p63α Mutations Lead to Aberrant Splicing of Keratinocyte Growth Factor Receptor in the Hay-Wells Syndrome
2003; Elsevier BV; Volume: 278; Issue: 26 Linguagem: Inglês
10.1074/jbc.m300746200
ISSN1083-351X
AutoresAlexey Fomenkov, Yi‐Ping Huang, Özlem Topaloglu, Anna Brechman, Motonobo Osada, T E Fomenkova, Eugene Yuriditsky, Barry Trink, David Sidransky, Edward A. Ratovitski,
Tópico(s)Virus-based gene therapy research
Resumop63, a p53 family member, is required for craniofacial and limb development as well as proper skin differentiation. However, p63 mutations associated with the ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome (Hay-Wells syndrome) were found in the p63 carboxyl-terminal region with a sterile α-motif. By two-hybrid screen we identified several proteins that interact with the p63α carboxyl terminus and its sterile α-motif, including the apobec-1-binding protein-1 (ABBP1). AEC-associated mutations completely abolished the physical interaction between ABBP1 and p63α. Moreover the physical association of p63α and ABBP1 led to a specific shift of FGFR-2 alternative splicing toward the K-SAM isoform essential for epithelial differentiation. We thus propose that a p63α-ABBP1 complex differentially regulates FGFR-2 expression by supporting alternative splicing of the K-SAM isoform of FGFR-2. The inability of mutated p63α to support this splicing likely leads to the inhibition of epithelial differentiation and, in turn, accounts for the AEC phenotype. p63, a p53 family member, is required for craniofacial and limb development as well as proper skin differentiation. However, p63 mutations associated with the ankyloblepharon-ectodermal dysplasia-clefting (AEC) syndrome (Hay-Wells syndrome) were found in the p63 carboxyl-terminal region with a sterile α-motif. By two-hybrid screen we identified several proteins that interact with the p63α carboxyl terminus and its sterile α-motif, including the apobec-1-binding protein-1 (ABBP1). AEC-associated mutations completely abolished the physical interaction between ABBP1 and p63α. Moreover the physical association of p63α and ABBP1 led to a specific shift of FGFR-2 alternative splicing toward the K-SAM isoform essential for epithelial differentiation. We thus propose that a p63α-ABBP1 complex differentially regulates FGFR-2 expression by supporting alternative splicing of the K-SAM isoform of FGFR-2. The inability of mutated p63α to support this splicing likely leads to the inhibition of epithelial differentiation and, in turn, accounts for the AEC phenotype. The recently discovered p53 homologue, p63, utilizes two promoters and an alternative splicing mechanism to encode six distinct protein isotypes (1Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Google Scholar, 2Trink B. Okami K. Wu L. Sriuranpong V. Jen J. Sidransky D. Nat. Med. 1998; 4: 747-748Google Scholar, 3Yang A. Kaghad M. Wang Y. Gillett E. Fleming M. Dotsch V. Andrews N. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Google Scholar). These isotypes (TAp63α, TAp63β, TAp63γ, ΔNp63α, ΔNp63β, and ΔNp63γ) differ in their ability to transactivate responsive genes, control the cell cycle, and induce apoptosis (1Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Google Scholar, 2Trink B. Okami K. Wu L. Sriuranpong V. Jen J. Sidransky D. Nat. Med. 1998; 4: 747-748Google Scholar, 4Yang A. Kaghad M. Caput D. McKeon F. Trends Genet. 2002; 18: 94-95Google Scholar). p63α is the longest isotype that contains a transactivation, DNA binding, and oligomerization domain and a sterile α-motif at the extreme carboxyl terminus (2Trink B. Okami K. Wu L. Sriuranpong V. Jen J. Sidransky D. Nat. Med. 1998; 4: 747-748Google Scholar). p63 functions as a transcriptional regulator involved in epidermal-mesenchymal interactions during embryonic development where it is required for regenerative proliferation of limb, for craniofacial and epithelial development, and for skin renewal (5Mills A. Zheng B. Wang X. Vogel H. Roop D. Bradley A. Nature. 