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First published online 27 January 2009
doi: 10.1242/jcs.035469

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Functional suppression of E-cadherin by protein kinase C

Department of Life Science and Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan

View larger version (57K): [in this window] [in a new window]   Fig. 1. Expression of PKC, but not PKC, decreases the immunofluorescence intensity of E-cadherins at cell-cell junctions of MDCK cells using the ECCD-2 anti-E-cadherin antibody. (A) GFP-PKC and GFP-PKC were stably expressed in MDCK cells and their expression levels were analyzed by immunoblotting with anti-GFP. (B) Parental MDCK cells (control) and those stably expressing GFP-PKC or GFP-PKC were grown as colonies. The micrographs were taken under a phase-contrast microscope. Scale bar: 40 µm. Note that the cells stably expressing GFP-PKC or GFP-PKC retain their ability to form colonies within which the cells are in contact with their neighbors. (C) MDCK cells stably expressing GFP-PKC were co-cultured with parental MDCK cells (control) and then grown to confluence. The cells were fixed and stained for adherens junctions, tight junctions and nuclei with anti-E-cadherin (clone ECCD-2), anti-ZO-1 and DAPI, respectively. (D) MDCK cells stably expressing GFP-PKC were co-cultured with parental MDCK cells (control) and then grown to confluence. The cells were fixed and stained as described in C. Dashed lines indicate the boundary between control cells and the cells expressing GFP-PKC. Scale bars: 25 µm.

View larger version (71K): [in this window] [in a new window]   Fig. 2. The ECCD-2 anti-E-cadherin antibody preferentially recognizes `active' E-cadherin. (A) MDCK cells stably expressing GFP-PKC were co-cultured with parental MDCK cells (control) and then grown to confluence. The cells were fixed and stained for E-cadherin with a rat monoclonal antibody (clone ECCD-2) and a mouse monoclonal antibody (clone 36). Scale bar: 25 µm. Note that the ECCD-2 antibody recognizes the extracellular domain of E-cadherin and the clone 36 antibody recognizes the intracellular domain of E-cadherin. (B) Detection of E-cadherin in MDCK cells by immunofluorescence staining with anti-E-cadherin antibodies under three conditions. The cells were grown to confluence (control) or cells were grown to confluence and then treated with 5 mM EGTA in serum-free medium for 30 minutes (Ca2+ depletion). After EGTA treatment, the medium was then replaced with fresh growth medium for 2 hours (Ca2+ repletion). Scale bars: 25 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 3. Expression of PKC weakens cell-cell adhesions. (A) Cell-dissociation assay. MDCK cells stably expressing GFP or GFP-PKC were allowed to grow as discrete colonies. The cell colonies were scraped in culture medium and suspended by repeated pipetting. Representative images at x100 magnification before (–) and after (+) pipetting are shown. Cell-dissociation index is expressed as the percentage of particles (cell clusters 3 cells) in total number of cells per dish. Values (means ± s.d.) are from three independent experiments. (B) Cell-aggregation assay. MDCK cells stably expressing GFP or GFP-PKC were grown to semi-confluence, trypsinized and suspended in culture medium. After 72 hours of constant shaking, cell aggregates (10 cells) were enumerated under a phase-contract microscope at x100 magnification. Values (means ± s.d.) are from three independent experiments (n=50). Representative images at 0 hours and after 72 hours of shaking are shown.

View larger version (48K): [in this window] [in a new window]   Fig. 4. The catalytic activity of PKC is required for suppression of the homophilic interactions between E-cadherins. (A) MDCK cells stably expressing GFP, GFP-PKC or its mutants (KD and RD) were grown to confluence and stained for E-cadherin (with the ECCD-2 antibody), ZO-1, β-catenin and actin filaments. The kinase-deficient (KD) mutant was generated by substitution of Lys376 with Arg. The RD mutant represents the regulatory domain (a.a. 1-298) of PKC. Arrowheads indicate the presence of GFP-tagged proteins at cell-cell junctions. Scale bars: 10 µm. The ratio of the fluorescence intensity of E-cadherin at cell-cell junctions (red boxed region) versus adjacent cytoplasm (yellow boxed region) was measured (n=50) (F.I., fluorescence intensity; AJ, adherens junctions; Cyto., cytoplasm). Note that PKC-mediated suppression of adherens junctions is accompanied by fewer organized β-catenin and F-actin at cell-cell junctions. (B) MDCK cells stably expressing the GFP-PKC KD mutant were stained with anti-E-cadherin and anti-ZO-1. White lines on confocal X-Y section images represent regions where confocal X-Z section images were taken. The X-Z images reveal that GFP-PKC colocalizes with E-cadherin, but not ZO-1, at cell-cell junctions. Arrowheads indicate the colocalization of GFP-PKC and E-cadherin at cell junctions. Scale bars: 5 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 5. The hinge region of PKC is required for its catalytic activity. (A) The diagram depicts the domain structure of GFP-PKC and its mutants used in this study. The mutants with deletion of the C2 domain (C2; a.a. 1-123), C1A domain (C1A; a.a. 159-208), C1B domain (C1B; a.a. 232-280) or hinge region (H; a.a. 280-347) were generated and stably expressed in MDCK cells. KD, kinase-deficient mutant; RD, regulatory domain. (B) GFP-PKC and its mutants were immunoprecipitated by anti-GFP antibody. The immunocomplexes were subjected to immunoblotting with anti-GFP antibody or an in vitro kinase assay using MBP as a substrate. The 32P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The phosphorylation of MBP was measured and expressed as a percentage relative to the level induced by GFP-PKC wild type, which is defined as 100%. Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay.

