled relative to the transactivation of wild-type p53, which was set to 100%. On the left the domain structure of the individual transcripts is indicated: TA (transactivation domain), DBD (DNA-binding domain), OD (oligomerization domain), QP (glutamine- and proline-rich domain), SAM (sterile alpha motif domain), TI (transactivation-inhibitory domain). TAp63 ${alpha}$ ${Delta}$ TID, residues 1 to 570; TAp63 ${Delta}$ ${alpha}$ , residues 1 to 397; TAp63 ${alpha}$ ${Delta}$ QP ${Delta}$ SAM, residues 1 to 397 and 568 to 641. (B) The lysates of p63-transfected cells used to measure relative transactivation (above) were resolved on an SDS-4 to 12% PAGE gradient gel, transferred to a PVDF membrane, and Western blotted with c-Myc-specific antibody 9E10. The bands that appear correspond to the expected molecular weights. The three species with high transactivation potential, TAp63 ${gamma}$ , TAp63 ${alpha}$ ${Delta}$ TID, and TAp63 ${Delta}$ ${alpha}$ , are not visible, while the inactive species, TAp63 ${alpha}$ and TAp63 ${alpha}$ ${Delta}$ QP ${Delta}$ SAM, are abundant. Numbers at right are molecular weights in thousands.

To investigate if this TI domain is both necessary and sufficientfor inhibition, we have deleted the entire ${alpha}$ C terminus and thenadded the TI domain immediately following the oligomerizationdomain (TAp63 ${alpha}$ ${Delta}$ QP ${Delta}$ SAM). This transcript, which lacks the SAM domainas well as a glutamine- and proline-rich domain (QP domain,also known as TAD2), shows very low activity in our transactivationassays (Fig. 1A). In contrast, a deletion mutant lacking the ${alpha}$ C terminus entirely (TAp63 ${Delta}$ ${alpha}$ ) is as active as TAp63 ${gamma}$ . To testthe possibility that the inactivity of TAp63 ${alpha}$ and TAp63 ${alpha}$ ${Delta}$ QP ${Delta}$ SAMis due to low protein expression or rapid degradation, we examinedthe protein levels by Western blot analysis (Fig. 1B). Interestingly,the inactive forms, TAp63 ${alpha}$ and TAp63 ${alpha}$ ${Delta}$ QP ${Delta}$ SAM, are well expressedwhile the active isoforms, TAp63 ${gamma}$ , TAp63 ${alpha}$ ${Delta}$ TID, and TAp63 ${Delta}$ ${alpha}$ , are barelydetectable, indicating that the differences in activity cannotsimply be explained by differences in protein levels. Our resultis in accordance with findings that transactivating p63 isoformshave a much higher turnover rate than do inactive isoforms (34).Overall, the data presented in Fig. 1 suggest that the TI domainis both necessary and sufficient for inhibiting the activityof p63.

The TI domain does not inhibit nuclear localization. In agreement with results from other laboratories (5), our nuclearmagnetic resonance investigation of the TI domain has shownthat the TI domain lacks secondary structure, as characterizedby a narrow range of proton chemical shifts. Unstructured sequencesoften contain signal peptides that target a protein to a certaincellular compartment. One possible mechanism by which the TIdomain could inhibit the transcriptional activity of p63 isby sequestering it in the cytoplasm. To investigate if the TIdomain prevents p63 from entering the nucleus, we have carriedout immunofluorescence assays by expressing TAp63 ${alpha}$ in BHK cells.The results, shown in Fig. 2, demonstrate that TAp63 ${alpha}$ is nuclear,suggesting that the TI domain does not influence the subcellularlocalization of p63. We obtained similar results by fluorescencemicroscopy with cyan fluorescent protein-TAp63 ${alpha}$ fusion proteinsin COS cells (data not shown).

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FIG. 2. The TI domain does not inhibit nuclear localization. Immunofluorescence was used to examine the cellular localization of TAp63 ${alpha}$ , as described in Materials and Methods. myc-tagged TAp63 ${alpha}$ was stained with the c-Myc-specific antibody 9E10 (left). The DNA was visualized with the fluorochrome Hoechst 33278 (right). The nuclear staining of TAp63 ${alpha}$ reveals that the TI domain does not inhibit nuclear localization of the p63 transcription factor.

