The Human PDZome: A Gateway to PSD95-Disc Large-Zonula Occludens (PDZ)-mediated Functions
2013; Elsevier BV; Volume: 12; Issue: 9 Linguagem: Inglês
10.1074/mcp.o112.021022
ISSN1535-9484
AutoresEdwige Belotti, Jolanta Polanowska, Avais M. Daulat, Stéphane Audebert, Virginie Thomé, Jean‐Claude Lissitzky, Frédérique Lembo, Karim Blibek, Shizue Omi, Nicolas Lenfant, Akanksha Gangar, Mireille Montcouquiol, Marie-Josée Santoni, Michaël Sebbagh, Michel Aurrand‐Lions, Stéphane Angers, Laurent Kodjabachian, Jérôme Reboul, Jean‐Paul Borg,
Tópico(s)Wnt/β-catenin signaling in development and cancer
ResumoProtein–protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRβ, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions. Protein–protein interactions organize the localization, clustering, signal transduction, and degradation of cellular proteins and are therefore implicated in numerous biological functions. These interactions are mediated by specialized domains able to bind to modified or unmodified peptides present in binding partners. Among the most broadly distributed protein interaction domains, PSD95-disc large-zonula occludens (PDZ) domains are usually able to bind carboxy-terminal sequences of their partners. In an effort to accelerate the discovery of PDZ domain interactions, we have constructed an array displaying 96% of the human PDZ domains that is amenable to rapid two-hybrid screens in yeast. We have demonstrated that this array can efficiently identify interactions using carboxy-terminal sequences of PDZ domain binders such as the E6 oncoviral protein and protein kinases (PDGFRβ, BRSK2, PCTK1, ACVR2B, and HER4); this has been validated via mass spectrometry analysis. Taking advantage of this array, we show that PDZ domains of Scrib and SNX27 bind to the carboxy-terminal region of the planar cell polarity receptor Vangl2. We also have demonstrated the requirement of Scrib for the promigratory function of Vangl2 and described the morphogenetic function of SNX27 in the early Xenopus embryo. The resource presented here is thus adapted for the screen of PDZ interactors and, furthermore, should facilitate the understanding of PDZ-mediated functions. Beyond enzymatic activities, cellular functions are largely mediated and coordinated by protein–protein interactions. These interactions build genuine protein networks that contribute to the organization of subcellular compartments and allow coordinated cellular functions to occur. Thus, signaling networks employ a broad range of proteins endowed not only with enzymatic activities but also with binding capacities for other proteins or lipids. Deciphering these protein networks is a prerequisite for understanding the principles of physiological and physiopathological cellular responses, but it is a tedious task because of the numerous and specialized interactions in which each protein can be engaged. Protein interactions are usually mediated by specialized domains presenting a spatial organization that defines their binding specificities. In some cases, binding of these domains to peptide sequences can be dependent on post-translational modifications such as phosphorylation (1Pawson T. Gish G.D. Nash P. SH2 domains, interaction modules and cellular wiring.Trends Cell Biol. 2001; 11: 504-511Abstract Full Text Full Text PDF PubMed Scopus (321) Google Scholar, 2Scott J.D. Pawson T. Cell signaling in space and time: where proteins come together and when they're apart.Science. 