The E3 ubiquitin ligase Itch controls the proteinstability of p63Mario Rossi*, Rami I. Aqeilan†, Michael Neale*, Eleonora Candi‡, Paolo Salomoni*, Richard A. Knight*, Carlo M. Croce†§,and Gerry Melino*‡¶

*Medical Research Council, Toxicology Unit, Leicester University, Leicester LE1 9HN, United Kingdom; †Department of Molecular Virology, Immunology,and Medical Genetics, Comprehensive Cancer Center, Ohio State University, Columbus, OH 43210; and ‡Biochemistry Laboratory, Istituto Dermopaticodell’Immacolata–Istituto di Ricovero e Cura a Carattere Scientifico, Department of Experimental Medicine and Biochemical Sciences, University of Rome‘‘Tor Vergata,’’ 00133 Rome, Italy

Edited by Peter K. Vogt, The Scripps Research Institute, La Jolla, CA, and approved July 5, 2006 (received for review April 27, 2006)

p63, a member of the p53 family of transcription factors, plays animportant role in epithelial development, regulating both cell cycleand apoptosis. Even though p63 activity is regulated mainly at theposttranslational level, the control of p63 protein stability is farfrom being fully understood. Here, we show that the Hect (ho-mologous to the E6-associated protein C terminus)-containingNedd4-like ubiquitin protein ligase Itch binds, ubiquitylates, andpromotes the degradation of p63. The physical interaction occursat the border between the PY and the SAM (sterile � motif)domains; a single Y504F mutation significantly affects p63 degra-dation. Itch and p63 are coexpressed in the epidermis and inprimary keratinocytes where Itch controls the p63 protein steady-state level. Accordingly, p63 protein levels are significantly in-creased in Itch knockout keratinocytes. These data suggest thatItch has a fundamental role in the mechanism that controls en-dogenous p63 protein levels and therefore contributes to regula-tion of p63 in physiological conditions.

keratinocytes � ubiquitination � p73 � AIP4

The major evidence linking p63 to the development of theepidermis comes from the analysis of the p63 knockout (KO)mouse phenotype. p63�/� mice die at birth with severe devel-opmental abnormalities, including limb appendage truncationsand defects in the epidermis (1–3). The surface epithelium isessentially absent. Consequently, p63 controls either the com-mitment of the immature ectoderm to epidermal lineages (1) orthe proliferative potential of the epidermal stem cells (2). p63deficiency also induces and accelerates cellular senescence andcauses accelerated aging phenotypes in the adult mouse in vivo(4). In addition, heterozygous germ-line mutations in the TP63gene result in human genetic syndromes involving defectivedevelopment of the limbs and�or ectodermal dysplasia, which ischaracterized by defects in the skin and associated structures (5).

p63 is a p53 homologue, sharing with p53 and p73 a similarmodular structure as well as a high degree of sequence homology(6), especially in the central DNA-binding domain. In additionto the full-length protein containing a transactivation domain(TAp63), the TP63 gene gives rise to several isoforms by usingan alternative promoter located in intron 3 (N-terminally deletedisoforms, �Np63) or by means of alternative splicing at the Cterminus (�, �, and � isoforms) (6). TAp63 is transcriptionallyactive and can induce cell cycle arrest and apoptosis (6, 7). Giventhat the transactivation activity resides in the protein’s N termi-nus, the �Np63 forms exhibit a degree of dominant-negativeeffect by means of competition for binding to p53 target pro-moters (8), similar to their homologue, �Np73 (9). However,recent studies indicate that �Np63 is transcriptionally competentper se (10–12) because of the presence of a second transactiva-tion domain located between exons 11 and 12 (10, 11). Thisobservation seems particularly relevant because �Np63 is thepredominant p63 isoform; it is expressed in a highly tissue-specific manner in the embryonic ectoderm and the basal

regenerative compartment of epithelial tissues, including theskin, teeth, and hair (3, 8, 13). �Np63 accounts for 100% of allp63 forms expressed up to embryonic day 9 and 99% atembryonic day 13. TAp63 expression starts at embryonic day 13and accounts for only 1% of the total p63 protein expressed atthis time. Thus, expression of the �Np63 isoform is predominantover that of TAp63 at a time before any epidermal stratificationoccurs (3, 13).