The TI domain of p63 binds to the N-terminal TA domain. Several transcription factors contain domains that control theirtransactivation through an intramolecular mechanism (11, 12),mainly by inhibiting DNA binding activity. To investigate whetherthe TI domain inhibits the activity of p63 through an intramolecularmechanism, we bacterially expressed and purified a GST and TIdomain fusion protein and conducted pull-down experiments onseveral different p63 isoforms ³⁵S labeled in an in vitro transcription-translationsystem. While TAp63 ${gamma}$ interacted with the GST-bound TI domain, ${Delta}$ Np63 ${gamma}$ did not (Fig. 3A), strongly suggesting that the TI domainin p63 binds to a region of the N-terminal TA domain. To furthertest this hypothesis, we conducted pull-down experiments withTAp63 ${alpha}$ , which contains both a TA and a TI domain. An intramolecularinteraction between the two domains, as it might occur in TAp63 ${alpha}$ ,should be more favorable than an intermolecular interactionwith the external GST-fused TI domain. Indeed, in the pull-downexperiment TAp63 ${alpha}$ bound very poorly. In accordance with thismodel, removal of the TI domain from TAp63 ${alpha}$ restored bindingto a level comparable to that for TAp63 ${gamma}$ , while a p63 form withthe QP and the SAM domain removed (TAp63 ${alpha}$ ${Delta}$ QP ${Delta}$ SAM) also failed tobind (Fig. 3A). Comparison of the relative transactivation (Fig.1A) with the results of these GST pull-down assays reveals astrong correlation that suggests that transactivation inhibitionis linked to binding between the TI and TA domains.

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FIG. 3. The TI domain of p63 binds to the N-terminal TA domain. (A) GST-TID pull-down assay conducted on different p63 isoforms and deletion mutants. The gray bars show the percentages of protein bound to beads loaded with a GST-TID fusion protein, and the black bars show the amounts of protein bound to beads loaded with equal amounts of GST alone. On the right are representative bands corresponding to the levels of ³⁵S-labeled protein bound to GST-TID, prior to quantification. Only p63 constructs with a free TA domain bind to the GST-TID. (B) GST-TID pull-downs of N-terminally truncated mutants of TAp63 ${alpha}$ ${Delta}$ TID. Removal of the first 15 amino acids results in a dramatic loss of binding, while removal of 17 or more amino acids destroys binding completely. The arrow indicates the approximate location of the truncations. (C) GST fusions to the first 26, 34, or 141 amino acids of TAp63 were used to assess binding to either ${Delta}$ Np63 ${gamma}$ (dark gray) or ${Delta}$ Np63 ${alpha}$ (light gray). GST alone bound to neither well, while GST-26 and GST-34 did not bind to ${Delta}$ Np63 ${gamma}$ and bound with moderate affinity to ${Delta}$ Np63 ${alpha}$ . GST-141 also failed to bind ${Delta}$ Np63 ${gamma}$ but bound with high affinity to ${Delta}$ Np63 ${alpha}$ . Thus, the first 34 residues are necessary for binding, but the affinity is greater upon inclusion of additional amino acids in the TA domain.

The TA region of p63, while poorly conserved overall with theTA of p53, contains a sequence that is homologous to the MDM2binding site (23, 31). In particular, the three hydrophobicresidues, F16, W20, and L23, which are deeply buried in a cleftin MDM2 in its complex with p53 (23), are conserved in p63 andp73 (Fig. 4). An interaction between MDM2 and the p73 proteinhas been reported in the literature, though, surprisingly, theinteraction does not lead to degradation of p73 but inhibitionof its transactivation potential (2, 8, 33, 48, 52). In thecase of p63, several conflicting reports have been published.While some research groups do not find interaction between MDM2and p63, others suggest that an interaction occurs that caneither suppress transcriptional activity or enhance it (3, 19,21, 25, 48). The sequence contacted by MDM2 has been shown elsewherefor p53 to be important for transactivation by binding to theTATA binding protein (14) as well as to several TATA bindingprotein-associated factors (TAF_II40 and TAF_II60) (26, 45). Theseobservations, together with our finding that the TI domain bindsthe TA domain, suggest a mechanism by which the TI domain bindsto the equivalent cofactor recruitment site in p63 and, by maskingit, inhibits transactivation. As an initial test of this model,we have created N-terminal truncation mutants that deleted thefirst 13, 15, 17, 20, or 23 amino acids from the TA domain ofTAp63 ${alpha}$ ${Delta}$ TID. In our TI domain interaction assay, the ${Delta}$ 13 mutantbound to the GST-TID fusion protein with an affinity comparableto that for p63 forms with the full-length TA domain (Fig. 3B).The ${Delta}$ 15 form bound with reduced affinity, and ${Delta}$ 17, which lacksF16, the first of the three putative key hydrophobic residues,does not bind. Further truncations, ${Delta}$ 20 and ${Delta}$ 23, also do not bind.These results suggest that the TI domain binds to the MDM2 bindingsequence in the N-terminal acidic TA domain of p63 and inhibitsactivity by masking important residues that presumably contactthe RNA transcriptional machinery.