2009; 326: 1220-1224Crossref PubMed Scopus (464) Google Scholar). More than 70 protein interaction domains are currently known. Among them, SH2 and SH3 domains bind, respectively, to short peptides containing phosphorylated tyrosines and enriched in proline residues. The early identification of the precise binding specificities of these domains has greatly simplified the discovery of numerous SH2 and SH3 partners and has facilitated the study of their roles in cell signaling and related cellular functions (3Pawson T. Dynamic control of signaling by modular adaptor proteins.Curr. Opin. Cell Biol. 2007; 19: 112-116Crossref PubMed Scopus (150) Google Scholar). Among the protein interaction domains, PSD95-disc large-zonula occludens (PDZ) 1The abbreviations used are:ELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinHAhemagglutininHPVhuman papillomavirusHRGheregulinHTRFhomogeneous time-resolved fluorescenceMAGUKmembrane-associated guanylate kinaseMEFmouse embryonic fibroblastMOmorpholino-modified antisense oligonucleotidePCPplanar cell polarityPDZPSD95-disc large-zonula occludensSNX27sorting nexin 27STREP-HAstreptavidine-hemagglutininY2Hyeast two-hybrid. 1The abbreviations used are:ELISAenzyme-linked immunosorbent assayGFPgreen fluorescent proteinHAhemagglutininHPVhuman papillomavirusHRGheregulinHTRFhomogeneous time-resolved fluorescenceMAGUKmembrane-associated guanylate kinaseMEFmouse embryonic fibroblastMOmorpholino-modified antisense oligonucleotidePCPplanar cell polarityPDZPSD95-disc large-zonula occludensSNX27sorting nexin 27STREP-HAstreptavidine-hemagglutininY2Hyeast two-hybrid. domains are the most widely distributed in genomes (4Nourry C. Grant S.G. Borg J.P. PDZ domain proteins: plug and play!.Sci. STKE. 2003; 2003 (RE7)PubMed Google Scholar, 5Tonikian R. Zhang Y. Sazinsky S.L. Currell B. Yeh J.H. Reva B. Held H.A. Appleton B.A. Evangelista M. Wu Y. Xin X. Chan A.C. Seshagiri S. Lasky L.A. Sander C. Boone C. Bader G.D. Sidhu S.S. A specificity map for the PDZ domain family.PLoS Biol. 2008; 6: e239Crossref PubMed Scopus (356) Google Scholar). PDZ domains can be present in one or several copies in proteins. Some proteins contain PDZ domains only, such as MUPP1, which includes 13 PDZ domains, whereas others exhibit a PDZ domain or domains associated with other functional domains, such as the membrane-associated guanylate kinase (MAGUK) protein family, which associates PDZ, SH3, and guanylate kinase domains (4Nourry C. Grant S.G. Borg J.P. PDZ domain proteins: plug and play!.Sci. STKE. 2003; 2003 (RE7)PubMed Google Scholar, 6Roh M.H. Margolis B. Composition and function of PDZ protein complexes during cell polarization.Am. J. Physiol. Renal Physiol. 2003; 285 (F377)Crossref PubMed Scopus (137) Google Scholar). PDZ domains are found in vertebrates, Drosophila, Caenorhabditis elegans, and yeast proteomes, emphasizing the conserved functionality of these domains along evolution (6Roh M.H. Margolis B. Composition and function of PDZ protein complexes during cell polarization.Am. J. Physiol. Renal Physiol. 2003; 285 (F377)Crossref PubMed Scopus (137) Google Scholar, 7Bilder D. PDZ proteins and polarity: functions from the fly.Trends Genet. 2001; 17: 511-519Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Protein interactions mediated by PDZ domains are involved in many biological processes, including clustering and targeting receptors to cellular membranes (8Kim E. Niethammer M. Rothschild A. Jan Y.N. Sheng M. Clustering of Shaker-type K+ channels by interaction with a family of membrane-associated guanylate kinases.Nature. 1995; 378: 85-88Crossref PubMed Scopus (895) Google Scholar), cell signaling, and cell architecture (6Roh M.H. Margolis B. Composition and function of PDZ protein complexes during cell polarization.Am. J. Physiol. Renal Physiol. 