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Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade
Biochimica et Biophysica Acta 1800 (2010) 591–598
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n
Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade Nibedita Mahata, Debasis Pore, Amit Pal, Manoj K. Chakrabarti ⁎ Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata-700010, West Bengal, India
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Article history: Received 21 October 2009 Received in revised form 8 March 2010 Accepted 10 March 2010 Available online 23 March 2010 Keywords: Escherichia coli heat-stable enterotoxin Cytoskeleton Cytochalasin D PKC-α COLO-205 cell line
a b s t r a c t Background: IP3-mediated calcium mobilization from intracellular stores activates and translocates PKC-α from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-α to membrane from cytosol in the Escherichia coli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205. Methods: Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole). Results: STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1 min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-α translocation to the membrane. Conclusions: These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-α movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane. General significance: In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-α to membrane. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Heat-stable enterotoxin (STa), a major cause of watery diarrhea in human, is secreted by enterotoxigenic Escherichia coli (ETEC) [1]. Two hundred eighty million episodes of diarrhea due to ETEC are estimated annually in children less than 5 years old in developing countries [2]. STa binds with cell surface receptor guanylyl cyclase C (GC-C) and leads to the rise of intracellular cGMP level. cGMP stimulates the cGMP-dependent protein kinases that regulate the activation of CFTR channels and Cl− secretion [3,4]. But the specific signaling pathway behind E. coli STa induced rise in cGMP activity and its secretory response is still not clear [5]. Previous reports from our laboratory [6–8] and others [9,10] suggested that besides cGMP, other signaling molecules have been anticipated in the STa-mediated
Abbreviations: cGMP, cyclic GMP; STa, E. coli heat-stable enterotoxin; PKC-α, protein kinase C-α; PMA, phorbol 12-myristate-13-acetate; BSA, bovine serum albumin; Ca2+, calcium ⁎ Corresponding author. Tel.: +91 33 2370-5533; fax: +91 33 2350 5066. E-mail address: [email protected] (M.K. Chakrabarti). 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.03.006
intestinal secretion. It has been reported earlier by our laboratory that STa causes an inositol-triphosphate (IP3)-mediated release of Ca2+ from intracellular store [6,7]. It is also reported that by perturbing the Ca2+ homeostasis, toxin may also affect the cytoskeletal architecture and alter the barrier function of epithelial cells, because of the critical role of Ca2+ in the regulation of cytoskeleton dynamics induced by bacteria [11–13]. However, the basis of this dynamic regulation of cytoskeleton remains to be defined [11]. Furthermore, it has also been reported by us that STa induces an increase in [Ca2+]i that leads to the activation and translocation of the protein kinase C-α (PKC-α) from cytosol to membrane, which might be required for the stimulation of a membrane-bound guanylyl cyclase [8]. However, the molecular mechanism behind the translocation of PKC-α is still not clear [14]. The present study has been aimed to investigate the reorganization of cytoskeletal proteins and their role in translocation of PKC-α to membrane in the E. coli STa-mediated signaling mechanism in a human colonic carcinoma cell line COLO-205. Our results clearly demonstrate that actin network is significantly rearranged in STatreated COLO-205 colonic carcinoma cell line and this rearrangement leads to the translocation of PKC-α.
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2. Materials and methods 2.1. Materials The chemicals used in the present study were obtained from the following sources: RPMI-1640 medium, fetal bovine serum (FBS), trypsin-EDTA, Hank's balanced salt solution, sodium bicarbonate, glucose and sodium pyruvate from Gibco BRL, USA; Dimethyl sulfoxide (DMSO), Phenylmethylsulphonylfluoride (PMSF), Dithiothreitol (DTT), Leupeptin, Aprotinin, E. coli heat-stable enterotoxin (STa), phorbol 12-myristate-13-acetate (PMA), penicillin, streptomycin sulfate, cytochalasin D, Nocodazole, taxol, PIPES, TRITC (tetramethylrhodamine B isothiocyanate)-conjugated phalloidin , and isoformspecific antibody against PKC-α from Sigma, USA; Nonidet P-40 from Pierce; anti-actin antibody from Santa Cruz Biotechnology; anti-tubulin antibody from Cell Signaling and secondary antibodies from Jackson ImmunoResearch. All other reagents were of analytical grade, and deionized water was used throughout the study. 2.2. Cell culture and preparation of viable cells and cell treatment COLO-205 cells, which were procured from National Centre for Cell Sciences (NCCS), Pune, India, were routinely cultured in tissue culture flasks and grown up to monolayer in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 2.4 g/l sodium bicarbonate, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate in humidified 5% CO2 atmosphere at 37 °C.Confluent monolayer were passaged using trypsin-EDTA. Viability of the cells was routinely checked by trypan blue exclusion. After reaching 70% confluence, cells were washed with Hank's balanced salt solution (HBSS) and cultured in serum-free RPMI 1640 medium for 24 h and then used for experimental purpose. For the dynamics study of the cytoskeleton, proteins COLO-205 cells were treated with or without 5nM STa in serum-free medium for different time periods. COLO-205 cells were exposed to different drugs at the following concentrations: 1 mM cytochalasin D, 1 mM nocodazole in serum free medium