Alexey Fomenkov1,*Edward A. Ratovitski1,*1Departments of Dermatology and 2Otolaryngology; The Johns Hopkins UniversitySchool of Medicine; Baltimore, Maryland USA
*Correspondence to: Alexey Fomenkov and Edward Ratovitski; Department ofDermatology; The Johns Hopkins University School of Medicine; 818 Ross Building720 Rutland Avenue; Baltimore, Maryland 21205 USA; Email: [email protected][email protected]
Received 09/28/04; Accepted 10/07/04
Previously published online as a Cell Cycle E-publication:http://www.landesbioscience.com/journals/cc/abstract.php?id=1290
KEY WORDS
p63, ubiquitin, SUMO-1, SHFM, split-hand/foot malformation, RUNX2, MINT
ACKNOWLEDGEMENTS
We thank Dr. Stefan Weger for providing uswith pGBT9-SUMO-1 and pCMV-EYFP-SUMO-1constructs.
This work was supported in part by grant R01-AI47224 from the National Institute of Arthritisand Infectious Diseases (E.A.R), grant R01-DE13561 from the National Institute for Dentaland Craniofacial Research (D.S., E.A.R. and B.T.),grant DE-015834 from the National Institute forDental and Craniofacial Research (A.F. and Y.H.),grant DS-00276-20S1 from the National Institutefor Deafness and Auditory Diseases (E.A.R.), and agrant from the Flight Attendant Medical Institute(A.F., Y. H., B.T., Z. G and E.A.R.).
Report
Altered Sumoylation of p63α Contributes to the Split-Hand/FootMalformation Phenotype
ABSTRACTp63 mutations have been identified in several developmental abnormalities, including
split-hand/foot malformation (SHFM). In this study, we demonstrate that the C-terminaldomain of p63α associates with the E2 ubiquitin conjugating enzyme, Ubc9. A p63αmutation, Q634X, which naturally occurs in SHFM modulated the interaction of p63αwith Ubc9 in yeast genetic assay. Furthermore, Ubc9 catalyzed the conjugation of p63αwith small ubiquitin modifier-1 (SUMO-1), which covalently modified p63α in vitro andin vivo at two positions (K549E and K637E), each situated in a SUMO-1 modificationconsensus site (φKXD/E). In addition, p63α mutations (K549E and K637E) abolishedsumoylation of p63α, dramatically activated transactivation properties of TAp63α, andinhibited the dominant-negative effect of ∆Np63α. These p63α mutations also affectedthe transcriptional regulation of gene targets involved in bone and tooth development(e.g., RUNX2 and MINT) and therefore might contribute to the molecular mechanismsunderlying the SHFM phenotype.
INTRODUCTIONSplit hand/foot malformation (SHFM) is a limb malformation involving the central
rays of the autopod (hand/foot). SHFM may present with syndactyly, median clefts of thehands and feet, and aplasia and/or hypoplasia of the phalanges, metacarpals andmetatarsals. In severe cases, the hands and feet have a lobster claw-like appearance.1 Failureto initiate the apical ectodermal ridge (AER) leads to truncations of all skeletal elementsof the limb (stylopod, zeugopod, autopod).1 Since SHFM only affects the autopod, thisprobably reflects a failure to maintain the normal function of the AER.1 A number of criticalmolecules involved in AER development are known, including fibroblast growth factors(FGF) and their receptors (FGFR), bone morphogenetic proteins (BMP), WNT signalingmolecules, homeobox-containing proteins (e.g., MSX1 and MSX2), and p63.1,2
p63 was found to play an essential role in epidermal-mesenchymal development duringembryogenesis.3-5 Studies with p63 null mice showed that p63 impairment leads toprofound defects in limb, craniofacial, and epithelial development and inhibits differenti-ation of stratified squamous epithelia.2-4 The forelimbs of p63 null mice are severelymalformed, lacking the radius and the complete autopod, while the hindlimbs do notdevelop at all.3,4 Therefore, p63 might function as an AER maintenance factor preservingthe proliferative activity in specialized ectodermal cells.1,2,5 The precise mechanisms andpathways by which p63 may execute this function remain to be established. Although p63is able to transactivate many target genes, it is not known whether these genes are of anyrelevance to normal limb development. A few of AER-specific target genes of p63 wererecently identified, including Jag1, Jag2 and REDD1.6,7
Even less is known about the upstream factors that control expression of p63. Recentstudies in zebrafish have established that the ∆Np63 promoter is a direct target of BMPsignaling,8,9 probably contributing to control of the integrity of the AER in limb bud out-growth. Interestingly, BMP4 and FGF8 were found to contribute to the syndactylyphenotype in Ft/+ mice.10
Twenty-seven mutations found in a number of ectodermal and craniofacial develop-mental disorders (ankyloblepharon, ectodermal dysplasia and clefting, AEC; ectodermaldysplasia, facial clefting; SHFM; limb-mammary syndrome; acro-dermato-ungual-lacrimal-tooth syndrome; and Rapp-Hodgkin syndrome) were mapped in distinct domainsof p63.2,11–17 Among the panel, two SHFM-associated mutations (K193E and K194E)were identified in the DNA-binding domain (DBD) of p63,13-15 whereas another mutation
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associated with SHFM (Q634X) was found in the C-terminaldomain of p63α isotypes containing a potential SUMO-1 targetconsensus φKXD/E motif (where φ is any hydrophobic residues andX is any amino acid residues), and likely to disrupt the transcriptioninhibitory domain (TID) of p63α.11,18
We hypothesized that p63 mutations may affect specific protein-protein complexes and therefore modulate p63 function towardspecific target genes. We postulated that these mutations mightaffect p63 association with protein modifiers (e.g., E2 or E3 ubiquitinligases). Recent genetic studies linked E3-like modifiers (e.g.,dactylin or Notch-associated Serrate-2) to SHFM and the syndactylyphenotype in mice.14,15,19,20 Thus, our studies elucidate the molec-ular mechanism underlying SUMO-1 modification of p63α isotypesand sumoylation effect on the stability and transactivation propertiesof both ∆Np63α and TAp63α toward certain gene targets (e.g.,RUNX2 and MINT), which may contribute to the SHFM phenotype.