2003; 285 (F377)Crossref PubMed Scopus (137) Google Scholar). As a classical example, the building, maintenance, and function of all epithelial tissues relies on the organized shaping of cells, the so-called cell polarity process, which engages a large number of PDZ proteins dedicated to the compartmentalization of proteins and lipids. The importance of these highly organized PDZ networks in epithelial homeostasis is demonstrated by the strong defects observed when PDZ functions are disrupted in pathological situations such as infectious diseases or cancers (9Javier R.T. Cell polarity proteins: common targets for tumorigenic human viruses.Oncogene. 2008; 27: 7031-7046Crossref PubMed Scopus (82) Google Scholar, 10Pearson H.B. Perez-Mancera P.A. Dow L.E. Ryan A. Tennstedt P. Bogani D. Elsum I. Greenfield A. Tuveson D.A. Simon R. Humbert P.O. SCRIB expression is deregulated in human prostate cancer, and its deficiency in mice promotes prostate neoplasia.J. Clin. Invest. 2011; 121: 4257-4267Crossref PubMed Scopus (134) Google Scholar, 11Zhan L. Rosenberg A. Bergami K.C. Yu M. Xuan Z. Jaffe A.B. Allred C. Muthuswamy S.K. Deregulation of scribble promotes mammary tumorigenesis and reveals a role for cell polarity in carcinoma.Cell. 2008; 135: 865-878Abstract Full Text Full Text PDF PubMed Scopus (317) Google Scholar). It is, for example, now well established that some PDZ proteins are targeted by certain classes of viruses that perturb their cellular functions, contributing to viral spreading and, in some cases, cellular transformation (5Tonikian R. Zhang Y. Sazinsky S.L. Currell B. Yeh J.H. Reva B. Held H.A. Appleton B.A. Evangelista M. Wu Y. Xin X. Chan A.C. Seshagiri S. Lasky L.A. Sander C. Boone C. Bader G.D. Sidhu S.S. A specificity map for the PDZ domain family.PLoS Biol. 2008; 6: e239Crossref PubMed Scopus (356) Google Scholar, 12Javier R.T. Rice A.P. Emerging theme: cellular PDZ proteins as common targets of pathogenic viruses.J. Virol. 2011; 85: 11544-11556Crossref PubMed Scopus (139) Google Scholar, 13Spanos W.C. Hoover A. Harris G.F. Wu S. Strand G.L. Anderson M.E. Klingelhutz A.J. Hendriks W. Bossler A.D. Lee J.H. The PDZ binding motif of human papillomavirus type 16 E6 induces PTPN13 loss, which allows anchorage-independent growth and synergizes with ras for invasive growth.J. Virol. 2008; 82: 2493-2500Crossref PubMed Scopus (106) Google Scholar, 14Thomas M. Narayan N. Pim D. Tomaic V. Massimi P. Nagasaka K. Kranjec C. Gammoh N. Banks L. Human papillomaviruses, cervical cancer and cell polarity.Oncogene. 2008; 27: 7018-7030Crossref PubMed Scopus (146) Google Scholar). enzyme-linked immunosorbent assay green fluorescent protein hemagglutinin human papillomavirus heregulin homogeneous time-resolved fluorescence membrane-associated guanylate kinase mouse embryonic fibroblast morpholino-modified antisense oligonucleotide planar cell polarity PSD95-disc large-zonula occludens sorting nexin 27 streptavidine-hemagglutinin yeast two-hybrid. enzyme-linked immunosorbent assay green fluorescent protein hemagglutinin human papillomavirus heregulin homogeneous time-resolved fluorescence membrane-associated guanylate kinase mouse embryonic fibroblast morpholino-modified antisense oligonucleotide planar cell polarity PSD95-disc large-zonula occludens sorting nexin 27 streptavidine-hemagglutinin yeast two-hybrid. Structurally, PDZ domains consist of 80 to 90 residues forming a packed structure of six β strands and two α helices (15Doyle D.A. Lee A. Lewis J. Kim E. Sheng M. MacKinnon R. Crystal structures of a complexed and peptide-free membrane protein-binding domain: molecular basis of peptide recognition by PDZ.Cell. 1996; 85: 1067-1076Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar, 16Morais Cabral J.H. Petosa C. Sutcliffe M.J. Raza S. Byron O. Poy F. Marfatia S.M. Chishti A.H. Liddington R.C. Crystal structure of a PDZ domain.Nature. 