PKD1 conserves the epithelial phenotype in normal mammary gland cells.

<p><b>A:</b> NMuMG cells were either left untreated or were treated with TGFβ1 (10 ng/ml) for 48 hours. Cell morphology was photographed (bar is 200 µm) and cells were harvested and analyzed for expression of epithelial (E-cadherin, cytokeratin) and mesenchymal (N-cadherin) markers by Western blotting with anti-N-cadherin, anti-E-cadherin, or anti-cytokeratin antibodies. Staining for actin (anti-actin) served as a loading control. <b>B:</b> NMuMG cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Endogenous PKD1 was immunoprecipitated (anti-PKD1) and analyzed for phosphorylation at its activation loop that correlates with its activity (anti-pS738/742-PKD), or samples were control stained for total PKD1 (anti-PKD1). <b>C:</b> Cells were stimulated with PMA (100 nM, 10 min), EGF (50 ng/ml, 10 min), Bradykinin (0.5 µg/ml, 10 min) or left untreated. Endogenous PKD1 was immunoprecipitated and subjected to an <i>in vitro</i> kinase assay using PKD substrate peptide. PKD1 activity is depicted relative to PMA-activated PKD1 (maximum activation). Equal immunoprecipitation was controlled by SDS-PAGE and immunoblot (anti-PKD1). <b>D:</b> NMuMG cells were either transfected with control vector or with active PKD1 (PKD1.CA, PKD1.S738E.S742E). 24 hours after transfection, cells were treated with TGFβ1 (10 ng/ml) for 24 hours. Lysates were analyzed for expression of N-cadherin, E-cadherin, expression of PKD1, or actin as a loading control. <b>E:</b> NMuMG cells were stably-transfected with vector control, wildtype PKD1 or kinase-dead PKD1.K612W (PKD1.KD) Cell morphology was analyzed by brightfield microscopy (bar is 200 µm). Expression of endogenous and overexpressed PKD1 was determined by Western blot analysis using an anti-PKD1 antibody. Immunoblotting for actin (anti-actin) served as loading control.</p

PKD regulates E-cadherin expression in epithelial cells.

<p><b>A:</b> NMuMG cells were transfected with GFP-tagged, kinase-dead PKD1 (PKD1.KD) and endogenous expression of E-cadherin was determined with immunofluorescence staining (anti-E-cadherin). DAPI staining served as a nuclear marker (bar is 50 µm). <b>B:</b> MCF-7 cells were transfected with vector control, HA-tagged PKD1 or kinase-dead PKD1 (PKD1.KD). After 48 hours, samples were analyzed by Western blot for E-cadherin expression (anti-E-cadherin) as well as expression of PKD1 (anti-PKD1). Staining for actin (anti-actin) served as loading control. <b>C:</b> MCF-7 cells were transfected with vector control, HA-tagged constitutively-active PKD1 (PKD1.CA) or kinase-dead PKD1 (PKD1.KD) as well as E-cadherin promoter luciferase gene reporter and renilla luciferase reporter. Induced luciferase activity was measured. Error bars shown represent standard deviations. The asterisks indicate statistical significance (p<0.05) as compared to vector control.</p

Active PKD1 directly phosphorylates SNAI1 at S11.

<p><b>A:</b> The amino-acids surrounding serine 11 in SNAI1 form a PKD consensus motif as it was described for S82 of Hsp27 and S978 of SSH1L. <b>B:</b> PKD phosphorylates SNAI1 at S11 in an <i>in vitro</i> assay. Bacterially-expressed and purified GST (negative control), GST-SNAI1 or GST-SNAI1.S11A were incubated in a kinase reaction with purified active PKD1. Substrate phosphorylation was detected using the pMOTIF antibody, which recognizes the phosphorylated PKD motif in PKD substrates <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030459#pone.0030459-Doppler1" target="_blank">[45]</a> or with the novel pS11-SNAI1 antibody specifically generated for this site. Control blots were performed for protein input (anti-PKD1, anti-GST). <b>C, D:</b> HeLa cells were transfected with combinations of vector control, active PKD1 (PKD1.CA) and SNAI1 or SNAI1.S11A mutant as indicated. PKD-mediated phosphorylation of SNAI1 was detected using the pMOTIF (C) or the pS11-SNAI1 (D) antibodies. <b>E, F:</b> HeLa cells were transfected with combinations of vector control, active RhoA (RhoA.CA) and PKD1 or PKD1.KD mutant (E) or control shRNA and shRNA specific for PKD1/2 (F) as indicated and FLAG-tagged SNAI1. PKD-mediated phosphorylation of SNAI1 was detected using the pS11-SNAI1 antibody. Samples were also control-stained for SNAI1 and PKD1 expression using anti-FLAG or anti-PKD1 antibodies, respectively. Anti-GST control staining for RhoA.CA and GST control are depicted in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030459#pone.0030459.s002" target="_blank">Figure S2</a></b>. <b>G:</b> NMuMG cells were treated with TGFβ1 (10 ng/ml) for 48 hours. Total cell lysates were analyzed for phosphorylation of endogenous SNAI1 at S11 (anti-pS11-SNAI1) or PKD1 activity (anti-pS738/742-PKD) or total PKD1 expression (anti-PKD1) as indicated. <b>H:</b> NMuMG cells were treated with CID755673 (25 µM, 4 hr) or left untreated as indicated. Total cell lysates were analyzed for phosphorylation of endogenous SNAI1 at S11 (anti-pS11-SNAI1) or SNAI1 expression (anti-SNAI1).</p

Phosphorylation of SNAI1 by PKD1 occurs in the nucleus and does not alter its localization.