1999; 398: 708-713Google Scholar, 6Yang A. Schweitzer R. Sun D. Kaghad M. Walker N. Bronson R. Tabin C. Sharpe A. Caput D. Crum C. McKeon F. Nature. 1999; 398: 714-718Google Scholar). In addition, p63 expression has been closely identified with keratinocyte stem cell proliferation (7Pellegrini G. Dellambra E. Golisano O. Martinelli E. Fantozzi I. Bondanza S. Ponzin D. McKeon F. Luca M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3156-3161Google Scholar). The proliferating cells of stratified squamous epithelia consist of stem cells and transient amplifying cells producing holoclones and paraclones, respectively. While p63 is abundantly expressed by epidermal and limbal holoclones, it is undetectable in paraclones (7Pellegrini G. Dellambra E. Golisano O. Martinelli E. Fantozzi I. Bondanza S. Ponzin D. McKeon F. Luca M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3156-3161Google Scholar). Several developmental craniofacial abnormalities (ectodermal dysplasias) are caused by p63 mutations in humans (8Celli J. Duijf P. Hamel B. Bamshad M. Kramer B. Smits A. Newbury-Ecob R. Hennekam R. Buggenhout G. Haeringen A. Woods C. Essen A. de Waal R. Vriend G. Haber D. Yang A. McKeon F. Brunner H. van Bokhoven H. Cell. 1999; 99: 143-153Google Scholar, 9McGrath J. Duijf P. Doetsch V. Irvine A. de Waal R. Vanmolkot K. Wessagowit V. Kelly A. Atherton D. Griffiths W. Orlow J. van Haeringen A. Ausems M. Yang A. McKeon F. Bamshad M. Brunner H. Hamel B. van Bokhoven H. Hum. Mol. Genet. 2001; 10: 221-229Google Scholar, 10Brunner H. Hamel B. van Bokhoven H. J. Med. Genet. 2002; 39: 377-381Google Scholar). Mutations in the p63 DNA binding domain are found in patients with ectrodactyly-ectodermal dysplasia-cleft lip/palate, split hand/foot malformation, and limb-mammary syndrome (8Celli J. Duijf P. Hamel B. Bamshad M. Kramer B. Smits A. Newbury-Ecob R. Hennekam R. Buggenhout G. Haeringen A. Woods C. Essen A. de Waal R. Vriend G. Haber D. Yang A. McKeon F. Brunner H. van Bokhoven H. Cell. 1999; 99: 143-153Google Scholar). Moreover a number of p63 mutations associated with the ankyloblepharon-ectodermal dysplasia-clefting (AEC) 1The abbreviations used are: AEC, ankyloblepharon-ectodermal dysplasia-clefting; ABBP1, apobec-1-binding protein-1; FGF, fibroblast growth factor; FGFR, fibroblast growth factor receptor; AD, activation domain; CMV, cytomegalovirus; X-gal, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; mut, mutant; wt, wild type; CT, carboxyl terminus; BD, binding domain; hnRNP, heteronuclear ribonucleoproteins; KGFR, keratinocyte growth factor receptor; MES, 4-morpholineethanesulfonic acid; GFP, green fluorescent protein; SD, synthetic dextrose; Ade, adenine; GST, glutathione S-transferase; RT, reverse transcription; SAM, sterile α-motif. syndrome (Hay-Wells syndrome) have been mapped inside the sterile α-motif (9McGrath J. Duijf P. Doetsch V. Irvine A. de Waal R. Vanmolkot K. Wessagowit V. Kelly A. Atherton D. Griffiths W. Orlow J. van Haeringen A. Ausems M. Yang A. McKeon F. Bamshad M. Brunner H. Hamel B. van Bokhoven H. Hum. Mol. Genet. 2001; 10: 221-229Google Scholar). Thus, p63 mutations found in the AEC syndrome represent a perfect model to study unique p63 protein-protein interactions (9McGrath J. Duijf P. Doetsch V. Irvine A. de Waal R. Vanmolkot K. Wessagowit V. Kelly A. Atherton D. Griffiths W. Orlow J. van Haeringen A. Ausems M. Yang A. McKeon F. Bamshad M. Brunner H. Hamel B. van Bokhoven H. Hum. Mol. Genet. 