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First published online 23 June 2009
doi: 10.1242/jcs.046979

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Rescue of atypical protein kinase C in epithelia by the cytoskeleton and Hsp70 family chaperones

¹ Department of Cell Biology and Anatomy, University of Miami Miller School of Medicine, Miami, FL 33136, USA
² CIEMAT, 28040 Madrid, Spain
³ University of Bonn Institute of Physiological Chemistry, 53115 Bonn, Germany
⁴ Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109, USA

View larger version (112K): [in this window] [in a new window]   Fig. 1. PKC colocalizes with intermediate filaments in the apical domain of CACO-2 cells. (A,B) Distribution of PKC in CACO-2 cells is not restricted to tight-junctional region. At 10 days after seeding, CACO-2 cells cultured on Transwell filters were fixed and processed with anti-PKC antibody (green channel), anti-PAR3 antibody (red channel), anti-K8 antibody (K8, blue channel, only in B) and DAPI (light blue), and analyzed by confocal microscopy. (A) XZ sections, apical side up. (B) XY confocal sections through the apical plane. (C) Triton-insoluble PKC colocalizes with intermediate filaments in the apical domain of CACO-2 cells. The cells were extracted with 0.5% TX-100, fixed, and processed with anti-K8 antibody (red channel) and anti-PKC antibody (green channel). High magnification of XY confocal section through the apical plane is shown in the upper panels. Lower panels represent XZ sections, apical side up (DAPI light blue). Scale bars: 10 µm. (D) PKC co-purifies with intermediate filaments in a highly insoluble fraction of CACO-2 cells. Equally cultured cells were extracted to obtain the three fractions: room temperature Triton-X-100-soluble fraction S1 (first supernatant); Triton-X-100-insoluble, 1.5 M KCl-soluble fraction S2 (second supernatant); and pellet P (Triton-X-100- and 1.5 M KCl-insoluble). Samples of 50 µg/lane of each fraction were seeded, separated by PAGE and blotted. Ponceau-S staining is shown as a loading control. Parallel blots were processed with the antibodies indicated at the top of each blot. Arrowheads on the right hand side of each blot indicate the expected relative molecular mass (Mr). Standards on the far left are 203, 126, 80 and 39x103.

View larger version (26K): [in this window] [in a new window]   Fig. 2. PKC in the intermediate filament fraction is capable of substrate phosphorylation. CACO-2 cells were grown to confluency in 75-mm plates for 10 days, serum starved for 24 hours, and subjected to the three-fraction (S1, S2 and P) preparation described in Fig. 1D. PKC activity was measured using a PKC kinase assay kit. Samples of 20 µg protein were diluted into the kinase assay dilution buffer and loaded on 96-well plates coated with a PKC substrate peptide. (A) Inhibitors were added to the appropriate wells in the following concentrations: 1 µM PKA inhibitor H-89, 1 µM GF10923X (at this concentration inhibiting only conventional and novel PKCs), 1 µM RO-31-8220 (inhibits more effectively atypical PKCs). Relative kinase activity was normalized per µg of protein. The data are means ± s.d. from three independent experiments (Student's t-test significance, *P<0.001). (B) A similar assay was performed from the three fractions obtained from cells expressing a specific anti-PKC shRNA or infected with empty vector lentiviral particles (contrl). After 10 days in culture and puromycin selection, the cells were fractionated in S1, S2 and P as described in Fig. 1D, and relative kinase activity was measured in each fraction as described above. The data are means ± s.d. from three independent experiments (t-test significance, **P<0.001). (C) In parallel experiments, S1, S2 and P fractions from cells expressing anti-PKC shRNA and controls were analyzed by immunoblot. Ponceau-S staining of the same membrane is shown as loading control. Mr standards: 194, 127, 87 and 39x103.