MATERIALS AND METHODSAntibodies. The rabbit polyclonal antibody against SUMO-1 (FL101,
1:500), mouse monoclonal antibody against green fluorescent protein (GFP,1:1000), which also recognizes yellow fluorescent protein (YFP), mousemonoclonal antibody against all p63 isotypes (4A4, 1:500) and the rabbitpolyclonal antibody against the C-terminus of p63α (H-129, 1:500) werepurchased from Santa Cruz Biotechnology. We also used a rabbit polyclonalantibody against the N-terminus of ∆Np63 isotypes (Ab-1, 1:500,Oncogene Science Inc.) and a mouse monoclonal antibody against Ubc9(1:1000, BD/Transduction Laboratories). The monoclonal anti-HA antibody(12CA5) was obtained from Roche Molecular Biochemicals, and the mon-oclonal antibody against SUMO-1 (21C7) was purchased from ZymedLaboratories.
Yeast 2-Hybrid Screening. A yeast 2-hybrid screen was performed usingfour 2-hybrid cDNA libraries from human fetal kidney, human fetal liver,human keratinocytes and HeLa cells (Clontech). Various bait plasmids wereconstructed including pGal4-BD-DBD (containing complete p63 DNA-binding domain, residues 70-411),21 pGal4-BD-p63CT (containing completeC-terminal part, residues 411-642), and pGal4-BD-p63SAM (containingsterile α-motif, SAM, residues 499-568).18 Plasmids were introduced intoSaccharomyces cerevisiae yeast strain AH109 (MATα, trp1-901, leu2-3,112, ura3-52, his3-200, gal4D, gal80D, LYS2 : : GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3 : : MEL1UAS-MEL1TATA-lacZ)as described elsewhere.21 The intermolecular interactions between bait andprey proteins were verified by subsequent transformation of both bait andprey plasmids into AH109 followed by α- and β-galactosidase assays accordingto the manufacturer’s instructions (Clontech).
Cells, Transfections and Luciferase Reporter Assays. Cells were tran-siently transfected for 24 h with desired plasmids (5–20 µg) usingLipofectamineTM-2000 (10–20 µl, Invitrogen) according to the manufac-turer’s protocol (HaCaT immortalized keratinocytes and primary mouseembryo fibroblast cells) or by a modified calcium-phosphate-mediatedprotocol (911 human embryonic retina cells) as described elsewhere.21 Theefficiency of transfection was monitored by cotransfection with apAdTrack-GFP vector and was ~80-90% for 911 cells and ~5-10% for HaCaTcells. Transfected HaCaT cells were separated by FACS analysis using GFPas a fluorescence marker. Functional activity of TAp63α was monitored bya luciferase-based assay using RGC: lux reporter22,37 in the presence ofpRL-TK for normalization. 24 h after transfection luciferase activity wasmonitored using the Dual Luciferase Assay Kit (Promega) in a MonolightTM–20 luminometer for 10 sec.
Protein Expression and Purification. The open reading frame for Ubc9was cleaved with EcoR I and Xho I from the pTOB7-Ubc9 plasmid(American Tissue Culture Collection) and subcloned into the pCMV-Sport6 vector resulting in the pCMV-Sport6-Ubc9 expression construct. ForpCMV-HA-SUMO-1 construction, the open reading frame for SUMO-1
was amplified from the HeLa cDNA library (Stratagene), Sal I and Kpn Irestriction sites were added using primers, and the fragment was cloned intothe corresponding restriction sites of the pCMV-HA vector (BD/Clontech).Both pGBT9-SUMO-1 and pCMV-EYFP-SUMO-1 constructs were obtainedfrom Dr. Stefan Weger (Department of Virology, Institute of InfectiousDiseases, Free University of Berlin, Berlin, Germany, ref. 23). The TAp63αand ∆Np63α expression constructs and their adenoviral and mutant deriv-atives were previously described.21,22
Both TAp63α and ∆Np63α were expressed in E. coli as GST-fusionpolypeptides. Using the following primers, sense, 5’-GGCCCGGGCCAT-GTCCCAGAGCACACA-3’ for TAp63, 5’-GGGATCCATGTTGTAC-CTGGAAAACAAT-3’ for ∆Np63, and antisense for both p63α isotypes,
SUMOYLATION OF p63
Figure 1. Interaction of the p63a C-terminus with protein candidate Ubc9 isaffected by SHFM-derived mutations in yeast. (A) Quantitative α-galactosi-dase assay. Samples: 1—pBD-p53 and pAD-SV40 T; 2—pBD with pAD; 3—pBD-p63CT (wild type) with pAD; 4—pBD-p63CTK637E with pAD; 5—pBD-p63CT∆639 with pAD; 6—pBD-p63CT∆634 with pAD; 7—pBD withpAD-Ubc9; 8—pBD-p63CT (wild type) with pAD-Ubc9; 9—pBD-p63CTK637E
with pAD-Ubc9; 10—pBD-p63CT∆639 with pAD-Ubc9; 11—pBD-p63CT∆634 with pAD-Ubc9; 12—pBD-p63SAM with pAD-Ubc9; 13—pBD-p63DBD with pAD-Ubc9. Yeast cells were cotransformed with appro-priate bait (BD) and prey (AD) plasmids. Cells obtained from positive cloneswere pelleted and supernatants were subjected to quantitative α-galactosidaseassay. Samples were normalized by equal amounts of yeast cells measuredat OD600. (B) 2-hybrid growth assay on selective media. Schematic presen-tation of positive colonies transformed with bait and prey plasmids grownon plates with indicated minimal media. SD/Leu-Trp-media supports growthof yeast clones bearing both plasmids. SD/His-Leu-Trp-media supportsgrowth of positive clones with interacting bait and prey plasmids.SD/Ade-His-Leu-Trp-media supports growth of the strong interaction betweenprey and bait plasmids. Positive colonies were selected on agar plates withSD/Trp-Leu-, SD/His-Trp-Leu- or SD/Ade-His-Trp-Leu-. All plates containedα-X-gal.
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5’-GGTCGACTCACTCCCCCTCCTCTTTGA-3’ (containing Bam HIand Sal I sites, respectively) we amplified both p63α open reading framesand subcloned them into the pGEX-4T-1 expression vector. The Not I/EcoR I fragment containing the entire open reading frame for Ubc9 wassubcloned from the pCMV-Sport6-Ubc9 vector into the pGEX-4T-1 plasmidfor E.coli expression.
