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

Abstract

Plakophilins (PKPs) are armadillo family members related to the classical cadherin-associated protein p120(ctn). 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 C alpha (PKC alpha) 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 PKC alpha 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.

Figure 1.

PKP2 is required for proper assembly of DP into desmosomes. (A and B) Impaired DP border localization in PKP2-deficient cultures. SCC9 cells (A, wide-field microscopy) or A431 cells (B, confocal microscopy) expressing pooled siRNAs against human PKP2 or nontargeting (NT) control, immunostained for endogenous DP and PKP2. Immunoblot analysis of SCC9 (A, right) or A431 (B, right) from NT or PKP2 siRNA-transfected cells to detect total levels of PKP2, PKP1, PKP3, DP, DSC2, DSG2, DSG3, and α-tubulin. PG protein levels were unaffected (not depicted). Arrows indicate the cell–cell border. (C) DP particles colocalize with keratin IF during PKP2 knockdown. Confocal images of SCC9 cells expressing PKP2 or NT siRNA immunostained for DP (green) and keratin (red). Panels b′ and d′ show magnified views of the boxed areas in a′ and c′, respectively. (D) DP assembly is impaired during PKP2 knockdown. Calcium switch of SCC9 cells transfected with siRNA against PKP2 or NT + siGlo, endogenous PKP2, or DP staining. DP border fluorescence intensity was measured, normalized to background, and plotted in D (right). Arrows indicate siGLO particles. Error bars represent SEM. (E) DP accumulates in a filamentous cytoplasmic pattern during PKP2 knockdown. Shown are stills from Videos 1 and 2 (available at http://www.jcb.org/cgi/content/full/jcb.200712133/DC1) depicting PKP2 or NT siRNA-transfected A431 DP-GFP cells. Arrows indicate where new borders are being formed. Red arrowheads show DP accumulation on IF. Time points indicate time lapsed from beginning of video. Bars, 10 μm.

Figure 2.

DPser2849 and PKC activity are required for proper DP border localization. (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 (Godsel et al., 2005). Fixed A431 cells expressing DP-GFP (a′) or DP^S2849G-GFP (b′) for comparison with D. (D, a′) Selected stills from Video 4 (available at http://www.jcb.org/cgi/content/full/jcb.200712133/DC1) 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^S2849G-GFP. Arrows indicate areas where new cell–cell contacts occur. Note the variability in the DP^S2849G-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.

Figure 3.

PKP2 regulates PKC signaling. (A) Enhanced PKC substrate phosphorylation during PKP2 knockdown. SCC9 cell lysates from nontargeting (NT) or PKP2 siRNA-transfected cells or cells treated with DMSO, 15 nM PMA, or 12.5 μM BIM for 30 min were probed for PKC substrates phospho-MARCKS or -adducin, total MARCKS, adducin, PKP2, or α-tubulin (loading control). Densitometry numbers below the blots represent ratios of phosphoprotein normalized to total protein from treatment samples relative to the NT siRNA sample. (B) PKCα coimmunoprecipitates the PKP2 N-terminal head but not the central armadillo repeat domain. FLAG-tagged PKCα and myc-tagged PKP2-Head or -Arm were cotransfected into HEK293 cells and subjected to anti-FLAG IP. Blots were probed with anti-myc or anti-FLAG antibodies. (bottom) A schematic of PKP2 constructs. 5% input blots represent 5% of triton lysate from which IP was performed, removed before IP for immunoblot analysis. (C) PKCα coimmunoprecipitates endogenous DP in HEK293 cells. FLAG-tagged wild-type PKCα or myristylated (constitutively active) PKCα were transfected into HEK293 cells and subjected to FLAG IP. Precipitates and lysates were probed for endogenous DP or FLAG. Data are representative of three independent experiments. (D) PKP2 is required for PKC–DP interaction. PKCα-FLAG was immunoprecipitated from HEK293 cells transfected with PKP2 or NT siRNA. Endogenous DP, PKP2, and FLAG were probed. Data are representative of three independent experiments. Densitometry (right) reveals 95% reduction in DP coimmunoprecipitated with PKCα.

Figure 4.

PKP2, PMA, or PKCα restores DP incorporation into desmosomes. (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.

Figure 5.

Model for role of PKP2 in PKC-regulated DP assembly. (A) Wild type. PKP2 recruits PKC to DP cytoplasmic complexes, where phosphorylation of DP at Ser2849 modulates its interaction with IF and allows DP assembly into cell–cell junctions. (B) PKP2 deficiency. PKC is no longer recruited to DP complexes and is free to phosphorylate other substrates, leading to aberrant accumulation of DP along IF and impaired DP assembly.