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FIG. 4. The alignment of the amino termini of p53, TAp63, and TAp73 is shown above the domain structure model. The alignment of TI domains of p63 ${alpha}$ and p73 ${alpha}$ is shown below. The three conserved residues important for p53 binding to MDM2 and transcriptional cofactors are highlighted as white on dark gray. Asterisks indicate p53 residues shown to be phosphorylated under certain conditions. The SUMO-1-modified lysine is highlighted as white on dark gray. Triangles indicate truncation points at the N and C termini used in assays depicted in Fig. 3 and 6.

To determine whether this conserved TA region is sufficientfor association with the TI domain, we created three GST fusionproteins with either the first 26, first 34, or first 141 aminoacids of the TA region of p63. All three fusions contained theconserved F, W, and L and were used to pull down either ${Delta}$ Np63 ${gamma}$ or ${Delta}$ Np63 ${alpha}$ (Fig. 3C). In accordance with our model of a TA-TI interaction,none of the GST fusions bound to the ${gamma}$ form of ${Delta}$ Np63 and all boundthe ${alpha}$ form. However, GST fused to the first 141 residues of TAp63bound to ${Delta}$ Np63 ${alpha}$ better than did either of the two shorter fusions,suggesting that the MDM2 interaction site identified throughhomology with p53 is necessary for association but that bindingimproves when sequences outside the MDM2 interaction site areincluded.

The conserved hydrophobic residues within the TA domain are important both for binding the TI domain and for transactivation. To address the role of the three putative key hydrophobic sidechains, F16, W20, and L23 (Fig. 5A), in binding the TI domain,we replaced each individually with alanine in TAp63 ${gamma}$ and performedpull-down assays with the GST-TI domain fusion protein. Theseexperiments demonstrated only a slight reduction in bindingaffinity for all three single mutants relative to that for wild-typeTAp63 ${gamma}$ (Fig. 5B). We also tested the transcriptional activityof all three mutants to examine whether binding and activityare correlated as in the previous domain deletion experiments(Fig. 1A and 3A), and we found that the activity of the singlemutants, like the binding affinity, was basically unchanged(Fig. 5C). Mutating all three residues simultaneously to alanine,however, had a significant effect on binding as well as on transactivation,reducing both by about 70%. Western blots show a slight accumulationof the single mutants and a very large buildup of the triplemutant, whereas the wild-type protein is barely detectable (Fig.5C). Based on these results, a much weaker interaction betweenthe triply mutated TA domain and the TI domain in TAp63 ${alpha}$ is expected.A weaker intramolecular interaction within TAp63 ${alpha}$ should resultin stronger binding to an external, unmutated TA domain. Indeed,in a GST pull-down assay with the first 141 amino acids of theTA domain (GST-141), the triply mutated TAp63 ${alpha}$ showed strongbinding while wild-type TAp63 ${alpha}$ did not bind (Fig. 5D). This triplymutated TAp63 ${alpha}$ is also completely inactive in the transactivationassay (data not shown). Thus, the same hydrophobic side chainsinvolved in promoting the transactivation of p63 in the absenceof a TI domain are also involved in the stability of the proteinand in binding the TI domain.

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FIG. 5. Conserved residues F16 and W20 are important both for binding to the TI domain and for transactivation. (A) A model of the putative MDM2 binding helix within the TA domain of p63. Hydrophobic residues on one face of the helix thought to be involved in binding to transcriptional cofactors are colored green. A pair of residues on the opposite face are colored blue. The structure is adapted from the crystal structure of MDM2 bound to a peptide from the TA domain of p53 (23). (B) GST pull-down assays on TAp63 ${gamma}$ containing TA domain mutations to alanine. The gray bar shows the percentage of wild-type TAp63 ${gamma}$ bound to beads loaded with a GST-TID fusion protein, the green bars show the percentages of bound mutant TAp63 ${gamma}$ , and the black bars show the amounts of TAp63 ${gamma}$ , wild type or mutant, bound to beads loaded with GST only. (C) The relative transactivation levels of wild-type TAp63 ${gamma}$ and TA domain mutants were compared. All values are scaled relative to the transactivation of wild-type TAp63 ${gamma}$ , which was set to 100% and appears gray. The transactivation levels of the mutants are shown in green. The lysates of transfected cells used to measure relative transactivation were resolved and Western blotted with the anti-p63 4A4 antibody. The bands are of the same size and correspond to the expected molecular weight. (D) GST pull-down assays on TAp63 ${alpha}$ containing a triple mutation in the TA domain. The gray bar shows the percentage of wild-type TAp63 ${alpha}$ bound to beads loaded with GST fused to the first 141 amino acids of the TA domain, while the green bar indicates the percentage of TAp63 ${alpha}$ with F16A, W20A, and L23A mutations bound. The black bars show the amounts of protein bound to beads loaded with GST only. (E) GST pull-down assays on TAp63 ${gamma}$ containing TA domain mutations to aspartate. The gray bar shows the percentage of wild-type TAp63 ${gamma}$ bound to beads loaded with a GST-TID fusion protein, the green and blue bars show the percentages of mutant TAp63 ${gamma}$ bound, and the black bars show the amounts of protein bound to beads loaded with GST only.