<p><b>A:</b> Immunofluorescence staining of NMuMG cells for endogenous PKD1 (anti-PKD1). The bar represents 10 µm. <b>B:</b> Immunofluorescence staining of NMuMG cells for S11-phosphorylated SNAI1 (anti-pS11-SNAI1) in absence or presence of competing phospho-S11-peptide and nuclei (DAPI). The bar represents 10 µm. <b>C:</b> HeLa cells were transfected as indicated and nuclear extracts were prepared and analyzed by Western blot for SNAI1 (anti-FLAG), pS11-SNAI1 (anti-pS11-SNAI1) and nucleolin (anti-nucleolin, loading control). <b>D:</b> NMuMG cells were transfected with GFP control, GFP-SNAI1, GFP-SNAI1.S11A or GFP-SNAI1.S11E mutants. Localization of GFP or GFP-tagged proteins was determined using immunofluorescence analysis (bar is 10 µm). <b>E:</b> NMuMG cells were transfected with FLAG-tagged wildtype SNAI1 or SNAI1.S11A mutant and GFP-tagged, active PKD1 (PKD1.CA) as indicated and localization of SNAI1 was determined by indirect immunofluorescence staining (anti-FLAG as primary antibody). The bar represents 10 µm.</p

Loss of nuclear PKD activity and SNAI1 phosphorylation at S11 are markers for invasive breast cancer.

<p>Tissue microarrays (TMAs) including 10 normal breast tissue samples, 40 invasive ductal carcinoma of the breast and 10 metastatic invasive ductal carcinoma samples from lymph nodes were H&E stained or analyzed for the expression of active PKD (anti-pY95-PKD), S11-phosphorylated SNAI1 (anti-pS11-SNAI1) and total SNAI1 (anti-SNAI1). Representative pictures of normal (<b>A–D</b>) and 3 tumor tissues (<b>E–P</b>) are depicted. Numbers indicate the position of the tissue on the TMA. The asterisk (sample #10) indicates tumor tissue form a region adjacent to the normal tissue (same patient). Inserts show enhanced area.</p

Phosphorylation of SNAI1 decreases its binding to Ajuba.

<p><b>A:</b> HeLa cells were co-transfected with MYC-tagged Ajuba and vector control, and FLAG-tagged wildtype SNAI1, SNAI1.S11A or SNAI1.S11E mutants as indicated. Ajuba was immunoprecipitated (anti-MYC) and precipitates were analyzed for co-precipitated SNAI1 (anti-FLAG). Samples were re-stained for Ajuba (anti-MYC) and lysates were control-stained for expressed SNAI1 (anti-FLAG). <b>B:</b> Proposed mechanism of how PKD1-mediated phosphorylation regulates SNAI1 function as a transcriptional repressor of E-cadherin gene expression.</p

PKD1-regulated SNAI1 binds to the E-cadherin promoter, but is ineffective in its function.

<p><b>A:</b> Hek293T cells were transfected with vector control, SNAI1 or active PKD1 (PKD1.CA) and SNAI1 as indicated. SNAI1/DNA complexes were immunoprecipitated (anti-FLAG) after crosslinking and precipitates were analyzed by PCR for the SNAI1-bound E-cadherin promoter. <b>B:</b> NMuMG cells were transfected with vector control, SNAI1, SNAI1.S11A or SNAI1.S11E mutants. SNAI1/DNA complexes were immunoprecipitated (anti-FLAG) after crosslinking and precipitates were analyzed by PCR for the SNAI1-bound E-cadherin promoter. <b>C:</b> NMuMG cells were treated with CID755673 (25 µM, 1 hr) or left untreated. Phospho-S11-SNAI1/DNA complexes were immunoprecipitated (anti-pS11-SNAI1) after crosslinking and precipitates were analyzed by PCR for the pS11-SNAI1-bound E-cadherin promoter. In experiments depicted in <b>A–C</b>, a PCR for the E-cadherin promoter using the input DNA as well as a ChIP using IgG instead of the anti-FLAG antibody served as controls. <b>D:</b> Hek293T cells were transfected with vector control, SNAI1 or SNAI1.S11A mutant, active PKD1 (PKD1.CA) or both and E-cadherin promoter luciferase reporter and renilla reporter plasmids. Induced luciferase activity was measured. Error bars shown represent standard deviations. <i>P</i> values were acquired with the <i>t</i> test, using GraphPad software. Asterisks indicate statistical significance.</p