Co-immunoprecipitation of SHP2 and CDCP1.

<p><b>A.</b> PC3 cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO₃). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 control antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. <b>B and C.</b> HCT 116 cells were transfected with an empty vector or wild-type SHP2 (SHP2-WT-HA) or dominant-negative SHP2 mutant (SHP2-C459S-HA) expression constructs, as indicated. Forty-eight hours after transfection, the cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO₃). The cells were lysed and the lysates were subjected to immunoprecipitation (IP) with anti-CDCP1 (A) or anti-HA (B) antibodies, as indicated. Total cell lysates and immunoprecipitates were analyzed by western blotting with the antibodies indicated. The position of the exogenously expressed SHP2 constructs that migrated more slowly, due to a fused HA-Tag, are indicated by an arrowhead. The immunoglobulin heavy chains (IgH) are indicated by asterisks (*). The results shown are representative of at least four independent experiments.</p

SHP2 regulates the phosphorylation and internalization of CDCP1.

<p><b>A.</b> HeLa cells stably transfected with an empty vector or with a WT-CDCP1 construct were stably transfected with a SHP2-targeting shRNA (D1 or D2), as indicated. Total cell lysates were prepared and analyzed by western blotting with the antibodies indicated. <b>B.</b> Stable HeLa-CDCP1 and HeLa-CDCP1-shSHP2 D1 cell lines (described above and in the experimental procedures) were first incubated with an anti-CDCP1 antibody at 4°C. The cells were washed and incubated at 37°C for the times indicated, to allow internalization of the CDCP1-antibody complexes. The cells were then incubated again at 4°C with the appropriate secondary antibody, and the amount of CDCP1 remaining at the cell surface was analyzed by flow cytometry. The results are indicated as a percentage of membrane CDCP1 ± SEM for three independent experiments. ns: p > 0.05 *: p = 0.03; ****: p = 10^–4 in non-parametric Student's <i>t</i> tests. The data shown are representative of at least three independent experiments performed in triplicate.</p

Phosphorylated CDCP1 is efficiently pulled down by recombinant SHP2 substrate trapping mutant or SHP2-SH2 domains.

<p>HeLa cells stably expressing CDCP1 were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO₃), as indicated. Cell lysates were subjected to GST-pull down assays with 5 μg of GST protein alone (GST only), GST fused to a SHP2 substrate trapping mutant (GST-DACS) or GST fused to the SHP2-SH2 domains (GST-SH2), as mentioned in A and B. The affinity-purified complexes were resolved by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies. In some conditions, particularly in HeLa cells, CDCP1 was detected as two species, the more slowly migrating species being tyrosine-phosphorylated (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123472#pone.0123472.g004" target="_blank">Fig 4A and 4B</a>, compare lower and middle panels, the arrowhead indicates the slower migrating species). The data shown are representative of more than eight independent experiments.</p

Primary sequence of intracellular CDCP1.

<p><b>A.</b> Amino acids are numbered from the first intracellular residue. Phosphorylatable tyrosine residues are shown in bold typeface and are numbered. The ITAM-like motif is shown in a box. <b>B.</b> Alignment of the ITAM-like motif of CDCP1 with the consensus sequence of an ITAM motif. Phosphorylatable residues are shown in bold typeface and are numbered according to the corresponding CDCP1 sequence.</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^Kip1 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

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

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