Mutating hydrophobic residues to aspartic acid instead of alaninecan have more dramatic effects since burying a charged sidechain in a hydrophobic environment is energetically highly unfavorable.Figure 5E shows that mutating F16 and W20 to aspartic acid reducesthe affinity by almost 50% while mutating L23 again does notaffect the binding. As expected, mutating V15 and H18, two residuesthat are located on the opposite face of the putative bindinghelix (Fig. 5A), does not affect the binding affinity either.All mutation data combined suggest that F16 and W20 are importantamino acids that contribute to binding of the TI domain, whileL23 as well as amino acids on the opposite face of the helix(V15 and H18) is not involved. The weak effect of mutating eitherF16 or W20 alone to alanine, however, also suggests that otherregions within the first 141 amino acids of TAp63 may be involvedin binding, in agreement with our previous findings (Fig. 3C).

The TI domain is a specific inhibitor of the p63 TA domain. Given the conservation of these three hydrophobic residues andtheir importance in the association with the TI domain, we createdchimeric proteins to test whether the TI domain might also acton p53. We began by adding the TI domain to the C terminus ofthe oligomerization domain of p53 (after removing p53's own26-amino-acid C-terminal regulatory domain). In transactivationassays this chimeric protein (p53TID) showed a >50% reductionof activity relative to that of p53 ${Delta}$ 26 (Fig. 6A). We tested ifthis partial inhibition is due to binding of the TI domain tothe p53 MDM2 binding domain by GST pull-down assays. These experimentsshowed no binding of p53 above that of the GST control (Fig.6B), suggesting that the interaction between the p63 TI domainand the p53 TA domain is weak at best. In contrast, a chimericprotein in which the TA domain of p53 has been replaced withp63's TA domain (TA^p63p53 ${Delta}$ 26) is readily pulled down by the GST-TID(Fig. 6B) and is highly active in the dual-luciferase transactivationassays (Fig. 6A). The double chimera of p53 with the TA domainof p63 and the TI domain of p63 (TA^p63p53TID), however, bindsto GST-TID only weakly (Fig. 6B) and is severely repressed intransactivation assays (Fig. 6A), similar to TAp63 ${alpha}$ . When Westernblot analyses are performed, all the chimeras are undetectablewith the exception of TA^p63p53TID, which is barely visible afterlong exposures (data not shown). These activity and bindingresults suggest that the TI domain of p63 preferentially interactswith the TA domain of p63, even if both are presented in thecontext of a different protein. The TA domain of p53, however,does not appear to interact with the inhibitory domain.

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FIG. 6. The TI domain is a specific inhibitor of the p63 TA domain. (A) Relative transactivation of different chimeras composed of swapped domains of p63 and p53. p53 ${Delta}$ 26, residues 1 to 361 of p53; p53TID, residues 1 to 361 of p53 and 568 to 641 of p63; TA^p63p53 ${Delta}$ 26, residues 1 to 127 of p63 and 92 to 361 of p53; TA^p63p53TID, residues 1 to 127 of p63, 92 to 361 of p53, and 568 to 641 of p63. Shaded segments represent p53 domains, and nonshaded segments represent p63 domains. (B) GST pull-down assay on different p53/p63 chimeras. The gray bars show the percentages of protein bound to beads loaded with a GST-TID fusion protein, and the black bars show the amounts of protein bound to beads loaded with GST only. (C) The relative transactivation levels of TAp63 ${alpha}$ with various C-terminal truncations were compared with that for a DNA-binding-incompetent mutant of p53. All values are scaled relative to the transactivation of TAp63 ${alpha}$ ${Delta}$ TID, which was set to 100%. TAp63 ${alpha}$ mutants lacking the last 8 ( ${Delta}$ 8), 16 ( ${Delta}$ 16), or 25 ( ${Delta}$ 25) amino acids show a moderate increase in activity relative to full-length TAp63 ${alpha}$ . ${Delta}$ 35, ${Delta}$ 45, and ${Delta}$ TID (missing the last 71 residues) have high activity. The arrow indicates the approximate location of the truncations. The lysates of transfected cells used to measure relative transactivation were resolved on an SDS-4 to 12% PAGE gradient gel, transferred to a PVDF membrane, and Western blotted with the anti-p63 4A4 antibody. The bands correspond to the expected molecular weights, gradually decreasing from left to right. (D) Results of GST-TID pull-down assays with C-terminally truncated forms of TAp63 ${alpha}$ . GST-TID fails to bind TAp63 ${alpha}$ presumably because the intramolecular association is more favorable than the same association in trans. Removal of the last 35 or more residues leads to a sudden and dramatic improvement in binding most likely caused by a disruption of the intramolecular TI-TA domain interaction. (E) GST pull-down assay with a truncated TI domain. The affinities of different p63 isoforms for a truncated form of the TI domain lacking the last 25 amino acids (GST-TID ${Delta}$ 25) were measured. The results are very similar to the results obtained with GST fused to the full-length TI domain.