View larger version (66K): [in this window] [in a new window]   Fig. 3. PKC and Hsp70 form a complex with intermediate filaments in vitro. (A,B) Non-phosphorylated PKC binds to K8. (A) The 6xHis- and V5-tagged human PKC was expressed in HEK293T cells and purified under non-denaturing conditions on Ni2+ columns. Run-throughs and 350 mM imidazole eluates were analyzed by immunoblot with anti-V5, anti-Hsc70 or anti-pT555-PKC antibodies. Of note, HEK293T cells do not express Hsp70; blots for this protein were negative (not shown). Mr of standards is x103. (B) For blot overlay, recombinant bacterially expressed keratins (5 µg/lane K8, K18 and K19) were separated in a 12% acrylamide gel and blotted onto nitrocellulose. The membrane was transiently stained with Ponceau-S red to show protein load (bottom panels). His-V5-tagged PKC, eluted from the Ni2+ column (A), desalted and concentrated by ultrafiltration, was added onto the casein-saturated keratin blots and incubated overnight at 4°C. The overlay was extensively washed and the membranes were processed as for a regular immunoblot with anti-V5 or anti-pT555-PKC antibodies. (C) Hsp70 and PKC do not coimmunoprecipitate in the soluble fraction of CACO-2 cells. Soluble fraction (S1) of 10-day-old CACO-2 cells was prepared as described in Fig. 1 and immunoprecipitated (IP) with anti-PKC (+) or with non-immune rabbit IgG (–). Samples of the extracts (input) and the eluates were probed with anti-PKC antibody (raised in mouse). The membrane was later re-probed with anti-Hsc70 and/or anti-Hsp70 antibody (raised in rabbit). (D-E) The amount of PKC pulled down with IF increases in the presence of Hsp70. Highly purified (>99.9%, purity control; see supplementary material Fig. S2A) native polymerized IFs were covalently coupled to CNBr-activated Sepharose beads and incubated overnight with gentle shaking at 4°C in PBS containing 1% TX-100 with recombinant PKC together with Hsp70 (input 1) or with the recombinant purified PKC alone (input 2). After extensive washes, the beads were eluted in sample buffer and analyzed by immunoblot (pull-down, IF). Incubation with Sepharose beads coupled to non-immune rabbit serum served as a negative control (pull-down, control). Notice that the K8 blot is shown at a much shorter exposure to avoid saturation. (E) Quantification of the result shown in D (pull-down). The bars represent the means ± s.d. of the ratio of densitometric values of the PKC bands relative to K8 bands in the same lane, in the pull-down with IFs without (left-hand bar) or in the presence of recombinant Hsp70 (right-hand bar) from three independent experiments. For all measurements, non-saturated images were used (Student's t-test significance, *P<0.01).

View larger version (87K): [in this window] [in a new window]   Fig. 4. Intermediate filaments are necessary for the apical expression of PKC and Hsp70 in CACO-2 cells. (A) CACO-2 cells were cultured on Transwell filters. At 4 days after seeding, the cells were transduced with lentiviral particles carrying shRNA against K8 (K8 shRNA) or empty vector particles (control). After 10 days in culture and puromycin selection, the cells were fixed and processed with anti-K8 antibody (blue channel), anti-PKC antibody (green channel), anti-Hsp70 (total, Hsp70 and/or Hsc70) antibody (red channel) and DAPI (light blue), and analyzed by confocal microscopy. XZ sections are shown with the apical side up. DAPI staining is shown in light blue. Scale bars: 10 µm. (B) CACO-2 cells were transduced as described above, but total SDS extracts were analyzed by immunoblot. Mr of standards is x103. (C) The reduction in band intensity was obtained from the ratios of band intensity in knockdown and control cells, normalized by the intensity of the actin band re-probed in the same lane. For all measurements, non-saturated images were used. The means ± s.d. from three independent experiments are shown.

View larger version (47K): [in this window] [in a new window]   Fig. 5. Hsp70 and/or Hsc70 are necessary to maintain a pool of pT555 PKC in steady state in CACO-2 cells. (A) Downregulation of Hsp70 by shRNA leads to the decrease in the amount of pT555 and total PKC protein in CACO-2 cells. At 7 days after seeding, cells were infected with two sets of lentiviral particles carrying two different shRNA constructs against Hsp70 (shRNA1, shRNA2). Two days after infection, cells were extracted and analyzed by immunoblot. Actin is shown as a loading control. (B) The reduction of each band was calculated as in Fig. 4C. (C) Apoptosis in Hsp70 shRNA infected cells was controlled using caspase-3 cleavage. A positive control of apoptosis was performed by incubating CACO-2 cells in 30 mM H2O2 for 2 hours (arrowhead, cleaved caspase 3). Mr of standards is x103. (D-I) CACO-2 cells were grown on Transwells and infected with lentiviral particles expressing shRNA1 as described above (E,G,I) or empty vector particles (D,F,H). The cells were probed by immunofluorescence using anti-Hsc70 antibody (green channel, D,E), anti-pT555 PKC antibody (red channel, H,I), and anti-K8 antibody (K8, purple channel, F,G). DAPI, light blue. Scale bar: 10 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 6. Hsc70 and PKC distribution depend on the integrity of intermediate filaments in small intestine crypts of K18R89C mice. Small intestines from K18+/–, K19–/–, hK18 R89C knockout/knock-in mice (C,D,I,J) (ko) or control littermates (A,B,E-H,K,L) (wt and control) were fixed in 10% TCA and frozen. Frozen sections were processed with anti-Hsc70 antibody (A-D, green) or anti-pT555 PKC antibody (G-J, green), and anti-K8 antibody (keratin, red), and counter-stained with DAPI (light blue). Notice that intestinal epithelium does not express Hsp70 when not stressed. Control sections were stained with non-immune IgG at the same dilutions (E,F,K,L). The images are single confocal optical sections. Scale bars: 10 µm.