Here, we found that p63 functionally associates with theubiquitin (Ub) protein ligase (E3) Itch, which belongs to theNedd4-like E3 family and, like the other members of the family,contains an N-terminal protein kinase C-related C2 domain,multiple WW domains, and a C-terminal Hect (homologous tothe E6-associated protein C terminus) Ub protein ligase domain(14). Itch is important for the regulation of murine epithelial andhematopoietic cell growth and is absent in the non-agouti-lethal18H (Itchy) mice that display profound immune defects andrepresent a well established Itch null mouse model (15, 16).Herein, we show that Itch binds, ubiquitylates, and determinesp63 degradation. We also show that, in Itch�/� primary keroti-nocytes, �Np63 protein levels are increased, suggesting a rele-vant role of Itch in regulating p63 in vivo.

Results and DiscussionItch Interacts with p63�. We have demonstrated that Itch acts asan E3 ligase of both TAp73� and �Np73� but not of p53 (17).Here, we set out to determine whether p63 also is regulated byItch. Similar to p73� isoforms, p63� contains a proline-rich motif(PPxY) at its C terminus that is present in the transactivating(TA) and �N isoforms (Fig. 1A) and is known to interact withWW domains. Because Itch contains four WW domains, weexamined whether Itch physically associates with TAp63� and�Np63� proteins by performing coimmunoprecipitation exper-iments. We transiently cotransfected HEK293 cells with eitherFlag-TAp63� or Flag-�Np63� and Myc-Itch. Cell lysates wereimmunoprecipitated with anti-Myc antibodies and then wereimmunoblotted with antibodies against Flag or Myc tags. Ourresults demonstrate that Itch interacts with both the TAp63� and�Np63� isoforms, even though there is a preference for thelatter (Fig. 1B, lanes 4 and 6). This preference may be due tostructural constrains of TAp63� caused by the intramolecular

Conflict of interest statement: No conflicts declared.

This paper was submitted directly (Track II) to the PNAS office.

Freely available online through the PNAS open access option.

Abbreviations: Ub, ubiquitin; KO, knockout.

§To whom correspondence may be addressed at: Comprehensive Cancer Center, Ohio StateUniversity, Wiseman Hall, Room 385K, 400 West 12th Avenue, Columbus, OH 43210.E-mail: carlo.croce@osumc.edu.

¶To whom correspondence may be addressed at: Apoptosis and Cancer Group, MedicalResearch Council, Toxicology Unit, Leicester University, Hodgkin Building, Lancaster Road,Leicester LE1 9HN, United Kingdom. E-mail: gm89@le.ac.uk.

© 2006 by The National Academy of Sciences of the USA

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interaction between the p63 transactivation domain and its Cterminus (18). We then tested whether mutation of the proline-rich domain results in loss of binding to Itch. This region isconserved in p73, where it is responsible for the interaction withItch (17). As shown in Fig. 1B (lanes 4 and 8 for TAp63 and lanes

6 and 10 for �Np63), a single amino acid substitution in the PYdomain of p63 (Y504F for TAp63; Y449F for �Np63) is suffi-cient to reduce its ability to interact with Itch. Although wecannot completely rule out that additional regions of p63 areinvolved in this interaction, these results confirm that the p63 PYdomain is the main binding site for Itch.

Itch and p63 Are Endogenously Coexpressed and Interact. To verifythat the interaction between Itch and p63 occurs at endogenouslevels, we performed coimmunoprecipitation experiments in animmortalized human keratinocyte cell line, HaCaT, which ex-presses abundant levels of �Np63�. Endogenous p63 was coim-munoprecipitated with Itch (Fig. 1C) but not with the negativecontrol p53. These results confirm that p63 associates with Itchat endogenous level.

Consistent with the biochemical results shown above, we alsofound that Itch and p63 are coexpressed in adult human normalskin (Fig. 1D). In particular, p63 tends to be more expressed inthe basal layer of the epidermis, whereas Itch expression ishigher in the outer layers.

Itch Ubiquitylates p63�. Having demonstrated that Itch interactswith p63�, we next assessed whether p63� can serve as a

Fig. 2. Itch induces ubiquitylation and affects the steady-state levels of p63�protein. (A) HEK293 cells were cotransfected with vectors expressing Myc-tagged WT Itch (Myc-Itch WT), enzymatically inactive Itch (Myc-Itch MUT),Flag-TAp63�, Flag-�Np63�, and HA-tagged Ub (Ub HA). Lysates were immu-noprecipitated with anti-Flag antibody and probed with anti-HA antibody.The top blot shows Ub-conjugated p63 proteins. The middle blot shows p63levels in the immunoprecipitates. The bottom blot shows Itch levels in theinput lysates. Experiments were carried out in the presence of the proteasomeinhibitor MG132 (20 �M). (B and C) HEK293 cells were transfected with theindicated combinations of plasmids and analyzed for p63 steady-state levelsby using anti-Flag antibody (top blots). The middle blots show Itch levels. Thebottom blots show tubulin levels as a loading control.