The TI domain contains two subdomains that inhibit transcription through different mechanisms. The modest reduction of transactivation of the p53TID chimera,in spite of the absence of a detectable interaction betweenthe TI domain and p53, prompted us to look into alternativemechanisms of transactivation repression. Since it remains possiblethat a weak association between p53 and the TI domain is sufficientto account for the loss of activity, we looked for a correlationbetween disruption of the intramolecular TA-TI domain interactionand relief of inhibition through a series of C-terminal truncationsof TAp63 ${alpha}$ . Removal of 8, 16, or 25 amino acids all led to a smallincrease in transcriptional activity relative to TAp63 ${alpha}$ . Furtherremoval of amino acids ( ${Delta}$ 35 and ${Delta}$ 45) restored the transactivationto levels comparable to those for TAp63 ${alpha}$ ${Delta}$ TID. Western blot analysisof these transactivation assays showed high protein levels forTAp63 ${alpha}$ and the ${Delta}$ 8, ${Delta}$ 16, and ${Delta}$ 25 deletion mutants, while ${Delta}$ 35, ${Delta}$ 45,and ${Delta}$ TID are hardly detectable (Fig. 6C), reconfirming the inversecorrelation of protein level with transcriptional activity observedearlier (Fig. 1).

To investigate the molecular mechanism by which the relief ofinhibition occurs in these C-terminal truncations, we performedpull-down assays (Fig. 6D). The ${Delta}$ 8, ${Delta}$ 16, and ${Delta}$ 25 deletion mutantsdid not show any increase in binding affinity to the GST-TIDfusion protein relative to TAp63 ${alpha}$ . Such an increase in bindingwould be expected if the C-terminal truncation disrupted thenative intramolecular TA-TI domain interaction, allowing theGST-TID to bind in trans. Removal of 35 or more amino acidsfrom the C terminus, however, restored binding affinity to levelscomparable to those for TAp63 ${alpha}$ ${Delta}$ TID. These results clearly demonstratethat the last 25 amino acids in the TI domain of p63 are unnecessaryfor binding to the TA domain. We confirmed these results byperforming pull-down assays with the ${Delta}$ 25 truncation mutant ofthe TI domain fused to GST (GST-TID ${Delta}$ 25). This construct was ableto bind TAp63 ${gamma}$ and TAp63 ${alpha}$ ${Delta}$ TID, but not ${Delta}$ Np63 ${gamma}$ or TAp63 ${alpha}$ , with relativeaffinities that are indistinguishable from those for GST fusedto the full-length TI domain (Fig. 6E). Thus, short C-terminaldeletions ( ${Delta}$ 8, ${Delta}$ 16, and ${Delta}$ 25) partially relieve inhibition, thoughthe intramolecular TA-TI domain interaction remains intact,implying that the TI domain may contain two subdomains withdifferent inhibitory mechanisms. This interpretation is furthersupported by our Western blot analysis of the protein levelsin the transactivation assays. In agreement with our earlierresults (Fig. 1), all p63 forms that are inactive due to bindingof the TI domain to the TA domain accumulate in the cell andcan be detected as strong bands in Western blots. In contrast,all active p63 forms in which the TA domain is not masked bythe TI domain are barely detectable, most likely due to rapidturnover of the protein. Further support for our model of twodifferent subdomains within the TI domain is based on sequencecomparison between p63 and p73. The sequence of p73 that showshigh homology to the TI domain of p63 has a polyglycine insertion25 amino acids from the C terminus (Fig. 4). This insertioncoincides with the position beyond which further removal ofamino acids eliminates the TA-TI domain interaction and maymark a boundary between these inhibitory subdomains.

