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First published online 12 February 2008
doi: 10.1242/jcs.016246

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Atypical protein kinase C (iota) activates ezrin in the apical domain of intestinal epithelial cells

¹ Department of Cell Biology and Anatomy
² Department of Pediatrics, University of Miami Miller School of Medicine, RMSB 4090–R124, 1600 NW 10th Avenue, Miami, FL 33135, USA

View larger version (130K): [in this window] [in a new window]   Fig. 1. Expression of active (pT555) PKC in the crypt-villus axis of the small intestine is synchronous with ezrin T567 phosphorylation. Frozen sections of mouse jejunum were stained with anti-T555-P (active) PKC antibody (A, green) and anti-T567-P ezrin antibody (C, red), or with the corresponding nonimmune IgGs (control, B, D). DNA was counterstained with DAPI (blue). (G, H) Phase-contrast images of the same sections overimposed with DAPI fluorescence. E, E', F show merged signals. E' is the field boxed in E showed at higher magnification. The images are shown with the bottom of the crypts to the left. Arrow in C, single isolated positive apical domain within a region of negative T567-P signal. Arrowheads show the lowest limit (earliest) of T567-P expression in the crypt. E' inset: higher magnification of the area blocked in E. Scale bar: 20 µm (A-H); 10 µm (E').

View larger version (43K): [in this window] [in a new window]   Fig. 2. PKC phosphorylates purified ezrin in T567 in vitro and in Sf9 insect cells. (A) 6xHis-tagged h-ezrin expressed in Sf9 cells and purified by Ni2+ columns was incubated in the absence (–) or presence of recombinant active PKC (+) (2 µg/ml) and [-32P]ATP (5 mCi/ml, 3000 Ci/mmol). After SDS-PAGE and blotting, the membrane was exposed to X-ray film for 4 hours. After autoradiography, the same membrane was subsequently probed with anti-ezrin antibody and chemiluminescence (ezrin, 5 second exposure). (B) An identical experiment was performed in the presence of 1 mM cold ATP. The immunoblot was performed sequentially on the same membranes using anti-p-T567 ezrin, then stripped, and reprobed with monoclonal anti-ezrin. (C) As in B, identical amounts of purified 6xHis-tagged ezrin purified on Ni2+ columns were phosphorylated (+) or not (–) with PKC and cold ATP for 30 minutes at 30°C. The reaction was stopped by dilution and the ezrin solutions were then incubated with Sepharose beads covalently bound to phalloidin-stabilized F-actin for 4 hours. After centrifugation, the supernatants were acetone precipitated and analyzed by immunoblot with anti-ezrin antibody (sup.). The beads were extensively washed, eluted in sample buffer and the eluates analyzed by immunoblot sequentially with anti-ezrin antibody and then with anti-actin antibody on the same membranes. Positions of molecular mass standards are indicated on left: 201, 133 and 85 kDa (top to bottom). (D) Sf9 cells were infected with baculovirus expressing h-ezrin, h-PKC, or double infected with both as indicated at the left. The efficiency of infection was approximately 90% for both viruses. After 48 hours the cells were fixed and labeled for PKC (blue channel), F-actin (phalloidin, green channel) and h-ezrin (red channel). The images are X-Y confocal sections at the transnuclear level. Arrows show cells with ezrin and F-actin recruited to the cell surface. (E) Scanning electron microscopy of Sf9 cells transduced with ezrin, or PKC-expressing baculovirus, or both. (F) Extracts from parallel Sf9 cultures of cells transduced with ezrin, or PKC-expressing baculovirus, or both were purified through a Ni2+ column, the eluates were separated in SDS-PAGE, blotted and probed sequentially with antibodies against ezrin or T567-P ezrin. (G) Genomic DNA from noninfected, singly infected or doubly infected Sf9 cells was separated in an agarose gel and compared with a ladder of 1.6 kb to 12 kb DNA standards (left lane). A positive control for DNA laddering typical of apoptosis was performed by incubating the cells in 10 mM H2O2 overnight. (H) Single and double infected Sf9 cultures and a H2O2-positive control done as mentioned above were stained with an Apopercentage kit to detect apoptosis. Scale bars: 10 µm (D); 1 µm (E); 100 µm (H).