2001; 10: 221-229Google Scholar). The sterile α-motif is a 65–70-amino acid residue sequence found in many diverse proteins from yeast to humans whose functions range from signal transduction to transcriptional repression. The sterile α-motifs have been implicated in mediating protein-protein interactions via the formation of homo- and heterotypic oligomers (11Schultz J. Ponting C. Hofmann K. Bork P. Protein Sci. 1997; 6: 249-253Google Scholar, 12Chi S. Ayed A. Arrowsmith C. EMBO J. 1999; 18: 4438-4445Google Scholar, 13Stapleton D. Balan I. Pawson T. Sicheri F. Nat. Struct. Biol. 1999; 6: 44-49Google Scholar, 14Kim C. Phillips M. Kim W. Gingery M. Tran H. Robinson M. Faham S. Bowie J. EMBO J. 2001; 20: 4173-4182Google Scholar). We sought to identify protein candidates that interact specifically with p63 via its sterile α-motif and to evaluate the effect of known AEC genetic alterations on this molecular interaction. We found that p63α binds apobec-1-binding protein-1 (ABBP1), a member of the RNA processing machinery, resulting in a shift of FGFR-2 alternative splicing toward the epithelial specific K-SAM isoform. However, mutant p63 harboring AEC-derived mutations failed to bind ABBP1. Thus, mutations mapped to p63 sterile α-motif may result in the AEC syndrome by abolishing the interaction with ABBP1 and modulating the FGFR-2 pathway leading to aberrant differentiation. Antibodies, Reagents, Cells, and Transfections—We used mouse monoclonal antibodies against all p63 isotypes (4A4, 1:200) and against the carboxyl terminus of p63α (H-129, 1:200, both from Santa Cruz Biotechnology, Inc.) and a rabbit polyclonal antibody against ΔNp63 (Ab-1, 1:200, Oncogene Research Products). A rabbit polyclonal antibody against CBF-A (homologue of ABBP1, 1:250) was obtained from Drs. Tomas Leanderson and Alaitz Aranburu (Lund University, Lund, Sweden). His-tagged p40 protein (residues 1–356, smallest p63 isotype) was obtained from Dr. Keiho Yamaguchi (The Johns Hopkins University, Baltimore, MD) and purified as described previously (34Ratovitski E. Patturajan M. Hibi K. Trink B. Yamaguchi K. Sidransky D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1817-1822Google Scholar). The human skin keratinocyte cell line (HaCaT, spontaneously immortalized) and human embryonic retina cell line (911, immortalized by adenovirus E1 expression) were obtained from American Tissue Culture Collection. Both HaCaT and 911 cells were grown in Dulbecco's minimal essential medium with 10% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin. All cultures were incubated in a humidified atmosphere of 5% CO2 at 37 °C. Primary head and neck squamous cell carcinoma and lung adenocarcinoma cell lines were isolated by us during 1985–1992 as described previously (33Hibi K. Trink B. Patturajan M. Westra W. Caballero O. Hill D. Ratovitski E. Jen J. Sidransky D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5462-5467Google Scholar). Skin tissue samples were obtained from p63-null mice (Ref. 5Mills A. Zheng B. Wang X. Vogel H. Roop D. Bradley A. Nature. 1999; 398: 708-713Google Scholar, Jackson Laboratories). Both p63+/– and –/– mice were bred at The Johns Hopkins University Animal Core Facility. Normal C57Bl6 mice were purchased from Charles River Breeding Laboratory. Preparation of Adenoviral Expression Vectors—Recombinant adenoviruses driving expression of wild type or mutant p63α proteins under control of a cytomegalovirus (CMV) promoter were prepared as described elsewhere (33Hibi K. Trink B. Patturajan M. Westra W. Caballero O. Hill D. Ratovitski E. Jen J. Sidransky D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5462-5467Google Scholar). cDNA for wild type or mutant p63α was subcloned into a pAdTrack-CMV shuttle vector and then introduced in viral backbone vector pAdEasy1 by homologous recombination in BJ5183 