1996; 382: 649-652Crossref PubMed Scopus (291) Google Scholar). The initial studies searching for PDZ domain partners revealed their affinity for carboxy-terminal peptide sequences and led to their classification in three main classes (17Harris B.Z. Lim W.A. Mechanism and role of PDZ domains in signaling complex assembly.J. Cell Sci. 2001; 114: 3219-3231Crossref PubMed Google Scholar, 18Songyang Z. Fanning A.S. Fu C. Xu J. Marfatia S.M. Chishti A.H. Crompton A. Chan A.C. Anderson J.M. Cantley L.C. Recognition of unique carboxyl-terminal motifs by distinct PDZ domains.Science. 1997; 275: 73-77Crossref PubMed Scopus (1219) Google Scholar). Class I PDZ domains were defined for their ability to bind T/S-X-Ψ (where T is threonine, S is serine, X is any amino acid, and Ψ is a hydrophobic residue) motifs. Class II and class III PDZ domains bind to ΨXΨ motifs and D/E-X-Ψ motifs (where D is aspartic acid and E is glutamic acid), respectively. Later on, many studies demonstrated that the mechanisms of PDZ interactions are more complex than initially thought, and some have questioned the early simplistic classification (19Giallourakis C. Cao Z. Green T. Wachtel H. Xie X. Lopez-Illasaca M. Daly M. Rioux J. Xavier R. A molecular-properties-based approach to understanding PDZ domain proteins and PDZ ligands.Genome Res. 2006; 16: 1056-1072Crossref PubMed Scopus (41) Google Scholar, 20Stiffler M.A. Chen J.R. Grantcharova V.P. Lei Y. Fuchs D. Allen J.E. Zaslavskaia L.A. MacBeath G. PDZ domain binding selectivity is optimized across the mouse proteome.Science. 2007; 317: 364-369Crossref PubMed Scopus (305) Google Scholar, 21te Velthuis A.J. Sakalis P.A. Fowler D.A. Bagowski C.P. Genome-wide analysis of PDZ domain binding reveals inherent functional overlap within the PDZ interaction network.PLoS One. 2011; 6: e16047Crossref PubMed Scopus (37) Google Scholar). Firstly, new models based on refined carboxyl terminus binding preferences classify PDZ domains in more subtle classes (5Tonikian R. Zhang Y. Sazinsky S.L. Currell B. Yeh J.H. Reva B. Held H.A. Appleton B.A. Evangelista M. Wu Y. Xin X. Chan A.C. Seshagiri S. Lasky L.A. Sander C. Boone C. Bader G.D. Sidhu S.S. A specificity map for the PDZ domain family.PLoS Biol. 2008; 6: e239Crossref PubMed Scopus (356) Google Scholar, 22Bezprozvanny I. Maximov A. Classification of PDZ domains.FEBS Lett. 2001; 509: 457-462Crossref PubMed Scopus (107) Google Scholar). Then, adding to their versatility, recent studies have shown that PDZ domains can also bind to internal motifs in their protein partners (23Lenfant N. Polanowska J. Bamps S. Omi S. Borg J.P. Reboul J. A genome-wide study of PDZ-domain interactions in C. elegans reveals a high frequency of non-canonical binding.BMC Genomics. 2010; 11: 671Crossref PubMed Scopus (31) Google Scholar, 24Penkert R.R. DiVittorio H.M. Prehoda K.E. Internal recognition through PDZ domain plasticity in the Par-6-Pals1 complex.Nat. Struct. Mol. Biol. 2004; 11: 1122-1127Crossref PubMed Scopus (105) Google Scholar, 25Zhang Y. Appleton B.A. Wiesmann C. Lau T. Costa M. Hannoush R.N. Sidhu S.S. Inhibition of Wnt signaling by Dishevelled PDZ peptides.Nat. Chem. Biol. 2009; 5: 217-219Crossref PubMed Scopus (126) Google Scholar, 26Zhang Y. Appleton B.A. Wu P. Wiesmann C. Sidhu S.S. Structural and functional analysis of the ligand specificity of the HtrA2/Omi PDZ domain.Protein Sci. 2007; 16: 1738-1750Crossref PubMed Scopus (48) Google Scholar), to other PDZ domains (27van den Berk L.C. Landi E. Walma T. Vuister G.W. Dente L. Hendriks W.J. An allosteric intramolecular PDZ-PDZ interaction modulates PTP-BL PDZ2 binding specificity.Biochemistry. 