Each pGEX-4T-1 expression derivative was transformed into E.coliBL21 strain (Novagen) and GST-fusion proteins were expressed and puri-fied according to the manufacturer’s recommendations. GST-fusion proteinseluted from glutathione-agarose column (Sigma) were further purified by afast-performance liquid chromatography on 5 ml MonoQ HR5/5 column(Pharmacia) using a 30 ml, 50–800 mM KCl gradient (1 ml/min) containing10 mM Tris-HCl (pH 7.4), 1 mM DTT, 1 mM EDTA, 5 mM MgCl2 and1% PMSF. About ~1.0–2.0 mg of fusion proteins were purified from 3–4 gof bacterial cells. GST-p63α/Ubc9 fusion proteins were digested by a thrombincleavage kit (Sigma) and purified again over a glutathione-agarose column.
Immunoblotting, Immunoprecipitation and 2-D Separation. Cellswere resuspended in lysis buffer [50 mM Tris (pH 7.5), 100 mM NaCl, 2mM EDTA, 0.5% Triton X-100, 0.5% Brij-50, 1 mM PMSF, 0.5 mM NaF,0.1 mM Na3VO4, 2X complete protease inhibitor cocktail], sonicated 5times for 10 sec time intervals, and clarified for 30 min at 15,000 x g.Supernatants (designated as total lysates) were resolved by 10% SDS-PAGEand then analyzed by immunoblotting (~30–60 µg/lane) or immunopre-cipitation (~150–200 µg/lane). Proteins transferred onto Hybond-ECLmembranes were blocked with 5% fat-free milk (BioRad) in PBS-0.2%Tween-20 for 1 h, then incubated for 2 h with primary antibodies, and afterwashing twice with PBS-0.5% Tween-20 were incubated for 1 h with sec-ondary antibodies [1:5000, goat anti-mouse or goat anti-rabbit immunoglob-ulins (IgG) coupled to horseradish peroxidase] and washed three times withPBS-0.2% Tween-20. Protein bands were visualized by an enhanced chemi-luminescence kit (Amersham). For immunoprecipitation, 500 µl of total celllysates were incubated with 10 µl of normal rabbit serum for 30 min and thenincubated with 50 µl of goat anti-rabbit IgG coupled to agarose for 30 min.Following centrifugation, 500 µl of precleared supernatant were incubatedfor 3 h with primary antibodies, then with 50 µl of a 50% suspension of goatanti-mouse/ anti-rabbit agarose) for 4 h at 4˚C, and washed three times with1 ml cold 20 mM Tris-HCl (pH 7.4), 125 mM NaCl, 1 mM Na3VO4, 50mM NaF, 1 mM EDTA, 0.2% Triton X-100, 0.2 mM PMSF. Samples wereboiled in 4x Laemmli buffer and fractionated by 10% SDS-PAGE followedby immunoblotting.
To detect different modifications of p63, we performed a 2-D gel separa-tion of the ∆Np63α immunoprecipitates. The first separation was performedon 11 cm IPG strips (pH 5.0–8.0, Bio-Rad) followed by the second separationon 4–15% SDS-PAGE. Modified ∆Np63α polypeptides were detected byimmunoblotting with 4A4 antibody.
Fluorescence Microscopy Analysis. Cells were grown on coverslips and transfected with appropriate expression constructsincluding EYFP-SUMO-1, Ubc9, ∆Np63α or TAp63α. Cellswere fixed in 4% formaldehyde in PBS (pH 7.4) for 30 min atroom temperature. Nuclei were stained with Hoechst dye. Forimmunofluorescence analysis, cells were permeabilized for 10 minin 1% Triton X-100 in PBS. Cover slips were washed twice inPBS and cells were incubated for 30 min at room temperaturewith the indicated primary antibodies diluted in 1% FBS in PBS.After three washes in PBS, cells were incubated for 30 min withsecondary antibody (Alexa350-labeled goat anti-rabbit IgG orAlexa546-labeled goat anti-mouse IgG as indicated. All secondaryantibodies were diluted 1:500 in 1% FBS in PBS. Cover slipswere washed three times in PBS and mounted with ProLongantifading agent (Molecular probes Inc). Confocal microscopywas performed at the JHU Core Facility.24
Gene Expression Profiling. Primary embryonic mouse fibrob-lasts from p63 null mice were prepared as described.25 Cells wereinfected with p63 expression adenoviral constructs (MO = 1)including TAp63α wild type, TAp63α double mutant (K549E/K637E), ∆Np63α wild type, or mutant ∆Np63α (K549/K637).Gene expression profiling by oligonucleotide-based Affymetrix
microarray analysis was performed and expression data between wild typeand mutant variants were compared.
Total RNA was isolated with RNA Easy Purification Kit (Qiagen) andbiotinylated (bio-) cRNA probes (15 µg/chip) were hybridized withAffymetrix Murine Genome U74Av2 arrays (9,400 genes) for 16 h at 45˚Caccording to the manufacturer’s instructions. To enhance the detection ofhybridized bio-RNA blots were first incubated with a streptavidin-phyco-erythrin conjugate followed by labeling with biotinylated goat antibodyagainst streptavidin (Vector Laboratories) and then stained again with thestreptavidin-phycoerythrin conjugate. Chips were then scanned using aHewlett Packard scanner by a photomultiplier tube through a 570 nm longpass filter. Digitized image data was processed using Genechip software(Affymetrix, Version 3.1).
Scaled data obtained from experimental conditions were normalized tothe data obtained from normal untreated conditions (NOMAD). To identifyspecific genes that were differentially expressed, a computer algorithm wasused to select genes exhibiting >2-fold expression change in all of our exper-imental samples relative to control (NOMAD). We used at least three bio-logical replicas for each of our samples and considered the genes that showeda common pattern of fold changes among the three individual experimentsfor further analysis. Statistical analysis of microarrays was used to identifysignificant gene targets. In addition, data was validated by ANOVA (corrected),PCA and hierarchical clustering analysis. All gene targets that overlappedbetween experiments were verified by RT-PCR.