View larger version (47K): [in this window] [in a new window]   Fig. 7. Hsc70 and PKC distribution depend on the integrity of intermediate filaments in small intestine villi of K8 knockout mice. Small intestines were harvested from K8-null mice (C,D,I,J) or from K18+/– littermates (A,B,E-H), fixed in 10% TCA and frozen. (A-D) Frozen sections were processed with anti-Hsp70 antibody (green) or (G-J) with anti-pT555 PKC antibody (green, arrowheads point at tight-junction compartment of PKC), and anti-K8 antibody (K8, red), and counter-stained with DAPI (light blue). Control sections were processed with non-immune IgG at the same dilutions (E,F). The images are single confocal optical sections. Scale bars: A-F, 20 µm; G-J, 10 µm. (K) Villus enterocytes from control mice (+/–; 1-3), and K8-null mice (–/–; 4-6) were isolated, extracted in SDS, run in SDS-PAGE and blotted. Immunoblots of the same membranes are shown for keratins, actin, Hsc70 and pT555. All the blots are shown in non-saturated exposures, except for Hsc70. In this case, the variability in protein expression was required to saturate the more intense bands to make the weaker bands visible. (L) The reduction in band intensity was calculated as in Fig. 4C (negative values indicate increase). Results are means ± s.d. from four knockout and four control animals normalized as percentage of reduction with respect to the average of bands for the same protein from control animals (+/–) run in the same gel. For all measurements, non-saturated images were used. Student's t-test statistics were performed directly on means ± s.d. of actin-normalized values (*P<0.025). (M) Texas-red Dextran 3000 (10 mM in 1:1 PBS:H2O) was perfused inside 10-cm loops of duodenum and the first part of jejunum in anesthetized K8-null (–/–) or control littermates (+/+) for 50 minutes. Blood samples were collected at time 0 (and pooled together because fluorescence values were indistinguishable) or at 50 minutes when animals were sacrificed. Samples of serum were analyzed by spectrofluorometry. The results are means ± s.d. from five animals in each group (Student's t-test significance, *P<0.05, **P<0.02).

