4 research outputs found
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 (PerVO<sub>3</sub>), 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
Co-immunoprecipitation of SHP2 and CDCP1.
<p><b>A.</b> PC3 cells were left untreated or were treated for 15 minutes with 25μM pervanadate (PerVO<sub>3</sub>). 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 (PerVO<sub>3</sub>). 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<sup>–4</sup> in non-parametric Student's <i>t</i> tests. The data shown are representative of at least three independent experiments performed in triplicate.</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