7 research outputs found
List of ICAT and β-catenin mutants created by site directed-mutagenesis.
<p>List of ICAT and β-catenin mutants created by site directed-mutagenesis.</p
Crystal structure of the β-catenin/ICAT complex.
<p><b>A</b> Crystal structure of ICAT bound to the core domain of β-catenin (PDB code 1LUJ,[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.ref022" target="_blank">22</a>]). ICAT is shown as yellow ribbons and β-catenin as purple cylinders. The secondary structures were calculated using the program STRIDE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.ref037" target="_blank">37</a>]. Residues mutated in this study are shown as hard spheres. ICAT residues are colored according to their characteristics: white for hydrophobic, green for polar, red for acidic and blue for basic residues. β-catenin F660 is in pink and the basic residues facing the C-terminal domain of ICAT are in cyan. <b>B</b>. Sequence alignment of the consensus peptide from several β-catenin binding proteins. The conserved acidic residues are in red and the aromatic residue in green. The first X residues, when they are hydrophilic, are boxed. <b>C</b>. β-catenin/ICAT complex showing the interaction between ICAT consensus peptide of the C-terminal domain (ribbon and sticks) and its facing β-catenin residues (surface). All residues are colored according to their characteristics. Figures were drawn using VMD software [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.ref038" target="_blank">38</a>].</p
ICAT negatively regulates the M-MITF promoter activity by competing with LEF1.
<p><b>A.</b> Mel501 cells were transfected with a <i>M-MITF</i>::<i>luciferase</i> vector in the presence of increasing amounts of <i>CMV</i>::<i>ICAT-WT</i> expression vector. Data are presented as means ± SEM of three independent experiments. <b>B.</b> qRT-PCR analysis of <i>M-MITF</i> mRNA levels in Mel501 cells transfected with empty or <i>ICAT-WT</i> expression vectors. <b>C.</b> WB analysis of MITF and ICAT proteins in Mel501 cells transfected with empty or <i>ICAT-WT</i> expression vectors. <b>β</b>-actin = loading control. <b>D.</b> WB analysis of MITF and p27<sup>Kip1</sup> protein levels in siRNA and ICAT-transfected Mel501 cells. SiMITF treatment and ICAT overexpression induce respectively a 42% and 35% increase of p27 protein amount; Scrb = control scrambled siRNA. <b>E</b>. Mel501 cells were transfected with <i>M-MITF</i>::<i>luciferase</i>, <i>LEF1</i> and <i>ICAT-WT</i> expression vectors. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p< 0.001; ****p<0.0001.</p
The lack of helix C in β-catenin does not prevent interaction with ICAT.
<p><b>A</b>. Schematic representation of WT and mutant Δ665 HA-tagged β-catenin-NLS proteins. <b>B</b>. Total cell lysates from Lu1205 cells transfected with WT or mutant β-catenin Δ665 were analyzed by WB or affinity immunoprecipitated with WT ICAT-GST recombinant protein and blotted with anti-HA and anti-ICAT antibodies. Numbers represent mean ± SD of normalized densitometry values from three independent experiments, *p<0.05.</p
Interactions between ICAT and β-catenin mutants, K312E, K435E and R386G in Lu1205 cell extracts: Consequences on <i>NEDD9</i> promoter activity.
<p><b>A</b>. Left: WB analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: Pull-down assay of HA-tagged WT and mutant β-catenin (K312E, K435E and R386G) by WT ICAT-GST recombinant protein. <b>B</b>. Left: WB analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: Pull-down assay of HA-tagged WT and mutant β-catenin (K312E and K435E) by LEF1-GST recombinant protein. <b>C</b>. Lu1205 cells were transiently transfected with <i>NEDD9</i>::<i>luciferase</i> and either <i>β-catenin-WT</i> or <i>β-catenin</i> mutants <i>(K312E</i>, <i>K435E and R386G)</i> expression vectors. <b>D</b>. Lu1205 cells were transiently transfected with <i>NEDD9</i>::<i>luciferase</i> vector in the presence of <i>CMV</i>::<i>LEF1</i>. Cells were also transfected with <i>β-catenin-WT</i> or <i>β-catenin</i> mutants <i>(K312E</i>, <i>K435E and R386G)</i> expression vectors. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.</p
The characteristics of the first X residue in the consensus peptide of several β-catenin binding proteins regulate their interactions with β-catenin.
<p><b>A</b>. Zoom is made on the first conserved Aspartate residue of the consensus peptide and its adjacent non conserved residues (shown as sticks) in ICAT, LEF1, TCF4, APC and E-cadherin (yellow ribbons) and the facing β-catenin Arm repeats 8 and 9 (purple cylinders). The first X residue of the consensus is encircled because residue numbering diverges between various β-catenin regulators, although they are facing the same β-catenin residues forming a basic patch. Hydrogen bonds between basic β-catenin residues and their counterpart in β-catenin regulators are presented as black dotted lines. In ICAT, V67 does not establish any hydrogen bond, whereas in LEF1 and TCF4/TCF7L2, E20 and E17, respectively make an H-bond with the facing β-catenin K508. In APC and E-cadherin, T1487 and S675 respectively form hydrogen bonds with the facing β-catenin R469. The color scheme of stick residues based on their characteristics is the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172603#pone.0172603.g001" target="_blank">Fig 1</a>. PDB codes: ICAT (1luj), LEF1 (3ouw), TCF3/TCF7L1 (1g3j), TCF4/TCF7L2 (1jdh), APC (1t08) and E-cadherin (1i7w).</p
β-catenin residue F660 is critical for ICAT anchoring to Arm repeat 12 but plays no role in the affinity for LEF1.
<p><b>A</b>. Left: Western blot (WB) analysis of lysates (Input) from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: pull-down assay of β-catenin WT and mutants F660S and F660A by WT ICAT-GST recombinant protein. <b>B</b>. Left: Input from Lu1205 cells transfected with WT or mutant HA-tagged β-catenin constructs; right: pull-down assay of β-catenin WT and mutants F660S and F660A by LEF1-GST recombinant protein. <b>C</b>. Lu1205 cells were transiently transfected with a <i>NEDD9</i>::<i>luciferase</i> vector. These cells were also transfected with either <i>NLS-</i>β<i>-catenin-WT</i> or <i>NLS-</i>β<i>-catenin-F660S</i> expression vectors in the absence (left) or presence (right) of exogenous LEF1. Data are presented as means ± SEM of three independent experiments. *p<0.05, **p<0.01, ***p<0.001, ns = not significant.</p