The last 25 amino acids, on the other hand, seem to inhibitthe activity of p63 by an additional mechanism that is differentfrom the binding of the TI domain to the TA domain. Interestingly,the last eight amino acids in p63 contain a conserved lysinethat becomes covalently modified by the ubiquitin homologueSUMO-1 in p73 (30) (Fig. 4). Covalent attachment of ubiquitinor SUMO-1 has been shown elsewhere to affect protein stabilityand can affect their turnover rate inside cells (6). Sumoylationmight, therefore, explain the inhibitory effect of the TI domainon p53 as well as the partial relief of inhibition by the ${Delta}$ 8, ${Delta}$ 16, and ${Delta}$ 25 mutants. Since the activities of the ${Delta}$ 8, ${Delta}$ 16, and ${Delta}$ 25truncation mutants are relatively similar, this additional inhibitoryeffect is most likely localized in the last eight amino acids.If this model is correct, removing the last eight amino acidsfrom the C terminus of the TI domain in the p53TID chimera shouldrelieve the partial inhibition with this chimera that we observedas shown in Fig. 6A. Indeed, in transactivation assays the p53TID ${Delta}$ 8construct showed a transcriptional activity that is indistinguishablefrom that of wild-type p53.

Thus, the combination of the results of the pull-down assays,transactivation assays, Western blot analysis, and sequencecomparison strongly suggests that the first $~$ 50 residues of theTI domain inhibit transactivation by binding to the TA domain,while the last 25 residues may be involved in associations withother proteins that modulate stability and activity of p63.Interestingly, a human patient with split-hand-split-foot malformationcaused by a nonsense mutation at Q634 has been identified elsewhere(47). This mutation creates a form of p63 ${alpha}$ that lacks the lasteight amino acids ( ${Delta}$ 8 form) and demonstrates, in accordance withour results, the importance of even just the last eight aminoacids for p63's biological function.

To investigate the exact mechanism by which binding of the TIdomain to the TA domain inhibits transcriptional activity, weinvestigated the interaction of the first 141 amino acids ofthe TA domain with several cofactors of the transcriptionalmachinery (TAF_II32, TAF_II40, TAF_II60, TAF_II70, TAF_II250, CBP,and p300) both by pull-down assays with GST-141 and by immunoprecipitation.None of these factors bound significantly above background levels,suggesting either that different cofactors are involved in p63transcriptional activity or that posttranslational modifications,in particular phosphorylation, might be necessary to establishan interaction.

The TI domain binds directly to the TA domain. The pull-down experiments described so far were performed inrabbit reticulocyte lysates as part of the in vitro transcription-translationkit used to label the p63 isoforms with ³⁵S. To explore thepossibility that there might be a rabbit protein in the lysatesthat mediates the TA-TI domain association, we conducted pull-downsexclusively with purified proteins made recombinantly in E.coli. The GST-TA fusions were used to assess binding to thefirst 49 residues of the TI domain (TID ${Delta}$ 25), and the resultswere assessed by Coomassie blue staining (Fig. 7). In agreementwith our results shown in Fig. 3C, the GST alone did not bindthe TI peptide, but fusions of the GST to TA peptides of increasinglength did. Once again, the GST fused to the first 141 residuesbound better than did GST fused to the first 26 or 34 residues.This experiment demonstrates that the TA-TI domain interactionis indeed direct and also suggests that no posttranslationalmodification is required for either domain to drive the association.

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FIG. 7. The TI domain binds directly to the TA domain. The GST-TA fusions used in Fig. 3C were used to assess binding to a recombinantly expressed and purified TID ${Delta}$ 25 peptide (residues 568 to 616). Lane 1 contains the purified TID ${Delta}$ 25 peptide. The doublet is due to the presence (higher molecular weight) or absence (lower molecular weight) of a C-terminal six-histidine tag as indicated by both mass spectrometry and nickel affinity (see Materials and Methods). The histidine tag does not appear to affect the affinity of the TID ${Delta}$ 25 peptide for the TA domain. The peptide was incubated with either GST, GST-26, GST-34, or GST-141 fusion proteins bound to glutathione-Sepharose, washed, eluted with hot SDS sample buffer, and run in lanes 2, 3, 4, and 5, respectively. The very high molecular weight bands correspond to the GST fusions and occur at roughly equivalent concentrations. The low-molecular-weight doublet indicates the fraction of TID ${Delta}$ 25 still bound to the fusions following the washes. GST alone does not bind a detectable amount of peptide, while GST-26 and GST-34 bind with moderate affinity and GST-141 binds with high affinity. Numbers at left are molecular weights in thousands.