Fig. 1. Itch physically interacts with p63. (A) Schematic representation of themodular structure of the TAp63� and �Np63� proteins. The main structuraldomains are indicated: transactivation domain (TA), DNA-binding domain,oligomerization domain (OD), second transactivation domain (TA2), sterile �motif (SAM), and transinhibitor domain (TI). The p63 region containing thePPxY motif is also shown (in red). (B) HEK293 cells were cotransfected withdifferent combinations of expression vectors for Myc-tagged Itch (Myc-Itch),Flag-tagged TAp63� (Flag-TAp63�), and Flag-tagged �Np63� (Flag-�Np63�)and their mutant versions, TAp63� Y504F (Flag-TAp63�Y504F) and �Np63�Y449F (Flag-�Np63�Y449F). Anti-Myc immunoprecipitates were probed withanti-Flag antibodies. The top blot shows coimmunoprecipitated proteins. Themiddle blot shows Itch levels in the immunoprecipitates. The bottom blotshows p63 levels in the input lysates. Experiments were carried out in thepresence of the proteasome inhibitor MG132 (20 �M). (C) Coimmunoprecipi-tation of endogenous p63 and Itch proteins. Human immortalized keratino-cyte HaCaT extracts were immunoprecipitated with monoclonal antibodiesagainst Itch, and Western blotting was performed with antibodies againstItch, p63, and p53. As a control, immunoprecipitation also was performed withan anti-p53 antibody (D01). (D) Human adult skin sections stained withmonoclonal mouse anti-Itch antibodies, secondary goat anti-mouse AlexaFluor 488, rabbit polyclonal anti-p63 (H129), and secondary goat anti-rabbitAlexa Fluor 568 and nuclei were counterstained with DAPI and analyzed byconfocal microscopy.

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substrate for the Ub protein ligase activity of Itch. To this end,we overexpressed Itch together with either TAp63� or �Np63�,and we HA-tagged Ub (Ub HA) and analyzed the appearanceof Ub-conjugated forms of p63 (Fig. 2A). Consistent with theresults of the coimmunoprecipitation experiments, Itch showedthe ability to ubiquitylate both �Np63� and TAp63� (Fig. 2 A,lanes 2 and 5). To verify that Itch-dependent ubiquitylationrequires its enzymatic activity, we examined whether the cata-lytic inactive mutant of itch (Itch MUT) is capable of ubiquity-lating p63. As shown in Fig. 2 A, Itch MUT is unable toubiquitylate p63, although it physically binds both p63 isoforms(data not shown). These results demonstrate that the Itch�p63interaction has direct functional consequences.

p63� Steady-State Levels and Half-Life Are Affected by Itch. Todetermine whether Itch-dependent ubiquitylation of p63� pro-motes p63� degradation, we measured steady-state levels of p63isoforms in the presence or absence of Itch. Both TAp63� and�Np63� steady-state protein levels were down-regulated whencoexpressed with Itch under the experimental condition tested.In contrast, p63 protein levels were not affected when the Itchinactive mutant was coexpressed (Fig. 2B). In addition, the levelsof the mutant version of �Np63� (�N63�Y449F, which does notefficiently interact with Itch) were only marginally affected bythe presence of Itch, thus indicating that direct binding to Itchis required for p63 degradation (Fig. 2C). To further prove thatthe Itch-dependent reduction in p63 protein levels was due to

Fig. 3. Itch reduces the half-life of the p63� protein. (A–C) Cycloheximide blockade. HEK293 cells were cotransfected with Flag-tagged TAp63� (A), �Np63�(B), and �Np63�Y449F (C) expression vectors in the presence of either control vector (Top) or Myc-tagged WT Itch expression vector (Middle). Twenty-four hoursafter transfection, cells were incubated with cycloheximide (20 �g�ml), and, at the indicated time points, cells were harvested, lysed, and analyzed for p63 levelsby using an anti-Flag antibody. In all cases, �-tubulin levels were measured as a loading control. (D–G) 35S pulse–chase: H1299 cells were transfected withHA-TAp63� (D), HA-�Np63� (E), Flag-�Np63� (F), or Flag-�Np63�Y449F (G) together with Myc-Itch or pCDNA-Myc expression vector. At 48 h after transfection,cells were labeled with 250 �Ci�ml Redivue PRO-MIX (L-[35S] in vitro cell-labeling mix). Unlabeled Met and Cys (1 mg�ml) were added, and cells were collectedat the indicated time points. Immunoprecipitation was performed with anti-HA (D and E) or anti-Flag (F and G) antibodies. Immunoprecipitates were washed,run on SDS�PAGE, and detected by autoradiography. Densitometry is shown in arbitrary units.