2007; 46: 13629-13637Crossref PubMed Scopus (50) Google Scholar), and to lipids (28Chen Y. Sheng R. Kallberg M. Silkov A. Tun M.P. Bhardwaj N. Kurilova S. Hall R.A. Honig B. Lu H. Cho W. Genome-wide functional annotation of dual-specificity protein- and lipid-binding modules that regulate protein interactions.Mol. Cell. 2012; 46: 226-237Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 29Zimmermann P. Meerschaert K. Reekmans G. Leenaerts I. Small J.V. Vandekerckhove J. David G. Gettemans J. PIP(2)-PDZ domain binding controls the association of syntenin with the plasma membrane.Mol. Cell. 2002; 9: 1215-1225Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). We have decided to create a collection of human PDZ domains readily amenable to high-throughput studies of PDZ-mediated interactions. Cloned PDZ domains arranged as an array were used in a straightforward yeast two-hybrid (Y2H) screen to successfully identify known and new interactors for the E6 oncoprotein encoded by human papillomaviruses and for PDGFRβ, BRSK2, PCTK1, ACVR2B, and HER4 protein kinases. These interactions were confirmed by means of peptide pull-down coupled to mass spectrometry identification, demonstrating the strength and robustness of the Y2H approach. We also screened this Y2H array with the carboxy-terminal sequence of Vangl2, a planar cell polarity receptor, and confirmed some of the identified interactions via mass spectrometry. Finally, we reveal the novel role of one of the Vangl2 PDZ partners in cell migration in vitro and in morphogenetic movements in vivo. Domain boundaries were obtained by cross-searching Interpro (V18), PFAM (V23), and SMART version 5.0 (30Schultz J. Copley R.R. Doerks T. Ponting C.P. Bork P. SMART: a web-based tool for the study of genetically mobile domains.Nucleic Acids Res. 2000; 28: 231-234Crossref PubMed Scopus (1040) Google Scholar). Each domain was extended on each side with a 10-amino-acid tail from the original protein to account for variations in border prediction and to ensure the integrity of the structure of the domain. In some cases, the sizes of these tails had to be slightly modified according to the position of the PDZ in protein (extreme end or start) or to ensure correct amplification. Primers containing Gateway® (Invitrogen) B1 and B2 recombination tails were designed using the OSP program as described elsewhere (31Hillier L. Green P. OSP: a computer program for choosing PCR and DNA sequencing primers.PCR Methods Appl. 1991; 1: 124-128Crossref PubMed Scopus (121) Google Scholar), including a stop codon before the B2 tail (supplemental Table S1). DNA fragments encoding each PDZ domain were amplified via polymerase chain reaction (Platinum HIFI polymerase, Invitrogen) from human cDNA libraries and cloned into a pDONRZeo Entry vector using the Gateway® cloning system as described elsewhere (32Hartley J.L. Temple G.F. Brasch M.A. DNA cloning using in vitro site-specific recombination.Genome Res. 2000; 10: 1788-1795Crossref PubMed Scopus (783) Google Scholar, 33Walhout A.J. Temple G.F. Brasch M.A. Hartley J.L. Lorson M.A. van den Heuvel S. Vidal M. GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes.Methods Enzymol. 