Structure-based mutational analysis of ICAT residues mediating negative regulation of β-catenin co-transcriptional activity

• Published in: PloS one 2017, Vol.12 (3), p.e0172603-e0172603
• Main Authors: Domingues, Mélanie J, Martinez-Sanz, Juan, Papon, Laura, Larue, Lionel, Mouawad, Liliane, Bonaventure, Jacky
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Anchoring; beta Catenin - metabolism; Binding; Binding, Competitive; Biology and Life Sciences; Cancer; Cell adhesion & migration; Cell growth; Cell Line, Tumor; Clonal deletion; Conserved sequence; Crystal structure; Down-regulation; Electrostatic properties; Gene Expression Regulation; Gene regulation; Genes; Humans; Hydrophobicity; Immunoprecipitation; Intracellular Signaling Peptides and Proteins - chemistry; Intracellular Signaling Peptides and Proteins - genetics; Intracellular Signaling Peptides and Proteins - metabolism; LEF/TCF protein; Life Sciences; Ligands; Lymphoid Enhancer-Binding Factor 1 - metabolism; Medicine and Health Sciences; Melanocytes - metabolism; Melanoma; Metastasis; Microphthalmia-associated transcription factor; Microphthalmia-Associated Transcription Factor - genetics; Microphthalmia-Associated Transcription Factor - metabolism; Models, Molecular; Mutation; Neutralization; Physical Sciences; Protein Binding; Protein Conformation; Protein Interaction Domains and Motifs; Proteins; Research and Analysis Methods; Residues; Superhelical DNA; Transcription; Transcription factors; Transcriptional Activation; β-Catenin
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0172603

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.