Escherichia coli cells (QBiogene). Resulting constructs were screened by PacI restriction digestion. PacI-linearized constructs for empty pAd, pAd-p40, pAd-TAp63α (wt), pAd-TAp63α (mut, L518F), pAd-ΔNp63α (wt), and pAd-ΔNp63α (mut, L518F) were introduced into 911 cells to generate adenoviruses. Recombinant adenoviruses were amplified by propagation in 911 cells. The CsCl-purified final viral preparations displayed a multiplicity of infection of ∼1012 plaque-forming units/ml monitored by titration and expression levels of the green fluorescent protein (GFP). Transfections—Cells (in a six-well plate) were transiently transfected for 24 h with the desired plasmids (5 μg) using LipofectAMINE 2000 (20 μl, Invitrogen) according to the manufacturer's protocol or by a modified calcium phosphate-mediated protocol (1Osada M. Ohba M. Kawahara C. Ishioka C. Kanamaru R. Katoh I. Ikawa Y. Nimura Y. Nakagawara A. Obinata M. Ikawa S. Nat. Med. 1998; 4: 839-843Google Scholar). 5 μg of plasmid DNA was mixed with 2× MES buffer, pH 7.05, and 2.5 m CaCl2 for 20 min and then added to 911 cells (in a six-well plate) for 24 h. For transfection of cells grown in a T-25 or T-75 flask, the amount of DNA and reagents was scaled up. The efficiency of transfection was monitored by co-transformation with a pAdTrack-GFP vector. The average transfection efficiency was ∼80–90%. Immunoblotting and Immunoprecipitation—Cells were resuspended in lysis buffer (50 mm Tris, pH 7.5, 100 mm NaCl, 2 mm EDTA, 0.5% Triton X-100, 0.5% Brij-50, 1 mm phenylmethylsulfonyl fluoride, 0.5 mm NaF, 0.1 mm Na3VO4, 2× complete protease inhibitor mixture), sonicated five times for 10-s time intervals, and clarified for 30 min at 15,000 × g. Supernatants (designated as total cell lysates) were resolved by 10% SDS-PAGE and then analyzed by immunoblotting (∼30–60 μg/lane) or immunoprecipitation (∼150–200 μg/lane). Proteins transferred onto polyvinylidene difluoride membranes were blocked with 10% fat-free milk (Bio-Rad) in 0.5% phosphate-buffered saline, Tween 20 for 1 h; then incubated for 2 h with primary antibodies diluted 1:500; washed twice with 0.5% phosphate-buffered saline, Tween 20; incubated for 1 h with secondary antibodies (goat anti-mouse or goat anti-rabbit immunoglobulins, both from Sigma) coupled to horseradish peroxidase diluted 1:5000; and finally washed three times with 0.5% phosphate-buffered saline, Tween 20. Protein bands were visualized by an enhanced chemiluminescence kit (Amersham Biosciences). For immunoprecipitation 500 μl of total cell lysates were incubated with 10 μl of normal rabbit serum for 30 min and then incubated with 50 μl of protein A-Sepharose 4B for 30 min (34Ratovitski E. Patturajan M. Hibi K. Trink B. Yamaguchi K. Sidransky D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1817-1822Google Scholar). Following centrifugation 500 μl of precleared supernatant was incubated for 3 h with primary antibodies, and then with 40 μl of a 50% suspension of protein A-Sepharose 4B (or goat anti-mouse agarose) for 4 h at 4 °C and washed three times with 1 ml of cold 20 mm Tris-HCl, pH 7.4, 125 mm NaCl, 1 mm Na3VO4, 50 mm NaF, 1 mm EDTA, 0.2% Triton X-100, 0.2 mm phenylmethylsulfonyl fluoride. Samples were boiled in 4× Laemmli buffer and then fractionated by 10% SDS-PAGE followed by immunoblotting (34Ratovitski E. Patturajan M. Hibi K. Trink B. Yamaguchi K. Sidransky D. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1817-1822Google Scholar). Yeast Two-hybrid Screening—A yeast two-hybrid screen was performed with the Matchmaker Gal4 system 3 using four two-hybrid cDNA libraries from human fetal kidney, human fetal liver, human keratinocytes, and HeLa cells (Clontech). As bait plasmids, we used pGal4-BD-p63CT (containing the complete carboxyl-terminal part, residues 411–642) or pGal4-BD-p63-SAM (containing the sterile α-motif, residues 499–568) of p63α. Appropriate DNA fragments (carboxyl terminus (CT), 1234–1926 base pairs; sterile α-motif (SAM), 1498–1704 base pairs) were amplified by PCR from TAp63α cDNA as a template using the following primers: for CT, sense, 5′-GGTCGCCCGGACCTCAATACAGTCTCCATCTTCA-3′; antisense, 5′-GGCTGTCGACTCACTCCCCCTCCTCTTTGATGCGCTG-3′; for SAM, sense, 5′-GGTCGCCCGGCCCCCACCTCCGTATCCCACAGAT-3′; antisense, 5′-GGCTGTCCAGGAGCTGCCGGTGGTCCAGGATGC-3′. PCR fragments were subcloned into a pBD-Gal4-Cm vector (Stratagene) in-frame with the Gal4 DNA binding domain using the SrfI and SalI restriction sites. The resulting pGal4-BD-CT or pGal4-BD-SAM plasmids were used as baits and introduced into Saccharomyces cerevisiae yeast strain AH109 (MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3, GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ). The resulting transformed yeast strains (CT+ or SAM+) were sequentially transformed with 50–100 μg of each cDNA library constructed into the pACT2 vector in-frame with the Gal4 transcription activation domain Gal4-AD. Transformed clones were selected on 20-cm SD/His–/Trp–/Leu–-agar plates. All clones were verified based on their ability to grow on different selective media, SD/Trp–/Leu–, SD/Trp–/Leu–/His– and SD/Trp–/Leu–/His–/Ade–, in the presence of X-gal to screen for ADE2, HIS3, and MEL1 expression. All positive clones were grown on complete YPAD medium, and total DNA was purified using glass beads (Sigma) and then used for retransformation into E. coli (XL blue strain). DNA from ampicillin-resistant colonies was purified and used for DNA sequencing using Gal4 DNA binding domain 5′- and 3′-specific primers (Clontech). The intermolecular interactions between bait and prey proteins were verified by subsequent transformation of both bait and prey plasmids into the AH109 yeast strain followed by α- and β-galactosidase assays according to the manufacturer's instructions (Clontech). Protein Expression—Two isoforms, ABBP1 and ABBP1Δex1/6, were expressed in bacteria as GST fusion recombinant polypeptides. The full-length cDNA for A/B heteronuclear ribonucleoprotein (hnRNP) (MGC 10739) was purchased from ATCC. The A/B hnRNP protein differs from the ABBP1 protein in that it lacks the carboxyl-terminal 47-amino acid residue insert (residues 263–311, see Fig. 3). The ABBP1 cDNA as a GST fusion construct was obtained from Dr. Paul P. Lau (Baylor College of Medicine, Houston, TX). Sequence analysis revealed that this cDNA has a BamHI deletion in the RNA binding domain at the amino terminus of ABBP1 (residues 101–186). Therefore, this plasmid was designated as pGST-ABBP1ΔBamHI. To restore the full-length ABBP1 sequence, we amplified this DNA fragment by PCR using A/B hnRNP cDNA as a template and the following primers: sense, 5′-GCGGATCCCAACACTGGACGGTCAAG-3′; antisense, 5′-GCGGATCCATTGGCAATTCAATGGCCT-3′. Both primers contained putative BamHI restriction sites allowing us to subclone this fragment into the BamHI site in the pGST-ABBP1ΔBamHI plasmid. The resulting construct was verified by sequencing and designated as ABBP1. Using the following PCR primers, sense, 5′-GCGCGAATTCATGTCGGAAGCGGGCGAGGAGCAG-3′, and antisense, 5′-GCGGCCGCTCAGTATGGCTTGTAGTTATTCTG-3′ (containing EcoRI and NotI sites, respectively), we amplified the open reading frames for ABBP1, ABBP1Δex1/6, and ABBP1ΔBamHI and then subcloned them into a pCMV-Sport6 vector for mammalian expression and into a pGEX-4T-1 for E. coli expression of GST fusion proteins. For mammalian expression each pCMV-Sport6 construct was transiently transfected into 911 or HaCaT cells. For bacterial expression, each pGEX-4T-1 construct was transformed into E. coli