2000; 328: 575-592Crossref PubMed Google Scholar), creating a collection of PDZ Entry clones. These clones were sequence-verified using PDONRZeoF primer 5′-GCAATGTAACATCAGAGAT. A pipeline was set up to reassay unsuccessful amplifications using different combinations of cDNA libraries and to obtain wild-type clones from isolated transformed bacterial colonies. PDZ Entry clones were used in a Gateway® LR reaction to transfer the DNA coding for the PDZ domain into the Y2H AD expression vector pACT2. All PDZ domains were transformed into the haploid Y187 yeast strain (MATα, ura3–52, his3–200, ade2–101, leu2–3, 112, gal4Δ, met-, gal80Δ, MEL1, URA3::GAL1UAS -GAL1TATA-lacZ) using a standard procedure. Similarly, E6 and Vangl2 DNA fragments encoding the 15 last residues of the proteins were cloned into the Y2H DB expression vector pGBT9, and the resulting constructs were transformed into the haploid AH109 yeast strain (MATa, trp1–901, leu2–3, 112, ura3–52, his3–200, gal4Δ, gal80Δ, LYS2::GAL1UAS-GAL1TATA-HIS3,GAL2UAS-GAL2TATA-ADE2, URA3::MEL1UAS-MEL1TATA-lacZ, MEL1). Interactions between each PDZ and E6 or Vangl2 were tested through mating of the two yeast strains. Briefly, an overnight culture in a selective medium of the bait and the PDZs expressing yeasts (of the opposite mating type) were grown together in liquid Yeast extract-Peptone-Dextrose (YPAD) supplemented with 10% PEG for 4 h at 30 °C under gentle agitation. After one wash in water, the yeast was spotted on a solid selective medium Tryptophan-Histidine-Leucine (WHL) for phenotypic assay. T47D, HEK 293T, Caco-2, and mouse embryonic fibroblast (MEF) cells were grown in accordance with ATCC recommendations. T47D cells were transfected with pEGFP, pEGFP-Vangl2, or pEGFP-Vangl2 mutants using lipofectamine 2000 reagent according to the manufacturer's instructions (Invitrogen). MEF nucleofection was done according to the manufacturer's protocol (AMAXA, Lonza, Basel/CH). Goat anti-Scrib (C20) antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse anti-GFP and rat anti-HA 3F10 were acquired from Roche. Rat anti-Vangl2 clone 2G4 was made in-house (34Belotti E. Puvirajesinghe T.M. Audebert S. Baudelet E. Camoin L. Pierres M. Lasvaux L. Ferracci G. Montcouquiol M. Borg J.P. Molecular characterisation of endogenous Vangl2/Vangl1 heteromeric protein complexes.PLoS One. 2012; 7: e46213Crossref PubMed Scopus (36) Google Scholar). Rabbit anti-βPIX antibody was obtained from Chemicon International Millipore/St. Quentin, France. Mouse anti-tubulin was obtained from Sigma. SNX27 antibody was purchased from Abcam, Cambridge, UK. The cDNA of SNX27 was kindly provided by Dr. Philippe Marin (35Joubert L. Hanson B. Barthet G. Sebben M. Claeysen S. Hong W. Marin P. Dumuis A. Bockaert J. New sorting nexin (SNX27) and NHERF specifically interact with the 5-HT4a receptor splice variant: roles in receptor targeting.J. Cell Sci. 2004; 117: 5367-5379Crossref PubMed Scopus (125) Google Scholar). Secondary antibodies coupled to horseradish peroxydase were acquired from Dako, Courtaboeuf, France. In pull-down experiments and mass spectrometry, E6 (MSCCRSSRTRRETQL) and Vangl2 (KSHKFVMRLQSETSV) amino-terminal peptides were incubated and analyzed as described elsewhere (36Audebert S. Navarro C. Nourry C. Chasserot-Golaz S. Lecine P. Bellaiche Y. Dupont J.L. Premont R.T. Sempere C. Strub J.M. Van Dorsselaer A. Vitale N. Borg J.P. Mammalian Scribble forms a tight complex with the betaPIX exchange factor.Curr. Biol. 2004; 14: 987-995Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). For immunoprecipitation, after preclearing with agarose beads and incubation with antibodies, protein G-agarose beads were added to the lysates, and bound immune complexes were recovered, washed three times in lysis buffer, and separated