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increased degradation, we analyzed the half-life of p63 in thepresence or absence of Itch by using two different methods. Bothpulse–chase (Fig. 3 A and B) and cycloheximide blockade (Fig.3 D and E) showed a marked decrease of TAp63 and �Np63half-lives in the presence of Itch. Under similar experimentalconditions, the half-life of the �Np63�Y449F mutant was onlymarginally affected (Fig. 3 C and G). Thus, our data show thatItch promotes the Ub-dependent degradation of p63�.

Itch Controls Endogenous �Np63� Protein Levels in Primary Keratin-ocytes. To further confirm the physiological role of Itch-mediated degradation of p63, we examined levels of endogenous�Np63�, the only detectable isoform, in primary mouse kera-tinocytes during differentiation. Ex vivo keratinocytes retain

their ability to differentiate morphologically (Fig. 4A), inducingspecific keratinocyte differentiation markers (Fig. 4B and datanot shown), and therefore represent a valid physiological modelfor studying the functional interaction between Itch and p63.Indeed, the cultured keratinocytes were able to differentiate inthe presence of high calcium concentration and express signif-icant amounts of involucrin (Fig. 4B). During keratinocytedifferentiation, Itch levels increase concomitantly with a de-crease in �Np63 levels in both mouse (Fig. 4B) and human (Fig.4C) cells, which is consistent with our observations of thedistribution of Itch and p63 in human skin (Fig. 1D).

We then compared the steady-state levels of �Np63� inkeratinocytes derived from non-agouti-lethal 18H Itch-deficient(KO) and WT mice, both of the same genetic background. Asshown in Fig. 4D (second blot from top), steady-state levels of�Np63� were higher in primary cells derived from Itch-deficientmice compared with WT. Upon induction of differentiation,�Np63� protein levels decreased more slowly in Itch KO cells(Fig. 4D). Furthermore, reintroduction of WT Itch into Itch KOkeratinocytes resulted in reduction of endogenous �Np63 pro-tein levels in these cells (Fig. 4E), confirming that Itch contrib-utes to the control of p63 steady-state protein levels in physio-logical settings.

Conclusionsp63 is expressed in the basal cell compartment of many differentepithelia, particularly in the epidermis, with a differential rolefor the TAp63� and �Np63� isoforms (12). Our findings indi-cate that Itch is coexpressed with p63 in the epidermis and is ableto control its Ub-dependent degradation. Consequently, eventhough our data do not exclude the presence of additional E3ligases controlling p63 protein levels, Itch is instrumental indown-regulating p63 in the epithelium. Most Itch substrates aretranscription factors [i.e., c-Jun (16, 19), JunB (16), Smad2 (20),and p73 (17)], which play major roles in different physiologicalpathways as well as in the responses to different types of stresses,thus implying a significant role for Itch itself. Among them,c-Jun, JunB, and Notch are expressed in the epidermis. We nowprovide evidence that another transcription factor expressed inthe epidermis, p63, is a physiological target of Itch. Interestingly,abnormal expression of the Itch substrates c-Jun and JunB hasbeen detected in psoriatic epidermis (21), raising the possibilitythat the deregulation of p63, c-Jun, and JunB by Itch could beone of the pathophysiological mechanisms involved. More re-cently, it has been reported that Itch activity is regulated at theposttranslational level (22–24), adding additional levels of com-plexity to the function of this enzyme.

Taken together, these data suggest an important role for thep63�Itch functional interaction in vivo and support the hypoth-esis that Itch could play an important role in the regulation ofepithelial homeostasis.

Materials and MethodsPlasmids. Myc-Itch plasmids and the HA–Ub construct (Ub HA)are described in ref. 17. Flag-TAp63� and Flag-�Np63� wereobtained by subcloning the cDNA TAp63� and �Np63� into theNheI and NotI sites of pCDNA3.1. Flag-TAp63�Y504F andFlag-�Np63�Y449F were obtained by using QuikChange II site-directed mutagenesis kits (Stratagene, La Jolla, CA).

