title: Structure-based mutational analysis of ICAT residues mediating negative regulation of β-catenin co-transcriptional activity.
abstract: ICAT (Inhibitor of β-CAtenin and TCF) is a small acidic protein that negatively regulates β-catenin co-transcriptional activity by competing with TCF/LEF factors in their binding to β-catenin superhelical core. In melanoma cells, ICAT competes with LEF1 to negatively regulate the M-MITF and NEDD9 target genes. The structure of ICAT consists of two domains: the 3-helix bundle N-terminal domain binds to β-catenin Armadillo (Arm) repeats 10-12 and the C-terminal tail binds to Arm repeats 5-9. To elucidate the structural mechanisms governing ICAT/β-catenin interactions in melanoma cells, three ICAT residues Y15, K19 and V22 in the N-terminal domain, contacting hydrophobic β-catenin residue F660, were mutated and interaction was assessed by immunoprecipitation. Despite the moderate hydrophobicity of the contact, its removal completely abolished the interaction. In the ICAT C-terminal tail consensus sequence, neutralization of the electrostatic interactions between residues D66, E75 and β-catenin residues K435, K312, coupled to deletion of the hydrophobic contact between F71 and β-catenin R386, markedly reduced, but failed to abolish the ICAT-mediated negative regulation of M-MITF and NEDD9 promoters. We conclude that in melanoma cells, anchoring of ICAT N-terminal domain to β-catenin through the hook made by residue F660, trapped in the pincers formed by ICAT residues Y15 and V22, is crucial for stabilizing the ICAT/β-catenin complex. This is a prerequisite for binding of the consensus peptide to Arm repeats 5-9 and competition with LEF1. Differences between ICAT and LEF1 in their affinity for β-catenin may rely on the absence in ICAT of hydrophilic residues between D66 and F71.
authors.affiliation: Institut Curie, PSL Research University, Bâtiment, Orsay, France.|University Paris-Sud, University Paris-Saclay, Orsay, France.|Inserm U1021, Normal and Pathological Development of Melanocytes, Orsay, France.|CNRS UMR 3347, Orsay, France.|Equipe Labellisée Ligue Contre le Cancer, Orsay, France.
authors.forename: Mélanie J
authors.initials: MJ
authors.lastname: Domingues
authors.affiliation: Institut Curie, PSL Research University, Bâtiment, Orsay, France.|University Paris-Sud, University Paris-Saclay, Orsay, France.|Inserm, U1196, Chemistry, Modelling and Imaging for Biology, Orsay, France.|CNRS, UMR 9187, Orsay, France.
authors.forename: Juan
authors.initials: J
authors.lastname: Martinez-Sanz
authors.affiliation: Institut Curie, PSL Research University, Bâtiment, Orsay, France.|University Paris-Sud, University Paris-Saclay, Orsay, France.|Inserm U1021, Normal and Pathological Development of Melanocytes, Orsay, France.|CNRS UMR 3347, Orsay, France.|Equipe Labellisée Ligue Contre le Cancer, Orsay, France.
authors.forename: Laura
authors.initials: L
authors.lastname: Papon
authors.affiliation: Institut Curie, PSL Research University, Bâtiment, Orsay, France.|University Paris-Sud, University Paris-Saclay, Orsay, France.|Inserm U1021, Normal and Pathological Development of Melanocytes, Orsay, France.|CNRS UMR 3347, Orsay, France.|Equipe Labellisée Ligue Contre le Cancer, Orsay, France.
authors.forename: Lionel
authors.initials: L
authors.lastname: Larue
authors.affiliation: Institut Curie, PSL Research University, Bâtiment, Orsay, France.|University Paris-Sud, University Paris-Saclay, Orsay, France.|Inserm, U1196, Chemistry, Modelling and Imaging for Biology, Orsay, France.|CNRS, UMR 9187, Orsay, France.
authors.forename: Liliane
authors.initials: L
authors.lastname: Mouawad
authors.affiliation: Institut Curie, PSL Research University, Bâtiment, Orsay, France.|University Paris-Sud, University Paris-Saclay, Orsay, France.|Inserm U1021, Normal and Pathological Development of Melanocytes, Orsay, France.|CNRS UMR 3347, Orsay, France.|Equipe Labellisée Ligue Contre le Cancer, Orsay, France.
authors.forename: Jacky
authors.identifier: http://orcid.org/0000-0002-1060-2078
authors.initials: J
authors.lastname: Bonaventure
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D001667:Binding, Competitive; D045744:Cell Line, Tumor; D005786:Gene Expression Regulation; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000378:metabolism; D008544:Melanocytes / Q000378:metabolism; D051739:Microphthalmia-Associated Transcription Factor / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D054730:Protein Interaction Domains and Motifs; D015533:Transcriptional Activation; D051176:beta Catenin / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C411982:CTNNBIP1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D051821:Lymphoid Enhancer-Binding Factor 1; D051739:Microphthalmia-Associated Transcription Factor; D051176:beta Catenin