on SDS-PAGE for Western blot analysis. Mass spectrometry analyses were performed using a MALDI-TOF Ultraflex instrument (Bruker Daltonics, Inc., Bremen, Germany) with reflector and positive modes, ion acceleration of 25 keV, a 5-Hz laser frequency, and a delay extraction of 110 ns. Six hundred shots were accumulated for each spectrum. Spectra were recalibrated using internal trypsin monoprotonated monoisotopic masses of 842.509, 1045.564, 2211.104, and 2283.180. Raw data were processed using Flex Analysis 3.0 and Biotool 3.1 software (Bruker Daltonics, Inc.). Protein searches were done using an in-house Mascot search engine (MatrixScience Ltd., London, UK) against the SwissProt database 2012_02 version, using "human" for taxonomy (20,247 sequences). Mascot search results from E6 and Vangl2 pull-down on Caco-2 and/or HEK 293T cell lysates are shown in separate folders. Each folder contains the whole set of protein bands that were analyzed and identified for a peptide pull-down. For each analyzed band, a Mascot search result folder containing a protein summary report, a protein view file, and the Mascot generic file is available in E6 and Vangl2_PullDown.7z files. Cells grown on coverslips coated with rat-tail collagen I (Roche) were stimulated with 1 nm heregulin (HRG), fixed in 4% paraformaldehyde in PBS for 20 min, permeabilized in 0.1% Triton X-100 for 5 min, and blocked with 10% fetal calf serum in PBS for 30 min before the addition of antibodies as described elsewhere (37Nola S. Sebbagh M. Marchetto S. Osmani N. Nourry C. Audebert S. Navarro C. Rachel R. Montcouquiol M. Sans N. Etienne-Manneville S. Borg J.P. Santoni M.J. Scrib regulates PAK activity during the cell migration process.Hum. Mol. Genet. 2008; 17: 3552-3565Crossref PubMed Scopus (80) Google Scholar). Images were acquired using a Zeiss Axiovert 200mot microscope linked to a Meta LSM 510 confocal module operated by LSM-FCS software (Carl Zeiss MicroImaging Inc., Jena, Germany) using a 63× oil-immersion objective (plan achromatic lens; numerical aperture = 1.3). Cell migration was evaluated using 8-μm pore polycarbonate membrane transwell chambers (Corning Costar, Amsterdam, NL). The bottom side of the membrane was coated with 25 μg/ml rat-tail collagen I for T47D and SUM149 cells, or 5 mg/ml human fibronectin for MEF cells. Cells were serum-starved for 16 h and then plated in the top chamber. Medium with or without 1 nm HRG was added to the bottom chamber, and cells were incubated for 12 h. Non-migrated cells were scraped from the top of the membrane. Migrated cells were fixed in 4% formaldehyde and stained with 0.1% crystal violet for counting. The interaction of E6, βPIX, or Vangl2 C-terminal peptides with PDZ domains was evaluated via homogenous time-resolved fluorescence assay (HTRF). To this end, we produced GST proteins fused to the indicated PDZ domains. Reaction mixtures consisted of GST-PDZ proteins at the indicated concentration in titration experiments or 2.5 × 10−9 m in IC50 experiments, an anti-GST-terbium antibody (1 × 10−9 m), streptavidin-d2 (4 × 10−8 m) (from Cisbio), a biotinylated C-terminal peptide (1.9 × 10−7 m), and the competing non-biotinylated homologous peptide at the indicated concentration in IC50 experiments. Upon excitation of the reaction mixture at 337 nm, a 615-nm fluorescence emission was produced by the donor terbium that in turn excited a 665-nm light emission by the acceptor streptavidin-d2 bound to the biotinylated peptide only if it resided in close vicinity of the donor. The long-lasting emission of the terbium cryptate allowed the fluorescence energy transfer to be measured