Cell Culture and Transfection. Human embryonic kidney cells(HEK293) and immortalized human keratinocytes (HaCaT) (agift from N. Fusenig, German Cancer Research Center, Hei-delberg, Germany) (25) were grown in DMEM (GIBCO-BRL,Carlsbad, CA). All cell lines were grown at 37°C in a humidifiedatmosphere of 5% (vol�vol) CO2 in air. Transient transfectionswere performed with Lipofectamine 2000 reagent (Invitrogen,Carlsbad, CA) according to the manufacturer’s instructions.

Fig. 4. Itch regulates �Np63� protein levels in primary keratinocytes. Primarymouse keratinocytes were cultured as indicated in Materials and Methods andinduced to differentiate in high calcium concentration (1.2 mM). (A) Phase-contrast micrographs were taken at indicated time points after induction ofdifferentiation. (B and C) The levels of Itch, p63, and involucrin were evaluated byWestern blot at the indicated time points after induction of differentiation inprimary mouse keratinocytes (B) and normal human epidermal keratinocytes (C).(D) Primary mouse keratinocytes were obtained from WT and Itch-deficient mice(KO) of the same genetic background and induced to differentiate. At theindicated time points after induction of differentiation, cells were harvested, andp63 and involucrin protein levels were analyzed. (E) Primary mouse keratinocytesobtained from Itch-deficient mice (KO) were infected with Itch WT or Itch MUTlentiviruses and collected 96 h later. Reintroduction of Itch WT, but not Itch MUT,resulted in �Np63 down-regulation. In all cases, �-tubulin levels were measuredas a loading control.

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Western Blot and Antibodies. Proteins were separated by SDS�PAGE and blotted onto nitrocellulose membranes. Membraneswere blocked with TBST (Tris-buffered saline and Tween 20)�5%nonfat dry milk and incubated with primary antibodies for 2 h atroom temperature (RT). After washing four times in TBST,membranes were incubated for 1 h at RT by using the appropriatehorseradish peroxidase-conjugated secondary antibody (BioRad,Hercules, CA). Detection was performed with the SupersignalWest Pico enhanced chemiluminescence system (Pierce, Rockford,IL). Endogenous Itch was detected with mouse monoclonal anti-body (BD Biosciences, San Diego, CA), �-tubulin was detected withsc-9104 (Santa Cruz Biotechnology, Santa Cruz, CA), p63 wasdetected with H129 (Santa Cruz Biotechnology), p53 was detectedwith FL-393 (Santa Cruz Biotechnology), the c-Myc-tagged con-structs were detected or immunoprecipitated with the sc-40 mono-clonal mouse antibody (Santa Cruz Biotechnology), and the Flag-tagged constructs were detected with the M2 monoclonal mouseantibody (Sigma, St. Louis, MO).

Immunoprecipitation. As described in ref. 26, HEK293 cells weretransiently transfected with the indicated mammalian expres-sion plasmids and harvested 48 h after transfection. Afterpreclearing for 1 h at 4°C, we performed immunoprecipitationby incubating 1.5 mg of whole-cell extracts with the indicatedantibodies and rocking at 4°C overnight. For immunoprecipi-tation of endogenous Itch, we used mouse anti-Itch (BDBiosciences) and, as an unrelated antibody, mouse anti-p53D01 (Santa Cruz Biotechnology).

Ubiquitylation Assays. HEK293 cells were transiently transfectedwith mammalian expression plasmids for HA-tagged Ub withthe indicated combination of plasmids. Forty-eight hours aftertransfection, cells were harvested, the insoluble fraction wasremoved by a high-speed spin, and 1 mg of total cellular proteinsof the clarified supernatant was subjected to immunoprecipita-tion by using anti-Flag antibodies. p63 Ub conjugates weredetected by using polyclonal rabbit antibody (Y-11) (Santa CruzBiotechnology).

Steady-State Protein Level Analysis. Levels of p63 proteins weredetermined 48 h after cotransfection with the indicated combi-nation of plasmids. Cell lysates were subjected to Westernblotting. p63 proteins were detected by using an anti-Flagantibody. The same blots were reprobed with anti-Myc antibodyto detect Itch and with anti-�-tubulin antibody as a loadingcontrol.

Measurement of p63 Half-Life. Cycloheximide (20 �g�ml) wasadded to HEK293 cells 24 h after transfection with the indicatedcombination of plasmids. Protein levels were determined bycollecting cells at the indicated time points and performingimmunoblotting as described above. The relative amount of p63protein was evaluated by densitometry and normalized on�-tubulin.