RT-PCR Assays for Differentially Expressed Genes. Total RNA wasisolated using Trizol reagent (Invitrogen). 1 µg of total RNA was used togenerate cDNA from each sample using a one-step RT-PCR kit (Qiagen) andcustom primers for the set of differentially expressed target genes, includingRUNX2, MINT, ELAV2 (HuB) and caspase-3, were used for PCR. Foreach gene analyzed, a cycle curve experiment was performed and the optimalnumber of PCR cycles for quantitative analysis was chosen within the linearrange of amplification. We used the following primers and number of cycles:Caspase-3: sense, 5’-ATGGAGAACACTGAAAACTCA-3’, antisense,5’-TTAGTGATAAAAATAGAGTTC-3’, 28 cycles; Elav-2 (HuB, HelN1):sense, 5’-ATGGAAACACAACTGTCTAAT-3’, antisense, 5’-TTAG-GCTTTGTGCGTTTTGTT-3’, 29 cycles; heat shock factor (HSF)-4:sense, 5’-ATGCAGGAAGCGCCAGCTGCG-3’, antisense, 5’-TCAGGG-AGAGGAGGGACTGGC-3’, 28 cycles; Sharp/HDAC (MINT): sense,5’-GCAAACAGCACGAGTGATTCG-3’, antisense, 5’-TTCCAGGCTT-CTCTGATGCG-3’, 31 cycles; Runx2: sense, 5’-ATGCTTCATTCGCCT-CAC-3’, antisense, 5’-CTCACGTCGCTCATCTTG-3’, 29 cycles; Msx2:sense, 5’-CGCCTCGGTCAAGTCGGAA-3’, antisense, 5’-GCC-CGCTCTGCTAGTGACA-3’, 31 cycles; parathyroid hormone receptor(PTHR): sense, 5’-ACCCCGAGTCTAAAGAGAAC-3’, antisense,
SUMOYLATION OF p63
Figure 2. Computer-prediction of sumoylation motifs (residues shown in bold) in TAp63αwith a high (1 and 2) and low (3 and 4) probability overlap with known mutationsidentified in SHFM syndrome (underlined bold residues).
As an internal loading control, a GAPDHregion was amplified using the following primers:sense, 5’-GAGAAGGCTGGGGCTCATTT-3’,antisense, 5’-CAGTGGGGACACGGAAGG-3’.PCR products were resolved on a 1.5% agarosegel and visualized with ethidium bromide. Toprovide a high degree of standardization, allexperiments were performed simultaneouslyusing the same reaction mixture, and GAPDHwas coamplified to confirm equal amounts ofstarting cDNA. RT-PCR amplification resultswere analyzed digitally by Kodak 1D 3.5 software(Kodak Scientific Imaging). The net intensities ofPCR products were measured and normalizedwith net intensities of GAPDH bands.
RESULTSTwo-Hybrid Screening for p63 Interacting
Proteins. Previous reports clearly demonstrated acritical role for p63 mutations found in AEC andSHFM.11,13,14,21 p63 mutations found in AECwere identified in the sterile α-motif (SAM, refs.11, 21), on the other hand, mutations detected inSHFM were mapped to the transcriptioninhibitory domain (TID, refs. 13-14) and theDNA-binding domain (DBD, refs. 13, 14). SAMdomain was found in various proteins from yeastto humans and shown to be involved in multiplephysical interactions with proteins implicated indifferentiation, development and morphogene-sis.26 We hypothesized, that protein-proteininteractions mediated by SAM, TID or DBD ofp63α isotypes may contribute to the molecularmechanisms underlying either the AEC or SHFMphenotypes.
For this purpose, we generated pGal4-BDyeast expression constructs harboring the C-ter-minus (CT) of p63α (residues 411-642), SAM(residues 499-568) or DBD (residues 114-349).Appropriate p63 sequences (CT, SAM or DBD)were subcloned in frame into the Gal4 DNAbinding domain (BD) and resultant bait plasmidswere designated as pGal4-BD-p63CT,pGal4-BD-p63SAM or pGal4-BD-p63DBD,respectively. Using pGal4-BD-p63CT orpGal4-BD-p63DBD as baits we screened four2-hybrid cDNA libraries from human fetal kidney,human fetal liver, human keratinocytes and HeLacells.21 We isolated several overlapping clones thatinteracted with both the DNA-binding domainand C-terminal domain of p63α isotypes.Sequencing analysis revealed that they representedthe N-terminal portion of Ubc9 fused to theGal4-activation domain (AD). Further analysisdemonstrated that the pGal4-AD-Ubc9 plasmidsbound to both pGal4-BD-p63CT and pGal4-BD-p63DBD but failed to bind to pGal4-BD-p63SAM (Fig. 1, samples 8, 12 and 13).
To establish whether mutations found in SHFM syndrome may affectprotein-protein interactions between p63α isotypes and Ubc9, we generatedbait expression cassettes with modified residues 634 or 639 (each replaced
by stop codon) leading to deletion of TID. These alterations representnaturally occurring mutations in SHFM. We also prepared a bait constructwith modified codon K637E, which alters a putative SUMO-1 binding
SUMOYLATION OF p63
Figure 3. Physical association of wild type or mutant p63 isotypes with Ubc9 and SUMO-1. (A and B)911 cells were cotransfected with expression cassettes for p63 isotypes and Ubc9. Samples: 1—TAp63α and Ubc9; 2—TAp63β and Ubc9; 3—TAp63γ and Ubc9; 4—∆Np63α and Ubc9; 5—∆Np63β and Ubc9; 6—∆Np63γ and Ubc9; 7—p40 and Ubc9. (A) Ectopic expression of p63 isotypes(upper panel) and Ubc9 (lower panel). (B) p63/Ubc9 complexes were precipitated with anti-Ubc9antibody and probed with 4A4 antibody (upper panel) or were precipitated (IP) with anti-p63 antibodies(H-129, and Ab-1) and probed with anti-Ubc9 antibody (lower panel). (C) Physical association ofTAp63α and its mutants (lacking SUMO-1 motif) with Ubc9 and SUMO-1. 911 cells were cotransfectedwith the expression cassettes for TAp63α (wild type), Ubc9 and HA-SUMO-1 (lanes 1, 5 and 9);TAp63α (K549E), Ubc9 and HA-SUMO-1 (lanes 2, 6 and 10); TAp63α (K637E), Ubc9 and HA-SUMO-1(lanes 3, 7 and 11); or TAp63α (K549E/ K637E), Ubc9 and HA-SUMO-1 (lanes 4, 8 and 12).TAp63α/Ubc9/SUMO-1 complexes were precipitated with H-129 antibody (lanes 1–4), anti-Ubc9antibody (lanes 5–8), or anti-HA antibody (lanes 9–12) and probed with anti-p63 antibody 4A4(lanes 1–12). The same samples were also precipitated with anti-p63α antibody (H-129) and blottedwith anti-Ubc9 antibody (D, lanes 1–4) or anti-HA-antibody (E, lanes 1–4). (F) Physical association of∆Np63α and its mutants (lacking SUMO-1 motif) with Ubc9 and SUMO-1. 911 cells were cotrans-fected with the expression cassettes for ∆Np63α (wild type), Ubc9 and HA-SUMO-1 (lanes 1, 5 and9); ∆Np63α (K549E), Ubc9 and HA-SUMO-1 (lanes 2, 6 and 10); ∆Np63α (K637E), Ubc9 andHA-SUMO-1 (lanes 3, 7 and 11); ∆Np63α (K549E/K637E), Ubc9 and HA-SUMO-1 (lanes 4, 8 and12). ∆Np63α /Ubc9/SUMO-1 complexes were precipitated with ant-p63α antibody, H-129 (lanes1–4), anti-Ubc9 antibody (lanes 5–8) or anti-HA antibody (lanes 9–12) and probed with anti-p63antibody, 4A4 (lanes 1–12). Complexes were also precipitated with anti-p63α antibody, H-129 (Gand H) and probed with anti-Ubc9 (G) or anti-HA (H) antibodies.
