Plakophilin 2: a critical scaffold for PKCα that regulates intercellular junction assembly

Plakophilins (PKPs) are armadillo family members related to the classical cadherin-associated protein p120ctn. PKPs localize to the cytoplasmic plaque of intercellular junctions and participate in linking the intermediate filament (IF)-binding protein desmoplakin (DP) to desmosomal cadherins. In response to cell–cell contact, PKP2 associates with DP in plaque precursors that form in the cytoplasm and translocate to nascent desmosomes. Here, we provide evidence that PKP2 governs DP assembly dynamics by scaffolding a DP–PKP2–protein kinase Cα (PKCα) complex, which is disrupted by PKP2 knockdown. The behavior of a phosphorylation-deficient DP mutant that associates more tightly with IF is mimicked by PKP2 and PKCα knockdown and PKC pharmacological inhibition, all of which impair junction assembly. PKP2 knockdown is accompanied by increased phosphorylation of PKC substrates, raising the possibility that global alterations in PKC signaling may contribute to pathogenesis of congenital defects caused by PKP2 deficiency

(A, left) PKC inhibition impairs DP border localization and induces a filamentous pattern

SCC9 cells treated with 12.5 μM BIM or DMSO for 30 min and immunostained for endogenous DP. (A, right) BIM-treated A431 DP-GFP cells. (B) PKA inhibition does not affect DP border localization. SCC9 cells in low calcium treated with 10 μM H-89 or DMSO and switched to high calcium for 3 h, immunostained for endogenous DP. Quantitative analysis of border fluorescence intensity confirmed that DP localization was comparable in H-89–treated cells. (C) Mutation of Ser2849 results in IF alignment (). Fixed A431 cells expressing DP-GFP (a′) or DP-GFP (b′) for comparison with D. (D, a′) Selected stills from Video 4 (available at ) of A431 DP-GFP cells treated with 12.5 μM BIM. The drug was added at beginning of video and maintained throughout the 90-min time course. (D, b′) Video 5 of untreated A431 DP-GFP. Arrows indicate areas where new cell–cell contacts occur. Note the variability in the DP-GFP pattern ranging from particulate to continuous filaments (compare with C). (E) Impaired DP border localization during PKCα knockdown. Calcium switch of SCC9 cells infected with PKCα small hairpin RNA retrovirus or empty virus immunostained for DP. DP border fluorescence was measured, normalized to background, and plotted on the right. Immunoblot analysis of PKC-deficient cells for DP, PKP2, DSG2, PKCα, or GAPDH loading control. Error bars represent SEM. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Plakophilin 2: a critical scaffold for PKCα that regulates intercellular junction assembly"</p><p></p><p>The Journal of Cell Biology 2008;181(4):605-613.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386101.</p><p></p

(A) PKP2 reexpression enhances DP border localization during PKP2 knockdown

Silencing-resistant FLAG-tagged PKP2 was coexpressed with PKP2 or nontargeting (NT) siRNA in SCC9 cells. Cells were stained for DP (right), FLAG, and PKP2 (using the same secondary antibody; left). Intensity of DP border fluorescence at borders between pairs of transfected cells or pairs of untransfected cells is shown on the far right. *, P < 0.001. (B) PKC activation rescues DP border localization during PKP2 knockdown. PKP2 siRNA or NT siRNA cells were treated with 15 nM PMA for 30 min and stained for DP. DP border fluorescence quantitation is shown on the bottom. **, P < 0.001. (C) PKC expression rescues DP border localization during PKP2 knockdown. SCC9 cells transfected with PKP2 or NT siRNA were infected with wild-type PKCα or constitutive active PKCα retrovirus and stained for PKP2 and DP. (C, top) Overexpression of PKCα rescues DP border localization. (C, bottom) DP localization at cell–cell borders was assessed for robustness by taking into account the percentage of occupied border, border continuity, and fluorescence intensity. Borders were scored on a graded scale of 1–5 and plotted as percentages of total borders counted. (C, bottom right) Representative borders scored from 1 to 5. 5 represents the most mature border, with most continuous and intense DP fluorescence, and 1 represents the least mature border, with minimal to no DP fluorescence. Graphs represent mean values from three independent experiments. NT and PKP2 siRNA experiments were performed at same time but separated in the graphs for clarity. Error bars represent SEM. Bars, 10 μm.<p><b>Copyright information:</b></p><p>Taken from "Plakophilin 2: a critical scaffold for PKCα that regulates intercellular junction assembly"</p><p></p><p>The Journal of Cell Biology 2008;181(4):605-613.</p><p>Published online 19 May 2008</p><p>PMCID:PMC2386101.</p><p></p