BL21 (Novagen). Overnight cultures of E. coli transformed with recombinant pGEX plasmids were diluted 1:10 in L broth with 50 μg/ml ampicillin and incubated at 37 °C to an A600 = 0.5. Isopropyl-β-d-thiogalactopyranoside was then added to a final concentration of 1 mm at 37 °C. After a further 2 h of growth, cells were pelleted at 5000 × g for 10 min at 4 °C and resuspended in a 1:5 (v/v) dilution of the original culture volume of NETN (0.5% Nonidet P-40, 1 mm EDTA, 20 mm Tris, pH 8.0, 100 mm NaCl) containing proteases inhibitors (Roche Applied Science). Cells were then sonicated and centrifuged at 10,000 × g for 5 min at 4 °C. GST fusion proteins were purified from 1-liter cultures using glutathione-agarose column chromatography (Sigma) according to the manufacturer's protocol. Fractions eluted from glutathione-agarose column were additionally purified by fast performance liquid chromatography on a 5-ml MonoQ HR5/5 column (Amersham Biosciences) using a 30-ml, 50–800 mm KCl gradient (1 ml/min) containing 10 mm Tris-HCl (pH 7.4), 1 mm dithiothreitol, 1 mm EDTA, 5 mm MgCl2, and 1% phenylmethylsulfonyl fluoride. Approximately 1.0–2.0 mg of fusion protein was purified from 3–4 g of bacterial cells. Site-specific Mutagenesis—Plasmid DNA preparations from mammalian (pCMV-Sport6) and yeast (pGal4-BD) expression vectors containing cDNAs for human TAp63α, ΔNp63α, and pGal4-BD-SAM were used as templates for site-directed mutagenesis using the QuikChange kit (Stratagene). To introduce the following point mutations (518, Leu to Phe; 518, Leu to Val; 526, Cys to Gly; 534, Gly to Val; 540, Gln to Leu; and 541, Ile to Thr) into p63-SAM we used the following 5′-phospho-oligonucleotides and their reverse complements: 518 (Leu to Phe), 5′-GTCAGTTTCTTTGCGAGGTTGGGC-3′; 518 (Leu to Val), 5′-GTCAGTTTCGTTGCGAGGTTGGGC-3′; 526 (Cys to Gly), 5′-TGTTCATCAGGTCTGGACTAT-3′; 534 (Gly to Val), 5′-ACGACCCAGGTGCTGACCACC-3′; 540 (Gln to Leu), 5′-ACCATCTATCTGATTGAGCAT-3′; 541 (Ile to Thr), 5′-ATCTATCAGACTGAGCATTAC-3′. The amplification of plasmids with a high fidelity Pfu Turbo DNA polymerase I was performed according to the manufacturer's protocol (Stratagene). The resulting mutated constructs were transformed into XL1-Blue supercompetent cells followed by sequencing. RT-PCR Assay—Total RNA was isolated from cells using Trizol reagent (Invitrogen). 1 μg of total RNA was used to generate cDNA from each sample using a one-step RT-PCR kit (Qiagen) and custom primers for the FGFR-2 common region or specific primers for either the K-SAM or BEK exons: FGFR-2com, sense, 5′-TGTTGAAAGATGCCGCCGTG-3′; FGFR-2com, antisense, 5′-CGTGTGATTGATGGACCCGTATTC-3′; FGFR-2Bek, antisense, 5′-GCGTCCTCAAAAGTTACATTCCG-3′; KGFRK-Sam, antisense, 5′-CGGTCACATTGAACAGAGCCAG-3′. As an internal loading control we amplified the GAPDH region using the following primers: sense, 5′-GAGAAGGCTGGGGCTCATTT-3′; antisense, 5′-CAGTGGGGACACGGAAGG-3′. PCR products were resolved by 2% agarose electrophoresis or 4–20% gradient non-denatured PAGE (Bio-Rad) and visualized with ethidium bromide. For each primer set the number of amplification cycles was predetermined to remain in the exponential phase. To provide a high degree of standardization all experiments including HaCaT and 911 were performed simultaneously using the same reaction mixture, and GAPDH was co-amplified to confirm equal amounts of starting cDNA. RT-PCR amplification results were analyzed digitally by Kodak 1D 3.5 software (Eastman Kodak Co.). The net intensity of PCR bands for the full-length FGFR-2-BEK and K-SAM (KGFR) were measured and normalized to net intensity of the control GAPDH bands. Two-hybrid Screening of p63-interacting Proteins—Previous reports clearly identified germ line mutations of p63 sterile α-motif in the AEC syndrome (9McGrath J. Duijf P. Doetsch V. Irvine A. de Waal R. Vanmolkot K. Wessagowit V. Kelly A. Atherton D. Griffiths W. Orlow J. van Haeringen A. Ausems M. Yang A. McKeon F. Bamshad M. Brunner H. Hamel B. van Bokhoven H. Hum. Mol. Genet. 