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motif. Baits of wild type pGal4-BD-p63CT or its mutant derivatives andpGal4-AD-Ubc9 prey plasmids were cotransformed into AH109 yeast andprotein-protein interactions were monitored by selective growth and α-andβ- galactosidase assays. Introduction of K637E mutation in the p63 TIDfailed to affect association between p63α with Ubc9 (Fig. 1, samples 8 and9), while deletion of two to eight C-terminal residues nearly completelyabolished the physical interaction between the C-terminus of p63α isotypes(TID) and Ubc9 (Fig. 1, samples 10 and 11).
p63α Isotypes Specifically Interact with Ubc9 and SUMO-1 throughTheir C-terminal Domains. Recent studies demonstrated that Ubc9 is akey enzyme mediating the conjugation of SUMO-1 to numerous protein
substrates.27-36 Sumoylation was also found to occur to p53family members (e.g., p53 and p73).37-39 However, theoccurrence of p63 sumoylation was still in question. Usinga sumoylation motif prediction program (www.abgent.com/cgi-bin/ tools.pl), we identified four potential sumoylationsites in the TAp63α sequence (Fig. 2). Two of them (with alow probability) were found in the DNA binding domainof TAp63α (positions K194 and K314). Interestingly, oneof these potential sumoylation sites overlapped with aregion that is naturally mutated in SHFM patients (K193Eand K194E). Another two SUMO modification motifs(with a high probability) were mapped to the C-terminalpart of TAp63α (K549, next to SAM and K637, in TID).Each potential sumoylation site is also found in ∆Np63α,since it shares the same sequence except first N-terminal1-69 residues. Therefore, our results suggested a correlationbetween p63α/Ubc9 association and occurrence of sumoy-lation sites found in the p63α C-terminus.
To confirm our data obtained from the yeast 2-hybridgenetic assay we performed immunoprecipitation analysis.Human embryonic retina 911 cells were transientlytransfected with expression cassettes for Ubc9 and one ofseven p63 isotypes (TAp63α, TAp63β, TAp63γ, ∆Np63α,∆Np63β, ∆Np63γ and p40). Protein levels were examinedby immunoblotting using the antibodies indicated (Fig. 3A,upper and lower panels). Consistent with our 2-hybridanalysis, we observed that both TAp63α and ∆Np63αspecifically associated with Ubc9, however other p63isotypes failed to form complexes with Ubc9 (Fig. 3B,upper and lower panels).
Next, we constructed p63 expression cassettes bearingmutations within potential SUMO-1 motifs (e.g., K549E,K637E and K549E /K637E). We then transiently trans-fected 911 cells with wild type or mutant p63α expressioncassettes in combination with either Ubc9 or SUMO-1expression constructs. We found that these mutations didnot affect p63 protein levels (Fig. 3A and B). Interestingly,the electrophoretic mobility of mutant proteins was fasterthan that of the wild type protein indicating a possibleabsence of certain modifications in mutant proteins (Fig.3C and F, lanes 1-4). All wild type and mutant p63αpolypeptides bound to Ubc9 (Fig. 3C and F, lanes 5–8,(Fig. 3D and G, lanes 1–4). However, the association ofTAp63α with SUMO-1 was completely abolished by themutations indicated (Fig. 3C, lanes 10–12 and Fig. 3E,lanes 2–4). SUMO-1 associated with both wild type andthe K549E mutant of ∆Np63α (Fig. 3F, lanes 9 and 10,(Fig. 3H, lanes 1 and 2), while its interaction with theK637E mutant and the K549E/K637E double mutant of∆Np63α was completely abolished (Fig. 3F, lanes 11 and 12,Fig. 3H, lanes 3 and 4). Since, p63α, Ubc9 and SUMO-1were ectopically expressed in 911 cells, we were not surprisedto observe ~100% of the wild type p63α proteins migratedas modified polypeptides (Fig. 3C and 3F). Thus, these dataindicate that both p63α isotypes associated with Ubc9 and
SUMO-1, while certain point mutations overlapping with the putativeSUMO-1 modification motif exclusively abolished physical interaction ofp63α isotypes with SUMO-1. Interestingly, only K637E mutation abol-ished binding of SUMO-1 to both p63α isotypes suggesting that K637E isa critical sumoylation site.