View larger version (43K): [in this window] [in a new window]   Fig. 3. PKC interacts with ezrin in vivo. Knockdown of PKC with shRNA. (A) CACO-2 cells were transfected with full-length ezrin cDNA (+) or mock transfected (–), and extracted in 0.5% Triton X-100 after 48 hours. Some extracts were immunoprecipitated (ip) with anti-ezrin (+) or with nonimmune IgG (–) as a control. Samples of the extracts (input) and the eluates were probed with anti-PKC antibody (raised in rabbit). The membrane in the middle panel was later reprobed with anti-ezrin antibody (right panel). Mol. mass standards: 85 and 31 kDa. (B-K) CACO-2 C2BBe cells were transduced with nonreplicating lentivirus particles carrying only a gene for puromycin resistance (empty) or an anti-hPKC shRNA sequence under a pol III promoter and the same puromycin-resistance gene (shRNA). (B, C) Confluent monolayers selected in puromycin were extracted in SDS sample buffer at 3, 4, 5, 7 and 10 days of culture. Samples of 30 µg protein were loaded in each lane, run in SDS-PAGE and blotted. The blots were sequentially probed with anti-PKC (B) and then reprobed with anti-tubulin (C) antibodies. Molecular mass standards: 200, 117, 85, 40 and 31 kDa. (D-K) The cells were transduced with empty lentivirus vector (D-G) or with the same vector carrying an anti-PKC shRNA sequence under a pol III promoter (H-K), and selected in puromycin. The cells were fixed and processed with anti-PKC (total protein) antibody (D,F,H,J, green), and with anti-pT555 PKC (active) antibody (E,G,I,K, red), and counterstained with DAPI (blue). D,E,H,I are X-Y projections of the five apical-most confocal sections (2 µm thick) comprising the entire apical region. F,G,J,K are 7-voxel thick X-Z sections of the entire confocal stack corresponding to the image above, shown with the apical side up. Scale bars: 10 µm.

View larger version (74K): [in this window] [in a new window]   Fig. 4. Remnant active ezrin after PKC knockdown localizes near the tight junction region. CACO-2 C2BBe cells were transduced with nonreplicating lentivirus particles as described in Fig. 3. (A,B) Confluent monolayers were extracted and the blots were sequentially processed with anti-T567-P ezrin (A) and then reprobed with anti-ezrin (total protein) (B) antibodies. Molecular mass standards: 200, 117, 85, 40 and 31 kDa. (C-M) Cells were transduced with empty lentivirus vector (C,E,J) or with the same vector carrying an anti-PKC shRNA sequence under a pol III promoter (D,F,G-I,K-M). The cells were fixed and processed with anti-ezrin (total protein) antibody (C,D, red), and anti-T567-P (active) ezrin antibody (E,F, green). C-F are X-Y projections of the apical domain as described in Fig. 3. (G-I) CACO-2 C2Bbe cells expressing anti-PKC shRNA were grown on filters for 8 days, fixed, and processed with anti-T567-P ezrin (green, G,I) and anti-ZO-1 (red, H,I) antibodies. Also, parallel monolayers grown on glass coverslips were processed for scanning electron microscopy of the apical surface (SEM) (J,K). The images are shown as X-Z 3D reconstructions of a confocal stack with the apical side up. (L,M) CACO-2 C2BBe cells were transduced with nonreplicating lentivirus particles (L) or lentiviral particles expressing anti-PKC shRNA (M). The cells were selected in puromycin and seeded on Transwell filters. After 6 days, the cells were fixed and processed for immunofluorescence with anti-PKA regulatory subunit IIβ (red) and counterstained with DAPI (blue). The arrows indicate cells with apical distribution of PKA regulatory subunit. Scale bars: 10 µm (C-I,L-M); 1 µm (J,K).

View larger version (125K): [in this window] [in a new window]   Fig. 5. Transfection with a dominant-negative PKC mutant abrogates ezrin phosphorylation in T567 in CACO-2 C2BBe cells. The cells were transfected with a V5-tagged K274W PKC mutant 4 days after seeding and fixed 1 day later. The monolayers were processed with anti-T567-P ezrin antibody (green, A,C), and anti-V5 tag antibody (red, B,D), and counterstained with DAPI. The nuclei of V5-positive cells with various levels of V5 expression are labelled 1-4. Scale bar: 10 µm.

View larger version (94K): [in this window] [in a new window]   Fig. 6. Transfection with a constitutively active PKC mutant increases ezrin T567 phosphorylation in CACO-2 cells. CACO-2 cells (A-H) were transfected under the same conditions described in Fig. 5 with a V5-tagged A120E PKC mutant, on day 4 and fixed on day 5 (A-D), or transfected on day 2 and fixed on day 3 (E-H) of culture. CACO-2 C2Bbe cells transduced with a lentivirus expressing the shRNA anti-PKC and selected in puromycin, were transfected with the same mutant on day 4 and fixed on day 5 after plating (I-M). The cells were processed with anti-T567-P ezrin antibody (green) and anti-V5 tag antibody (red), and counterstained with DAPI (blue). The images shown in a X-Y confocal plane at the apical level (A,B,E), at the basal level (F) or in X-Z 3D reconstructions (C,D,G,H,L,M). I-K are projections of the entire confocal stack separated in the individual channels. The red channel was omitted in A,C,H,J,M to show T567-P signal alone. Arrows indicate transfected cells. Arrowheads point to the basal surface in X-Z reconstructions. Scale bars: 10 µm.

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