View larger version (79K): [in this window] [in a new window]   Fig. 6. The hinge region of PKC is required for the targeting of cell-cell junctions. MDCK cells stably expressing the GFP-PKC mutants (C1A, C1B, C2 and H) were grown to confluence and then stained for E-cadherin (with the ECCD-2 antibody), ZO-1, β-catenin and F-actin. Arrowheads indicate the presence of the GFP-tagged proteins at cell-cell junctions. Boxed regions represent the areas selected for measurement of the ratio of the fluorescence intensity of E-cadherin at cell-cell junctions versus adjacent cytoplasm. Scale bars: 10 µm. The ratio of the fluorescence intensity of E-cadherin at cell-cell junctions versus adjacent cytoplasm was measured (n=50) (F.I., fluorescence intensity; AJ, adherens junctions. Cyto., cytoplasm). Values are mean ± s.d. Note that E-cadherin suppression by the C1A, C1B or C2 mutant is associated with fewer organized β-catenin and actin filaments at cell-cell junctions.

View larger version (49K): [in this window] [in a new window]   Fig. 7. Localization of PKC to cell-cell junctions is essential for adherens junction suppression. (A) The diagram depicts the domain structures of GFP-PKC mutants (H, H280-323 and H324-347) with deletion in the hinge region. (B) GFP-PKC or its mutants were stably expressed in MDCK cells and their kinase activities were analyzed by an in vitro kinase assay using MBP as a substrate. The 32P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The phosphorylation of MBP was measured and expressed as a percentage of the level induced by GFP-PKC wild-type, which is defined as 100%. Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay. (C) MDCK cells stably expressing GFP-PKC or its mutants (H, H280-323 and H324-347) were grown to confluence. The cells were stained for E-cadherin and nuclei. Arrowheads indicate the presence of the GFP-tagged proteins at cell-cell contacts. Scale bar: 10 µm. The ratio of the fluorescence intensity of GFP-tagged proteins at cell-cell junctions (red boxed region) versus adjacent cytoplasm (yellow boxed region) was measured (n=50) and is shown below the micrographs (F.I., fluorescence intensity; AJ, adherens junctions; Cyto., cytoplasm). (D) MDCK cells stably expressing GFP-PKC or its mutants, C1A, C1B and H280-323, were sparsely cultured, treated with 100 nM PMA for 20 minutes, and stained with DAPI. Arrowheads indicate the presence of GFP-tagged proteins at the plasma membrane. Scale bar: 10 µm. The ratio of the fluorescence intensity of GFP-tagged proteins at the plasma membrane (red boxed region) versus adjacent cytoplasm (yellow boxed region) was measured (n=50) and is shown beneath the micrographs. Note that GFP-PKC and H280-323, but not the C1A or C1B mutants, translocate to the plasma membrane upon PMA treatment.

View larger version (61K): [in this window] [in a new window]   Fig. 8. Overexpression of GFP-PKC expedites HGF-induced scatter of MDCK cells. (A) MDCK cells stably expressing GFP, GFP-PKC wild-type, the KD mutant or the H280-323 mutant were allowed to grow as colonies and then treated with 20 ng/ml HGF to induce cell scattering. Time-lapse micrographs were taken by cooled CCD under a differential interference contrast microscope. Representative images at the indicated time points are shown. Scale bar: 20 µm. (B) At various lengths of time after HGF stimulation, the percentage of scattered colonies in all counted colonies was determined. A colony is judged as `scattered' when the half of the cells have lost contact with their neighbors and exhibit a fibroblast-like phenotype. Values (means ± s.d.) are from three independent experiments (n=100).

View larger version (48K): [in this window] [in a new window]   Fig. 9. siRNA-mediated knockdown of endogenous PKC in MDCK cells delays the disruption of cell-cell adhesions induced by HGF. (A) The MDCK cells that stably express PKC-specific siRNA upon addition of doxycycline (Dox) were incubated with (+) or without (–) doxycycline (Dox) for 72 hours. The expression level of PKC was analyzed by immunoblotting with anti-PKC. (B) 72 hours after induction of PKC-specific siRNA, the cells were treated with 20 ng/ml HGF. The time-lapse micrographs were taken by cooled CCD under a differential interference contrast microscope. Representative images at the indicated time points are shown. Scale bar: 20 µm. (C) Quantitative result of HGF-induced cell scattering. At various lengths of time after HGF stimulation, the percentage of scattered colonies in all counted colonies was determined. Values (means ± s.d.) are from three independent experiments (n=100). (D) 72 hours after induction of PKC-specific siRNA, the cells were treated with 20 ng/ml HGF and then stained for E-cadherin with the ECCD-2 antibody at the indicated time points. White lines represent the regions of interest used to measure fluorescence intensity. (E) 2 hours and 4 hours after HGF treatment, the profiles of the E-cadherin fluorescence intensity along the white lines drawn in D were shown in line graphs (blue; without PKC siRNA induction; red, with PKC siRNA induction; a.u., arbitrary units). The line-scan measurements of the fluorescence intensity were recorded by Zeiss LSM imaging software.

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