∆Np63α is Modified by SUMO-1 in Two C-terminal Positions InVitro and In Vivo. To analyze the sumoylation mechanism of p63, wedesigned an in vitro SUMO-1 modification assay. We used purifiedunmodified GST-∆Np63α (as a substrate), GST-Ubc9 (used as E2 ligase)fusion polypeptides expressed in E. coli and HA-SUMO-1 protein ectopicallyexpressed in 911 cells (obtained from total lysate depleted for endogenous
SUMOYLATION OF p63
Figure 4. Mutations of SUMO-1 motif affect sumoylation of ∆Np63α. Bacterially expressedunmodified GST-∆Np63α (5 µg, M.W. = 95 kDa) was mixed with 100 µg total lysate (911cells expressing empty vector and depleted for SUMO-1 and Ubc9 or 911 cells expressingHA-SUMO-1 and depleted for Ubc9) and/or with increasing amounts of bacteriallyexpressed GST-Ubc9 in 200 µl of reaction buffer [10 mM Tris HCl (pH 7.4), 1 mM MgCl2,50 mM KCl, 1 mM ATP]. (A) Samples: 1, GST-∆Np63α (50 µg) plus E1; 2, GST-∆Np63α (50µg) plus E1 and HA-SUMO-1; 3, GST-∆Np63α (50 µg) plus GST-Ubc9 (1 µg), E1 andHA-SUMO-1; 4, GST-∆Np63α (50 µg) plus GST-Ubc9 (5 µg), E1 and HA-SUMO-1; 5,GST-∆Np63α (50 µg) plus GST-Ubc9 (10 µg), E1 and HA-SUMO-1. The reaction mix wasincubated at 300C for 30 min and stopped with 10 mM N-ethylmaleimide. GST-∆Np63α waspurified with 50 µl of 50% slurry glutathione-agarose beads and binding proteins were elutedwith 50 µl of loading buffer and boiled for 5 min. Resulting proteins was separated by a 10%SDS-PAGE and probed with ant-p63 antibody, 4A4 (recognizes all p63 isotypes) and anti-HAantibody for SUMO-1. (B) Effect of SUMO-1 motif associated mutations in p63α on in vitrosumoylation. GST-∆Np63α (wild type, WT) or mutant (K549E, K637E or K549E/K637E)were incubated with lysates from HA-SUMO-1 transfected 911 cells (100 µg) and 10 µg ofGST-Ubc9. P63 sumoylation was probed with anti-HA antibody. (C) In vivo sumoylation of∆Np63α by Ubc9 and EYFP-SUMO. T75 flasks of 911 cells (~80% confluency) were cotrans-fected with either ∆Np63α (WT) or mutant (K549E/K637E) and with expression cassettes forUbc9 and EYFP-SUMO-1 for 24 h. Cells were washed and collected in 1 x PBS containing10 mM of N-ethylmaleimide. Total lysates were prepared and ∆Np63α (WT) or mutantvariants were precipitated with H-129 antibody or anti-SUMO-1 antibody. ∆Np63α proteinswere probed with 4A4 antibodies and SUMO-1 modified proteins were probed with anti-GFPantibody, which also recognizes YFP (Clontech). Note: M.W. of HA-SUMO-1 is ~11.5 kDa,while M.W. of EYFP-SUMO-1 is ~39 kDa.
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Ubc9 by immunoprecipitation with anti-Ubc9 anti-body). As a source for E1 ligase, we used total lysates of911 cells transfected with an empty pCMV vecor andsimilarly depleted for endogenous Ubc9 and SUMO-1with corresponding antibodies.
5 µg of GST-∆Np63α (95 kDa) were mixed with100 µg of cell lysate expressing empty pCMV vector orpCMV-HA-SUMO-1 (Fig. 4, lanes 1 and 2, respec-tively). Indicated reaction mixtures were also supple-mented with 30 µg of GST-Ubc9 (45 kDa, Fig. 4A,lanes 3–5). Assay reactions were incubated for 5, 15and 30 min at 30˚C. The modified proteins werepulled down with glutathione agarose beads, andproteins were visualized with 4A4 antibody for p63 oranti-HA antibody for SUMO-1 (Fig. 4A, left and rightpanels, respectively). We found that ∆Np63α wasexclusively sumoylated in the presence of exogenousUbc9 in a time- and dose-dependent fashion (Fig. 4A).Since the anti-HA antibody recognized two p63αbands precipitated with H-129 antibody, we suggestthat ∆Np63α could be modified by SUMO-1 at leastin two positions (Fig. 4A).
To further map the ∆Np63α sumoylation sites, weused mutant ∆Np63α polypeptides fused to glu-tathione-S-transferase (GST-∆Np63α-K549E, GST-∆Np63α-K637E, and GST-∆Np63α-K549E/K637E)as substrates for in vitro sumoylation assay (Fig. 4B).Both K637E mutation alone and K549E/K637Edouble mutation completely abrogated the initialSUMO-1 modification of ∆Np63α, while K549Emutation alone affected only the second modificationevent (Fig. 4B), suggesting that the primary sumoylationsite is localized at the ∆Np63α TID.
To further examine sumoylation of ∆Np63α, weectopically expressed wild type or double mutantK549E/K537E of ∆Np63α in combination withEYFP-SUMO-1 and Ubc9 in 911 cells (Fig. 4C). Usingimmunoprecipitation analysis, we found that ∆Np63αharboring two mutations (K549E and K637E) failed tobe sumoylated supporting the notion that the wild type∆Np63α could be modified by SUMO-1 at two posi-tions (Fig. 4C).
Immortalized normal human keratinocytes, HaCaT,are known to express high amounts of ∆Np63α in thenucleus.41,42 To examine whether forced expression of Ubc9 or SUMO-1affects in vivo ∆Np63α modifications, we used HaCaT cells transientlytransfected with pCMV-HA-SUMO-1 and increasing amounts ofpCMV-Ubc9. Endogenous ∆Np63α was precipitated with H129 antibodyand protein levels were monitored by immunoblotting with antibodies top63 (4A4), SUMO-1 (anti-HA), and Ubc9. We observed that endogenous∆Np63α was indeed modified by SUMO-1 in the Ubc9-depdendent man-ner (Fig. 55A, up to 10% of total ∆Np63α was sumoylated as shown inright panel).
To further analyze whether mutations in the p63α C-terminus wouldaffect its modification pattern we performed a 2-D gel separation of the wildtype and mutant (K549E, K637E and K549E/K637E) ∆Np63α polypep-tides ectopically expressed in 911 cells. ∆Np63α polypeptides were precipi-tated with H-129 antibody and the resulting pellet was separated by 2D gelelectophoresis. We detected up to 9–10 modified ∆Np63α spots (Fig. 6).Among them we identified a spot for a very positively charged form that wasabundant in cells expressing wild type ∆Np63α and dramatically decreasedin cells expressing mutant variants (Fig. 6, upper panel). We also found thatmutations used in these experiments led to accumulation of completelyunmodified ∆Np63α isoforms, whereas there were no detectable unmodi-fied isoforms (Fig. 6, lower panel).