DISCUSSION

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Discussion

Our experiments demonstrate that p63 and p53 employ differentmechanisms to regulate transcriptional activity. p53 also containsa regulatory domain at its extreme C terminus which is, in contrastto the TI domain of p63, only 26 amino acids long. More importantly,the mechanism of inhibition seems to be different, as the inhibitorydomain of p53 is involved in regulating the sequence-specificbinding affinity of the DNA-binding domain (15, 16, 24) whilethe TI domain of p63 binds to the N-terminal TA domain. Theclosely related protein p73 shows a very similar domain structure,including a TI domain and a SAM domain in its ${alpha}$ C terminus. Thishigh sequence homology between the two proteins (Fig. 4) predictsthat the results shown here are relevant not only for p63 butalso for p73.

${Delta}$ Np63 ${gamma}$ and ${Delta}$ Np63 ${alpha}$ display a similar and moderate dominant-negativeeffect toward p53 but radically different effects toward transactivatingTAp63 ${gamma}$ (49). TAp63 ${gamma}$ is, if at all, only weakly repressed by ${Delta}$ Np63 ${gamma}$ but very strongly repressed by ${Delta}$ Np63 ${alpha}$ , the major p63 isoform incells. In light of our results, these different effects of p63isoforms on p53 and TAp63 ${gamma}$ can now be interpreted. The TI domainof ${Delta}$ Np63 ${alpha}$ cannot interact intramolecularly since it lacks itsown TA domain. However, p63 tetramerizes (7), and formationof hetero-oligomers between TAp63 ${gamma}$ and ${Delta}$ Np63 ${alpha}$ could allow the TIdomain to interact with the TA domain of another protein withinthe tetramer. A 2:2 complex of ${Delta}$ Np63 ${alpha}$ and TAp63 ${gamma}$ would, therefore,reduce the number of active TA domains in the tetramer to zero(Fig. 8A), powerfully inhibiting the activity of TAp63 ${gamma}$ . ${Delta}$ Np63 ${gamma}$ ,however, lacks the TI domain, and a 2:2 complex with TAp63 ${gamma}$ willleave two active TA domains (Fig. 8B). Thus, by controllingthe ratios of the different isoforms through alternative splicing,the activity of p63 may be fine-tuned.

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FIG. 8. Models of p63 heterotetramers. (A) In a tetramer consisting of two TAp63 ${gamma}$ (green) and two ${Delta}$ Np63 ${alpha}$ (red) monomers, the TI domains of ${Delta}$ Np63 ${alpha}$ can interact with and inhibit both TA domains of the TAp63 ${gamma}$ monomers. Activation of this tetramer could occur by recruiting another protein, e.g., a kinase that disrupts the TI-TA domain interaction. Binding of kinase or the other factor, illustrated as "X," could be facilitated by the SAM domain. (B) Tetramerization of two TAp63 ${gamma}$ monomers (green) with two ${Delta}$ Np63 ${gamma}$ monomers (yellow) leaves both TA domains active.

With respect to p53, ${Delta}$ Np63 ${gamma}$ and ${Delta}$ Np63 ${alpha}$ show very similar levelsof inhibition. Earlier results have suggested that p53 and p63cannot form hetero-oligomers (7), and the inhibitory effectsof ${Delta}$ Np63 isoforms on p53 were attributed to competition for DNA-bindingsites (49). Other research laboratories have reported a directinteraction between p53 and p63 in their DNA-binding domains(38). In addition, Pozniak et al. have shown that ${Delta}$ N isoformsof p73 play an antiapoptotic role and are direct antagonistsof p53 in sympathetic neurons (37). They attribute this antagonisticrole of p73 to direct binding to p53. While our results suggestthat the TI domain of p63 cannot directly bind to the TA domainof p53 and inhibit its transactivation potential, they are consistentwith a model based on competition for DNA-binding sites anddo not contradict a possible association through the DNA-bindingdomains. The combined results of all research groups are alsoconsistent with a model in which p63 and p73 are involved inregulatory networks with p53 and control p53's apoptotic functionin neurons and epithelial stem cells. Most likely, however,the TI domain is used only for the direct regulation of thetranscriptional control of p63 and maybe p73, but not in interactionswith p53.