at a time when all nonspecific light emission that followed the 337-nm illumination had faded. After the incubation (18 h, 4 °C), the intensity of light emission (A) at 615 and 665 nm was measured in a Polarscan Omega (BMG Labtech, Champigny, France) microplate reader equipped for HTRF. For each condition, the A665/A615 R ratio was obtained and a delta F value (DF) was computed as follows: [(RSample − RNSB)/RNSB] * 100, where RNSB is the light emission produced by the reaction mixture without GST-PDZ protein. IC50 values were determined by measuring the inhibition of the DF0 obtained with the biotinylated peptide by the homologous non-biotinylated peptide (DF) in dose-response and computing the obtained DF/DF0 using Prism software (log inhibitor) versus the response-variable slope (four parameter subprogram). Values with R2 better than 0.99 were considered. Each of the 96 Maxisorb wells (Nunc) was sequentially coated with biotinylated BSA (200 ng), streptavidin (100 ng), biotinylated E6 (2E-7M) in PBS, and finally PBS/BSA 1% w/v. The GST-PDZ domains or GST used as a negative control at the indicated concentration in PBS/BSA were incubated on the coats overnight (4 °C). After rinsing and further incubation for 3 h on ice with peroxidase-conjugated anti-GST antibody 1/5000 (Ab 3416, Abcam), anti-GST amounts bound to the coats were evaluated after rinsing with the tetramethylbenzidine chromogenic HRP substrate. Absorbance (optical density: 450 nm) was determined on an ELISA POLARstar reader. Eggs obtained from Xenopus laevis females (NASCO, Fort Atkinson, WI) were fertilized in vitro, dejellied, cultured, and injected as described elsewhere (38Marchal L. Luxardi G. Thome V. Kodjabachian L. BMP inhibition initiates neural induction via FGF signaling and Zic genes.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 17437-17442Crossref PubMed Scopus (95) Google Scholar). The morpholino-modified antisense oligonucleotide (MO) targeting SNX27 translation initiation (5′-TCCCCCTCCTCGTCCGCCATCTTTT-3′) was purchased from GeneTools LLC (Philomath, OR), resuspended in sterile water to a concentration of 10 mg/ml, and further diluted prior to injection. The MO targeting Xenopus Vangl2 was reported in Refs. 39Mitchell B. Stubbs J.L. Huisman F. Taborek P. Yu C. Kintner C. The PCP pathway instructs the planar orientation of ciliated cells in the Xenopus larval skin.Curr. Biol. 2009; 19: 924-929Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar and 40Heasman J. Kofron M. Wylie C. Beta-catenin signaling activity dissected in the early Xenopus embryo: a novel antisense approach.Dev. Biol. 2000; 222: 124-134Crossref PubMed Scopus (462) Google Scholar. The cDNA encoding Xenopus laevis SNX27 (Image clone 6945202) was purchased from GenomeCube® (Source BioScience, Nottingham, UK). Embryos were processed for whole-mount in situ hybridization with SNX27 and Sox2 digoxygenin-labeled probes (Roche) as described elsewhere (38Marchal L. Luxardi G. Thome V. Kodjabachian L. BMP inhibition initiates neural induction via FGF signaling and Zic genes.Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 17437-17442Crossref PubMed Scopus (95) Google Scholar). We have set up a rational approach to test the potential interactions of a given protein against an array of almost all human PDZ domains. This approach, based on a single Y2H assay, has been shown to be effective in probing the entire set of C. elegans PDZ domains for interacting proteins (23Lenfant N. Polanowska J. Bamps S. Omi S. Borg J.P. Reboul J. A genome-wide study of PDZ-domain interactions in C.
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