Nuclear Colocalization of SUMO-1 and ∆Np63α. To test whetherSUMO-1 and ∆Np63α are colocalized in the nucleus and whether mutationsin ∆Np63α affect this colocalization, we introduced the pEYFP-SUMO-1expression cassette in the presence or absence of wild type or mutant∆Np63α into 911 cells (negative for p63 expression). Cells were grown oncover slips and stained for p63 (H-129), while YFP-SUMO-1 was readilydetectable with direct fluorescence imaging (Fig. 7). 911 cells transfectedwith SUMO-1 alone showed no ∆Np63α expression (Fig. 7A), and demon-strated an association of SUMO-1 with PML-associated intranuclear forma-tions (Fig. 7B and C), which was previously reported.23,32-36 We also detectedan exclusive nuclear localization of wild type and mutant ∆Np63α in trans-fected 911 cells (Fig. 7D, F, G and I). However, wild type ∆Np63αcompletely disrupted the PML-associated localization of SUMO-1 and ledto widespread YFP-SUMO-1-staining throughout the entire nucleus(Fig. 7E), while SUMO-1 retained its association with the PML bodies inthe presence of mutant ∆Np63α-K549E/ K637E (Fig. 7H). Thus, forcedexpression of wild type but not mutant ∆Np63α dramatically affectedSUMO-1 localization, suggesting that wild type ∆Np63α is tightly associatedwith SUMO-1, while mutant ∆Np63α is not, thereby failing to retrieveSUMO-1 from intranuclear structures such as the ND10 domain of PMLbodies.32-36
SUMOYLATION OF p63
Figure 5. Ubc9-dependent sumoylation of endogenous ∆Np63α in HaCaT cells. T75 flasks of~50% confluent HaCaT cells were cotransfected with an expression cassette for HA-SUMO-1(50 µg) and the indicated amount of Ubc9 expression cassette (transfection efficiency was~5-10% as monitored by fluorescence). After 24 h cells were collected in 1x PBS with 10 mMN-ethylmaleimide and total lysates were prepared. Samples: 1, Control HaCaT cells transfectedwith an empty pCMV vector (50 µg); 2, HaCaT cells cotransfected with 50 µg of Ubc9 and 50 µgof HA-SUMO-1 plasmids. 3, HaCaT cells cotransfected with 25 µg of Ubc9 and 50 µg ofHA-SUMO-1 plasmids; 4, HaCaT cells cotransfected with 12 µg of Ubc9 and 50 µg ofHA-SUMO-1 plasmids. (A) ∆Np63α was precipitated with H-129 antibody and probed withanti-HA antibody for SUMO-1 or 4A4 antibody for p63. (B) ∆Np63α was precipitated withH-129 antibody and probed with anti-Ubc9 antibody. (C) HA-SUMO-1 was precipitated withanti-SUMO-1 antibody and probed with anti-HA antibody. Note: M.W. of Ubc9 is 18 kDa, whileM.W. of HA-SUMO-1 is ~11.5 kDa. M.W. of unmodified ∆Np63α is ~68 kDa, and M.W. ofdouble-sumoylated ∆Np63α is ~91 kDa.
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Ubc9 and SUMO-1 Modulate the p63Transcriptional Network. To examine whetherSUMO-1 modifications affect transcriptional regulationmachinery mediated by p63α isotypes, we employed aluciferase-based reporter assay to examine TAp63αtransactivation abilities. Recent observations by ourgroup showed that TAp63α, known as a weak tran-scription factor, is still capable of activating or repressingspecific sets of target genes.21,51 To test the effects ofUbc9 and SUMO-1 on transactivation abilities ofTAp63α, we used the p53-dependent RGC: luxreporter construct.37 911 cells were cotransfected withwild type TAp63α or mutant TAp63α in the presenceor absence of Ubc9 and SUMO-1. We observed thatcoexpression of Ubc9 or SUMO-1 with wild typeTAp63α or mutant TAp63α (K549E) failed to affecttheir transactivation abilities (Fig. 8A). However,mutations K637E or K549E/K637E dramaticallyactivated the transactivation of the RGC promoter byTAp63α (Fig. 8A) even in the presence of both Ubc9and SUMO-1. Moreover, we found that mutationsK637E or K549E/ K637E also decreased the ∆Np63αinhibitory effect on TAp63α transcriptional activity(data not shown). Therefore, the increase in TAp63αtransactivation shown above (Fig. 8A) could be due toTID removal from TAp63α42 and for diminishing ofthe dominant-negative inhibition imposed by∆Np63α.43,44
To further investigate the effect of SHFM muta-tions in the p63α C-terminus on the transcriptionalregulatory network, we performed profiling analysis fordifferentially expressed target genes affected by wildtype or mutant TAp63α (K549E/K637E) in primaryp63 null mouse embryonic fibroblasts. Primary fibrob-lasts from p63 null mice were infected with recombinant
adenoviruses for wild type TAp63α or mutant TAp63α(K549E/K637E) for 12 h (Fig. 8B). In control experiments(wild type TAp63α vs. wild type TAp63α, or mutant TAp63αvs. mutant TAp63α), we failed to detect any significant differencesbetween the expressed gene profiles (data not shown). However,gene expression profiling of mutant TAp63α vs. wild typeTAp63α produced ~150 hits of differentially expressed genesout of 10,000 probes (data not shown). A number of differentiallyexpressed genes were confirmed by semi-quantitative RT-PCR(Fig. 8C). Indeed, SHFM-associated mutations in the TAp63αC-terminal domain mediated upregulation and downregulation
SUMOYLATION OF p63
Figure 6. (Above) Effect of potential sumoylation site mutations on ∆Np63α modification pattern.∆Np63α wild type (A) and mutants K637E (B), K549E (C) and K637E/K549E (D) were intro-duced into 911 cells. ∆Np63α polypeptides were precipitated from total lysates with H-129 anti-body and separated by 2-D gel. Modified P63α polypeptides were detected by immunoblottingwith 4A4 antibody (* denotes a very abundant positively charged isoform found in wild type∆Np63α samples). (E) Bacterially expressed unmodified ∆Np63α was obtained from theGST-∆Np63α fusion polypeptide by thrombin digestion and separation from the GST tag on glu-tathione-agarose beads. Phosphomodifications were identified by incubation of pelleted proteinswith a set of tyrosine (YOP)- and serine/threonine/tyrosine (1-PPase)-specific protein phos-phatases. Dephosphorylation pattern was detected by the shift in IEF mobility between phospho-rylated and dephosphorylated proteins (data not shown).
Figure 7 (Left). Colocalization of ∆Np63α (wild type orK549E/K637E mutant) with EYFP-SUMO-1 in p63 null primarymouse fibroblasts. Primary mouse fibroblasts were grown oncover slips and transiently transfected with the expressioncassette for EYFP-SUMO-1 alone (A–C) or with a combinationof wild type ∆Np63α (D–F) or K549E/K637E mutant ∆Np63α(G–I). Cells were fixed and subjected to direct fluorescenceimaging analysis for detection of EYFP-SUMO-1 (B, E and H) orimmunofluorescence analysis for ∆Np63α (A, D and G) withH-129 antibody followed by goat anti-rabbit Alexa 350 conju-gated IgG (blue in D, F, G and I) or for β-actin antibody followedby goat anti-mouse Alexa 546 conjugated IgG (red in C, F and I).EYFP-SUMO-1 when expressed alone is localized only atnuclear speckles. Coexpression of SUMO-1 with wild type∆Np63 led to de-localization of EYFP-SUMO-1 throughout theentire nuclei, while mutations that abolished sumoylation of∆Np63 failed to affect the EYFP-SUMO-1 association with thenuclear speckles.(A and C) show DAPI nuclear staining (blue).