View larger version (61K): [in this window] [in a new window]   Fig. 8. Keratins and Hsp70 are required for rephosphorylation of mature dephosphorylated PKC. S1, S2 and P fractions were prepared from CACO-2 cells as described in Fig. 1 except that anti-phosphatase cocktails were omitted. The phosphorylation state of PKC turn motif was examined using anti-pT555-PKC antibodies (pT555). (A) To induce the activity-dependent dephosphorylation of PKC, S1 was incubated in the presence of 150 µM of PKC substrate peptide and 1 mM ATP at 30°C with gentle shaking for 5 hours, resulting in S1 with dephosphorylated PKC (S1*). After treatment, the peptide was removed from the solution by ultrafiltration. Subsequent incubation of that fraction (S1*) with 1 mM ATP for 4 hours (S1*+ATP) did not lead to rephosphorylation of PKC. (B) Incubation of the dephosphorylated pellet fraction (P*) alone with 1 mM ATP for 4 hours (P*+ATP) failed to show PKC rephosphorylation. (C) 50 µg of protein in S1* was then incubated with 20 µg of protein in the equally dephosphorylated pellet (P*) fraction or (D) with 15 µg of purified native filamentous keratins (IF) in the presence or absence of 1 mM ATP at 30°C for 4 hours. Pan-keratin antibodies (pan-keratin) were used to show keratin load. (E) Only filamentous keratins are able to rephosphorylate PKC. S1* fraction was supplemented with purified native filamentous keratins (IF) in the presence (S1*+IF+ATP) or absence of 1 mM ATP (S1*+IF) as in D, or with the recombinant K8 (S1*+K8+ATP), K18 (S1*+K18+ATP) or K19 (S1*+K19+ATP) in the presence of 1 mM ATP for 4 hours. (F-H) PKC rephosphorylation in the presence of purified IFs is Hsp70-dependent. (F) Immunodepletion by incubation with anti-Hsc70 and/or anti-Hsp70 antibodies resulted in substantial decrease in the amount of Hsp70 protein in S1 fraction (Hsp70-depleted). Incubation of S1 fraction with normal rabbit serum served as a control (control). The activity-dependent dephosphorylation of PKC in the presence of the substrate peptide was unaffected by either Hsp70 immunodepletion or incubation with the normal rabbit serum (S1*). However, reduction of Hsp70 protein in S1 fraction resulted in the abrogation of mature PKC rephosphorylation in the presence of intermediate filaments and 1 mM ATP (S1*+IF+ATP; Hsp70-depleted versus control *). (G) Addition of the recombinant Hsp70 protein to the immunodepleted S1* fraction restores PKC rephosphorylation. 50 µg of protein from the dephosphorylated Hsp70-immunodepleted S1* fraction was incubated with 15 µg of purified native filamentous keratins (IF) and supplemented (+) or not (–) with 2 µg of the recombinant Hsp70 protein in the presence of 1 mM ATP at 30°C for 4 hours. (H) Hsp70 bound to IFs is sufficient for PKC rephosphorylation. 50 µg of protein from the dephosphorylated Hsp70-immunodepleted S1* fraction was incubated with 15 µg of purified native filamentous keratins (+IF, +IF+ATP) or with 20 µg of the equally dephosphorylated pellet (+P*, +P*+ATP) fraction in the presence or absence of 1 mM ATP at 30°C for 4 hours. The blots are typical of very consistent experiments repeated at least three times.

View larger version (68K): [in this window] [in a new window]   Fig. 9. Subcellular distribution of PKC and Hsc70 modified by overexpression and redistribution of keratins. Frozen sections of duodenum from genetically unmodified FVB/n mice (A-D,I-L) or transgenic K8 overexpressors (K8 oe; E-H,M-P) were processed with anti-K8 antibody (K8; red channel), and anti-pT555 PKC antibody (A,B,E,F), anti-Hsc70 antibody (I,J,M,N) or non-immune rabbit IgG (control; C,D,G,H,K,L,O,P) (green channel). Notice that intestinal epithelium does not express Hsp70 when not stressed. In each case, controls were performed on parallel sections from the same block from which the images with specific antibody were obtained (shown above the corresponding control). DNA was counterstained with DAPI (light blue). The images are representative of sections from six overexpressor animals and five FVB/n littermate controls and are confocal optical sections obtained at 0.9 Airy units maintaining the same gains in the red and green channels. Arrows: B, pT555 signal consistent with tight junction distribution; E, mislocalized K8 signal; F, pT555 signal both in the normal expected distribution (apical junctions) and in the region of abnormally localized IFs. Scale bars: 10 µm. (Q) Duodenum and jejunum enterocytes were isolated from five HK8-4 K8 transgenic overexpressors (K8 oe) or four control littermates (FVBn), and analyzed by immunoblot (30 µg/lane total protein), reprobing the same membranes with anti-atypical PKC phospho-turn motif antibody (Epitomics) (pT555), and anti-keratin and anti-actin (loading control) antibodies. For the pT555 signal, bands in lanes 5, 6, 7 and 8 are saturated to show visible bands in lanes 1 and 4 at the same exposure. (R) The relative intensity of pT555 signal respect to the actin loading control signal was measured in non-saturated exposures of the same blots. Significance was calculated by the Student's t-test (*P<0.001).

View larger version (52K): [in this window] [in a new window]   Fig. 10. PKC acquires an inactive conformation after normal activity and can be rescued from ubiquitinylation and degradation by a Hsp-70- and keratin-dependent mechanism. The model is based on data originally published by Newton and coworkers for conventional PKC (Gao et al., 2006) and on the results in this work. TJ, tight junction; AJ, adherens junction.

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