Our results explain why TAp63 ${alpha}$ shows a low level of transactivationrelative to that of TAp63 ${gamma}$ and explain the strong dominant-negativeeffect of ${Delta}$ Np63 ${alpha}$ on transactivating p63 isoforms. Based on ourdata, it is now possible to integrate a wealth of other studiesabout p63 regulation into one consistent model. In this model,a high concentration of ${Delta}$ Np63 ${alpha}$ immediately inactivates any smallconcentration of active p63 isoforms and keeps them in an inhibitedcomplex. Complex formation of active p63 isoforms with ${Delta}$ Np63 ${alpha}$ increases the lifetime of the TA domain-containing forms whilekeeping them in an inactive state. To activate the transcriptionalpotential of p63, only the inhibitory interaction between theTI and the TA domains has to be relieved, thereby avoiding time-consumingprotein expression. In this way, differential splicing of thep63 gene can create a transcriptional regulatory complex thatallows the cell to control the activity of p63 in a fast, switch-likemanner.

Several lines of evidence support such a model. First, ${Delta}$ Np63 ${alpha}$ is the most abundant isoform found in epithelial cells, andit has been demonstrated that even a mutant ${Delta}$ Np63 ${alpha}$ incapable ofbinding DNA retains the strong dominant-negative effect towardactive p63 isoforms, while losing its dominant-negative effecton p53. This suggests that the inhibition of active p63 isoformsby ${Delta}$ Np63 ${alpha}$ is not due to competition for DNA-binding sites butoccurs through direct interaction between the isoforms. Second,several reports have demonstrated that p63 isoforms with anactive TA domain are barely detectable by immunochemistry (40,49), and the TA domain has been implicated in the responsibilityfor fast degradation (34). In contrast, ${Delta}$ Np63 isoforms, whichlack the TA domain, seem to be more stable. Further supportingthese observations, mutations made in the putative MDM2 bindinghelix of p63 dramatically increase stability of the protein(Fig. 5C), and notably these same residues are involved in anassociation with the TI domain (Fig. 3B and C, 5B, and 7). Thus,when sites within the TA domain responsible for fast degradationare masked by the TI domain, protein stability and protein levelsshould increase, and indeed, our results indicate that TA isoformscontaining the TI domain accumulate to significantly higherlevels than those without (Fig. 1B and 6C). According to ourmodel, this masking and stabilization could also occur in transin hetero-oligomeric complexes containing ${Delta}$ Np63 ${alpha}$ and TAp63 ${gamma}$ /ß.Finally, the central, biological role that the ${alpha}$ C terminus playsin p63 is emphasized by the analysis of p63 mutations in humanpatients. Surprisingly, patients with mutations in the ${alpha}$ C terminusthat partially abolish the inhibitory function of the TI domainand create transcriptionally active p63 ${alpha}$ forms show very similarphenotypes as do patients with mutations in the DNA-bindingdomain that abolish DNA binding of all p63 isoforms (4). Whilethe mechanism of how loss of inhibition in the ${alpha}$ isoforms andloss of DNA binding in all isoforms lead to similar phenotypesis currently unknown, it shows that the inhibitory functionin the TI domain is of paramount importance.

The question remains how p63 becomes active. Our results indicatethat disruption of the inhibitory interaction between the TIand the TA domains would trigger p63's transactivation potential.Such a trigger could be phosphorylation of either of the twodomains, and several known sites of phosphorylation in the TAdomain of p53 are conserved in p63 (Fig. 4). As discussed above,potential regulatory sites for the TA-TI interaction might belocated in the last 25 amino acids of the TI domain. Alternatively,since SAM domains are protein-protein interaction modules thatare found in a large variety of different protein classes, theSAM domain that is located directly N-terminal to the TI domaincould recruit, for example, kinases (Fig. 8). A recent analysisof mutations found in the SAM domain of p63 in human patients(28) has suggested a binding site for a potential interactionpartner that is different from the SAM-SAM domain dimerizationinterface found in several SAM domain structures so far (41,42, 44). This opens the possibility that the p63 SAM domainmight interact with a binding partner that is not another SAMdomain. Studies to address these questions are being carriedout.

ACKNOWLEDGMENTS

We thank Nilesh Shah for assistance with the cyanogen bromidecleavage reactions, Alessander Guimaraes for generating theluciferase reporter, and Holly Ingraham for helpful discussions.

Financial support was provided by an institutional researchgrant from the American Cancer Society (AC-02-8), the UCSF Innovationsin Basic Sciences Award, and the Sandler Family supporting foundation.Z.S. was supported by the Burroughs Wellcome Fund. H.C.L., H.D.O.,and A.E.K. were supported by an NIH training grant (GM08284).

FOOTNOTES

* Corresponding author. Mailing address: Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, CA 94143. Phone: (415) 502-7050. Fax: (415) 476-0688. E-mail: volker{at}picasso.ucsf.edu.

REFERENCES

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