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of several critical genes [e.g., Elav-2 (HuB), heat shockfactor (HSF)-4, RUNX2, osteocalcin, caspase-3,RanBP2, and MINT] contributing to the regulation ofcell differentiation and limb morphogenesis.45-58
of lysine residues in transcription factors and otherprotein substrates modulate their stability, activityor localization.59-61 These modifications are medi-ated by an enzymatic cascade involving ubiquitin-activating and -conjugating enzymes (e.g., E1, E2and E3 ligases) targeting transcriptional factorsinto a 26S proteasome degradation pathway (ubiq-uitinylation) and also modulating their transacti-vation activity by sumoylation.59,60,62-66 SUMO-1can be conjugated to a large number of cellularproteins, including transcription factors.27-39,62-66
Sumoylation does not lead to degradation butinstead appears to regulate protein/protein interac-tions and intracellular localization as well as protectsome modified targets from ubiquitin-dependentdegradation.27-40,63-67
A key component of this enzymatic machinery,the SUMO-1 conjugating enzyme, Ubc9, an E2ligase, is shown to carry out the sumoylation ofseveral protein substrates (e.g., RanGAP1, p53 andIκBα, refs. 30–32). Several candidates for theSUMO-1-specific E3 ligase were found to modulateSUMO-1 conjugation by increasing the bindingbetween Ubc9 and protein substrates.27-29
Sumoylation was previously shown to occur inother p53 family members including p53 andp73,38-40 but was not yet demonstrated in p63.Although, p53 was shown to be sumoylated in thepresence of SUMO-1 E3 ligase, PIAS,28 our initialmass spectroscopy analysis of p63-interactingprotein candidates identified that another SUMO-1specific E3 ligase, RanBP229 physically associatedwith ∆Np63α in vivo (data not shown).
We found that the p63α C-terminal domainassociates with Ubc9 in a variety of experimentalconditions. We observed that the p63α C-terminaldomain associated with the E2 ubiquitin conjugat-ing enzyme, Ubc9. A p63α mutation (Q634X)naturally occurring in SHFM modulated the inter-action of the p63α C-terminal domain with Ubc9in a yeast 2-hybrid assay. Furthermore, Ubc9 catalyzed the conjuga-tion of ∆Np63α with small ubiquitin modifier-1 (SUMO-1), whichcovalently modified ∆Np63α in vitro and in vivo at two positions(K549E and K637E), within SUMO-1 modification consensus site(φKXD/E). In addition, the p63α mutations (K549E and K637E)abolished sumoylation of p63α, dramatically activated transactivationproperties of TAp63α, and inhibited the dominant-negative effect of∆Np63α. These p63α mutations also affected transcriptional regu-lation of gene targets involved in bone and tooth development (e.g.,RUNX2 and MINT) and therefore might contribute to the molecularmechanism underlying the SHFM phenotype.
By applying 2-hybrid screens for p63 interacting proteins alongwith p63-induced gene expression profiling carried out in primary
fibroblasts from p63 null mice we demonstrated a deregulation ofexpression of various transcription factors directly involved in bonedevelopment and osteoblast differentiation. Among them,RUNX2,54,55 ATF455 and MINT56-59 are involved in regulation ofbone formation during embryonic development. We found thatRUNX2 and MINT were downregulated ~3.0 and 4.5 x fold,respectively, in response to mutated TAp63α. The RUNX2 transcrip-tional factor is absolutely required for osteoblast differentiation sincemutations in this protein were shown to be a major cause for clei-docranial dysplasia.68 Meanwhile, MINT, also known as MSX2-binding protein, inhibits MSX2 transactivation of the osteocalcinpromoter through its RNA binding domain.57-59 Inactivation of MSX2itself causes certain craniofacial disorders (e.g., Parietal Foramina),
SUMOYLATION OF p63
Figure 8. SHFM-derived p63 mutations modulate transcriptional regulation of p63 target genes.(A) SHFM-derived mutations increase TAp63α transactivation abilities. The expression cassettesfor wild type TAp63α (0.4 µg) or mutant TAp63α (K549E, K637E, or K549E/K637E, 0.4 µg)were cotransfected into 911 cells in the presence of an equal amount of mock (1), Ubc9 (2),EYFP-SUMO-1 (3) or both Ubc9 and EYFP-SUMO-1 (4) expression plasmids in the combinationswith RGC: lux reporter plasmid (0.5 µg) for TAp63α and pRL-TK normalization plasmid (0.5 µg).(B) Semi-quantitative RT-PCR analysis of expression of TAp63α (wild type and K594E/K637E)and ∆Np63α (wild type and K594E/K637E). (C) Semi-quantitative RT-PCR analysis of selectedgenes regulated by SHFM-associated p63 mutations. Confluent p63 null primary mousefibroblasts were infected with wild type TAp63α or mutant TAp63α recombinant adenoviruses(infection efficiency was ~95-99% as monitored by fluorescence). Sets of upregulated (> 2-fold)and downregulated (> 2-fold) genes as detected by microarray analysis were chosen for RT-PCR.The PCR products were analyzed by 1.5% agarose electrophoresis and visualized with ethidiumbromide. GAPDH was coamplified to confirm equal amount of starting cDNA.
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while gain of function mutation P148H leads to craniosyntosis.69-72
Thus, we suggest that p63 can modulate expression of genesinvolved I bone development, such as RUNX2 and MINT, whichmay contribute to the developmental limb syndactyly phenotype.
Our studies indicate a critical role for sumoylation/ubiquitinylationin p63 function and regulation. Furthermore, we identified addi-tional protein candidates interacting with p63α implicated in proteinstability (Jab1, UbB, data not shown) and observed that TAp63αwith SHFM-derived mutations inhibited expression of genes associ-ated with the proteasome degradation pathway (e.g., UbP21, UbP6,UbE3A/Hect, data not shown). We have previously reported thatRACK1 containing WD40 motifs associates with the p63α C-ter-minal domain and might serve as E3 Ub ligase for degradation ofp63α isotypes.73 Thus, dysregulation of the proteasome degradation/modification network by p63 mutants might also contribute to themolecular mechanisms underlying SHFM and other developmentaldisorders.
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