2001; 10: 221-229Google Scholar). We hypothesized that protein interactions mediated by the sterile α-motif of p63α would contribute to the AEC phenotype. We thus sought to identify protein candidates that interact with the p63 sterile α-motif by means of two-hybrid screens. We generated yeast expression constructs containing the CT of p63α (residues 411–642) or the SAM (residues 499–568). Both sequences (CT or SAM) were subcloned in-frame to the Gal4 DNA binding domain (BD), and resulting bait plasmids were designated as pGal4-BD-p63CT or pGal4-BD-p63-SAM, respectively. Using pGal4-BD-p63CT as bait, we screened four two-hybrid cDNA libraries from human fetal kidney, human fetal liver, human keratinocytes, and HeLa cells. After screening a total 6.9 × 106 yeast transformants, we identified 48 positive clones. All clones were verified to grow on different selective media, SD/Trp–Leu–, SD/His–Trp–Leu–, and SD/Ade–His–Trp–Leu– in the presence of X-gal to screen for ADE2, HIS3, and MEL1 expression. This high stringency screen virtually eliminates false positive interactions but preserves low affinity interactions. As controls, we used yeast expression constructs with a p53/SV40 pair (positive) or a p53/lamin C pair (negative) (data not shown). In addition, the intermolecular interactions between bait and prey proteins were verified by subsequent transformation of both plasmids into the AH109 yeast strain followed by α- and β-galactosidase assays. These individual clones were sequenced and found to encode the following human proteins: RACK1 (G-binding protein, protein kinase C receptor, 16 clones), Ral guanine nucleotide exchange factor (four clones), ABBP1 (seven clones), Era guanine trinucleotide phosphohydrolase A (five clones), Scaf4/rA4 RNA-splicing protein (nine clones), β-catenin (four clones), and β-microglobulin (three clones). All pGal4-AD plasmids isolated from identified positive yeast colonies were also found to interact with pGal4-BD-SAM (data not shown). Qualitative and quantitative assays showed that the interactions between p63 and RACK1, ABBP1, or Scaf4/rA4 proteins are specific (Fig. 1). To establish whether mutations found in the AEC syndrome affect any of these protein interactions, we generated a bait p63 construct with a mutation in the sterile α-motif at position 518 (Leu to Phe) representing the most common genetic alteration in AEC. Pairs of wild type and mutant pGal4-BD-p63-SAM baits and appropriate prey plasmids were retransformed into the AH109 yeast strain, and protein interactions were monitored by selective growth and α- and β-galactosidase assays. As shown, introduction of the L518F mutation dramatically decreased the association of p63 with ABBP1 and Scaf4/rA4, while interactions with the RACK1 prey plasmid were unaffected (Fig. 1). The interaction of p63 with other prey plasmids (Era, Ral, β-catenin, and β-microglobulin) was also unaffected by this mutation (data not shown). p63-SAM Specifically Associates with ABBP1—We decided to focus our efforts on further study of the ABBP1-p63 interaction. Sequence analysis of the prey cDNA that interacted with the p63 sterile α-motif identified predominantly the carboxyl-terminal domain of human ABBP1 (Fig. 2). Two isoforms of the A/B hnRNP family were identified previously in mammalian cells including humans (ABBP1 and A/B hnRNP, Refs. 15Khan F. Jaiswal A. Szer W. FEBS Lett. 1991; 290: 159-161Google Scholar and 16Lau P. Zhu H. Nakamuta M. Chan L. J. Biol. Chem. 1997; 272: 1452-1455Google Scholar). They share the same sequence except for a 47-residue region, which is present in ABBP1 but missing in the alternatively spliced isoform A/B hnRNP (17Champion-Arnaud P. Ronsin C. Gilbert E. Gesnel M. Houssaint E. Breathnach R. Oncogene. 