35S Pulse–Chase. H1299 cells were transfected with a total of 2.5�g of the indicated plasmids in a 1:2 ratio of p63�Itch. At 48 hafter transfection, cells were starved for 30 min in DMEM withdialyzed serum and then labeled with 250 �Ci�ml (1 Ci � 37GBq) Redivue PRO-MIX (L-[35S] in vitro cell-labeling mix)(Amersham, Piscataway, NJ) for 60 min. Unlabeled Met and Cyswere added, and cells were collected in RIPA buffer (200 mM

Tris, pH 8�150 mM NaCl�0.5% sodium deoxycholate�0.1%SDS�1% Nonidet P-40�0.2 mM EDTA) at the indicated times.Immunoprecipitations were performed with 150 �g of totalprotein lysate and 4 �l of anti-HA (Santa Cruz Biotechnology)or 1 �l of anti-Flag (Sigma). Immunoprecipitates were washedsix times in RIPA buffer and six times in NET gel (50 mMTris�HCl, pH 7.4�0.25% gelatin�1 mM EDTA�150 mM NaCl�0.1% Nonidet P-40), run on SDS�PAGE, and detected byautoradiography.

Primary Mouse Keratinocyte and Normal Human Epidermal Keratin-ocyte (NHEK) Differentiation. Primary mouse keratinocytes wereobtained from newborn mice and cultured by following stan-dard procedures (27). Brief ly, they were prepared and seededin low-calcium medium, and 1.2 mM CaCl2 was added topromote differentiation. Cryopreserved NHEK cells obtainedfrom BioWhittaker (Berkshire, U.K.) were grown in calf skincollagen-coated dishes (Sigma) in serum-free keratinocytemedium (BioWhittaker) at 0.05 mM calcium and supple-mented with Single-Quots (BioWhittaker) containing 7.5mg�ml bovine pituitary extract, 0.5 mg�ml insulin, 0.5 mg�mlhydrocortisone, and 0.1 mg�ml human EGF. Third-passagecells were differentiated by adding 1.2 mM calcium to theculture and kept in culture for 3 days. Protein extracts wereprepared at the indicated time points after induction ofdifferentiation, and endogenous protein levels were analyzedas described.

Lentiviral Infection. Lentiviral particles for Itch WT and Itch MUTwere prepared as reported in ref. 28. Briefly, 293T cells weretransfected with the three packaging plasmids and the vectorexpressing the gene of interest. The supernatant was harvested,and virus was concentrated by ultracentrifugation. Lentiviralparticles for the expression of Itch WT or MUT were then addedfor 8 h to primary mouse keratinocyte cells derived fromItch-deficient mice. All of the experiments were performed atleast 96 h after infection.

Immunofluorescence. Normal adult skin samples were prepared asdescribed in ref. 29. Briefly, adult knee skin samples wereparaffin-embedded and cut into sections that were 5 �m thick.Sections were permeabilized by microwaving in citrate buffer.Sections were blocked with 5% goat serum in PBS and thenincubated with mouse anti-Itch antibodies and anti-p63. Afterwashing three times in PBS, sections were incubated for 1 h withsecondary antiserum (goat anti-mouse Alexa Fluor 488 or goatanti-rabbit Alexa Fluor 568), and the nuclei were counterstainedwith DAPI. Sections were then mounted by using MowiolAntifade reagent (Calbiochem, Darmstadt, Germany) and an-alyzed with a confocal laser microscope (LSM 510; Zeiss,Oberkochen, Germany).

We thank Valentino Parravicini and Rose Zamoyska (Medical ResearchCouncil, London, U.K.) for providing 18H Itch-deficient mice and G. M.Cohen for his help in revising the manuscript. This work was supportedby grants from Telethon (GGP02251 to E.C. and GGPO4110 to G.M.);a Kimmel Scholar Award (to R.I.A.); Associazione Italiana per laRicerca sul Cancro Grant 2743; European Union Grants QLK-CT-2002-01956-Impaled, LSGBH-2005-019067-Epistem, and Blandino-2004-LSHC-CT-2004-503576-Active p53; Genomica Funzionale COMETA;Ministero dell’Università e della Ricerca Grants FIRB-2001-RBNE01KJHT (G. Marino) and FIRB-2001-RBNE01NWCH (G. Ro-tilio); and MinSan (G.M.).

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