1991; 6: 979-987Google Scholar, 18Gatto F. Breathnach R. Mol. Cell. Biol. 1995; 15: 4825-4834Google Scholar, 19Sarig G. Weisman-Shomer P. Fry M. Biochem. Biophys. Res. Commun. 1997; 237: 617-623Google Scholar). To evaluate the specific region of ABBP1 that mediates its association with the p63 sterile α-motif, we carried out two-hybrid yeast expression studies between pGal4-BD-p63-SAM and either pGal4-AD-ABBP1, ABBP1ΔBamHI, or pGal4-AD-ABBP1Δex1/6 (Fig. 2). We found that only ABBP1 was capable of interacting with p63-SAM, while ABBP1Δex1/6 failed to show binding to the p63 sterile α-motif in yeast (data not shown). Moreover the mutated p63-SAM baits (L518F, L518V, C526G, G534V, Q540L, and I541T) also failed to maintain binding between p63 and ABBP1 (data not shown). These observations suggest that these residues are critical for maintaining the interaction between the p63 sterile α-motif and ABBP1. To confirm the results of two-hybrid screens by independent techniques, we generated pCMV-Sport6 expression cassettes for ABBP1, ABBP1ΔBamHI, and ABBP1Δex1/6 driven by CMV promoter. The resulting constructs were transiently transfected into 911 cells together with the p63 expression constructs (ΔNp63α, wild type or mutant), and expression was monitored by RT-PCR (Fig. 3, A and B, respectively). Protein levels of recombinant polypeptides were evaluated by immunoblotting with the antibodies indicated (Fig. 3, C and D). We observed that p63α exclusively associated with the full-length ABBP1 and ABBP1ΔBamHI, while it failed to associate with ABBP1Δex1/6 (Fig. 3E). However, all ABBP1 isoforms failed to associate with the p63α mutant (L518F) emphasizing the importance of this residue in forming physical complexes between p63α and ABBP1. To further examine the association of p63α and ABBP1, we generated GST fusion polypeptides for ABBP1, ABBP1Δex1/6, and ABBP1ΔBamHI. The BamHI deletion of ABBP1 produces an isoform lacking a major part of the RNA binding domain, and deletions of exons 1 and 6 produce isoforms lacking the p63α binding domain. We also expressed the wild type and mutant p63α using expression constructs and recombinant adenoviruses. Total cell lysates expressing p63 isotypes were mixed with purified GST fusion ABBP1 isoforms, and protein complexes were precipitated with an antibody to CBF-A and blotted with an antibody to p63 (Fig. 4). As a control, we used the His-tagged p40 protein (the smallest p63 isotype lacking the extreme carboxyl-terminal of p63α). Our data demonstrate that p63α forms a physical complex with ABBP1 or ABBP1ΔBamHI but not with ABBP1Δex1/6 (Fig. 4C). As expected, His-p40 failed to bind either of the ABBP1 polypeptides (Fig. 4C). Mixing of total lysates of 911 cells expressing p63 polypeptides with GST fusion ABBP1 protein showed that both TAp63α and ΔNp63α associate with ABBP1, whereas p40 and all tested p63α proteins bearing AEC-derived mutations failed to form complexes with GST-ABBP1 (Fig. 4D). Thus, we propose that the 47-residue insert of ABBP1 (residues 263–311) likely functions as a binding site for the p63 sterile α-motif. The primary structures of the p63 and p73 sterile α-motifs are very similar, and based on p73 (12Chi S. Ayed A. Arrowsmith C. EMBO J. 1999; 18: 4438-4445Google Scholar), a homology model for the p63 sterile α-motif has been proposed (3Yang A. Kaghad M. Wang Y. Gillett E. Fleming M. Dotsch V. Andrews N. Caput D. McKeon F. Mol. Cell. 1998; 2: 305-316Google Scholar). According to this model all AEC-a
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