for 1 h at 37 °C. 2.3. Preparation of microtubules Microtubules were prepared from COLO-205 cells (2 ×106 cells/ml) according to Solomon et al. (1986) [15] with some modification. The cells were treated with 1 mM taxol for 2 h before extraction of microtubules, which in turn stabilizes microtubules without promoting polymerization. The culture medium was then aspirated and replaced with 0.1 M PIPES buffer(PIPES buffer containing 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, 0.04 TIU/ml aprotinin, 2 mM phenylmethylsulfonylfluoride, and 1 mM benzamidine, pH 6.9) containing 1 mM taxol. Cells were scraped from the substratum with a cell scraper and centrifuged at 1000×g for 5 min at room temperature. The cell pellet was then resuspended with PIPES buffer (0.1 M PIPES buffer containing 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, 0.04 TIU/ml aprotinin, 2 mM phenylmethylsulfonylfluoride, and 1 mM benzamidine, pH 6.9) containing 1% Nonidet P-40 and 1 mM taxol and incubated for 15 min at 37 º C. After the incubation, the suspension was centrifuged at 1000 ×g for 5 min at 37 °C. The pellet consisting of the microtubules was then solubilized in 125 mM Tris buffer, pH 6.8, containing 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol and heated to 100 °C for 5 min. After centrifugation at 10,000 ×g for 5 min, the supernatant was stored at −20 º C for subsequent Western blot analysis. For total cell extracts, same number of cells was taken and washed twice with HBSS buffer and solubilized as described above. 2.4. Preparation of microfilaments Microfilament-enriched preparations were extracted from COLO205 cells (2×106 cells/ml) as described by Phillips et al. (1980) [16,17]. Culture medium was aspirated and changed to HBSS buffer. Cells were scraped from the tissue culture flasks with a cell scraper. Cells were
centrifuged at 100 ×g for 5 min at room temperature. The pellet was dissolved in 500 µl of Triton solution (1% Triton X-100, 10 mM EGTA, and 0.1 M Tris–HCl, pH 7.4) and kept on ice for 10 min. The preparation was then centrifuged at 8000 ×g for 5 min at room temperature. Tritonsoluble G-actin fraction was contained in the supernatant. The pellet, which corresponds to the Triton-insoluble fraction, was solubilized in 2% SDS-2% 2-mercaptoethanol (v/v) by boiling at 100 °C for 10 min. 2.5. Preparation of cytosolic and membrane-bound PKC-α Cytosolic and membrane-bound PKC-α were prepared according to Datta Gupta et al. (2005) [8] with little modification. Confluent monolayers of COLO-205 cells (2× 106 cells/ml) were treated in the presence and absence of STa or with disruptors for 1 h before stimulation of STa for 1 min. The reactions were terminated using the ice-cold Hank's balanced salt solution (HBSS). Cells were washed twice in 20 mM Tris–HCl (pH 7.5), suspended in the same buffer containing PMSF, and leupeptin and then homogenized with a motor driven Teflon homogenizer (REMI Udyog, India). The homogenate was centrifuged for 60 min at 30,000 rpm at 4 °C. The Supernatant was separated from pellet and used for measuring the cytosolic PKC-α [18]. The pellet was then resuspended in 20 mM Tris–HCl (PH 7.5) containing PMSF and leupeptin, homogenized briefly in the presence of 1% Triton X-100 to solubilize the membrane-bound proteins and recentrifuged as before (for 60 min at 30,000 rpm at 4 °C). The resulting supernatant served as the source of membrane-bound PKC-α [19]. 2.6. Isolation of cytosolic and membrane fraction Membrane and cytosolic fractions were obtained by using the method of Cote et. al. (1996) [20]. Briefly, the confluent monolayer of COLO-205 cells (2×106 cells/ml) were treated with STa (5nM) and incubated at 37 °C for 0–120 s, cells were washed twice with HBSS buffer and then with 10 mM ice-cold Tris–HCl buffer (containing 0.5 mM EDTA, 1 mM MgCl2, 1 µg/ml leupeptin, 0.21 µg/ml aprotinin1mM PMSF, and10 µM benzimidine, pH 8.0). The cells were then scraped and homogenized in the same buffer. Cell extracts were centrifuged at 800 ×g for 5 min, and the supernatant was then centrifuged at 15,000 ×g for 30 min. After centrifugation, the resulting supernatant was used as cytosolic fraction. The pellet was then resuspended in 50 mM Tris–HCl buffer (containing 2 mM EDTA, 5 mM MgCl2, and 250 mM sucrose) to obtain the membrane fractions. 2.7. Immunoprecipitation Immunoprecipitation was performed according to Ron et al. (1999) [21] with little modification. Each membrane fraction (prepared as described above) was pre-cleaned by incubation with 50 μl of protein A/G plus agarose (Santa Cruz Biotechnology) for 2 h at 4 °C. The samples were centrifuged at 200×g for 1 min, to collect the supernatant, and protein content was measured. Immunoprecipitation was performed overnight at 4 °C with 5 μg of actin antibody per 500 μg protein diluted with an equal volume of immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris–HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 0.4 mM sodium vanadate, 1 mM PMSF, leupeptin and aprotinin). Fifty microliters of protein A/G plus agarose was then added, and the samples were incubated at 4 °C for 2 h. Finally, the samples were centrifuged at 14,000×g for 10 min, the supernatant was discarded, and the pellet was resuspended in SDS protein gel loading solution. It was then subjected to immunoblotting by using anti-PKC-alpha antibody. It was also subjected to immunoblotting by using anti-PKC-βII antibody to exclude unspecific binding to actin. 2.8. Immunoblotting and densitometric analysis Western blot analysis was done to detect the actin, tubulin, and PKC-α by using anti-actin, anti-tubulin, and anti-PKC-α antibodies.
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Samples from a same number of cells were compared in each experiment. Samples were added to SDS-PAGE buffer and kept at boiling bath for 5 min to solubilize the proteins [22]. Then the samples were subjected to 12.5% SDS-polyacrylamide gels for separation and then transferred to nitrocellulose membrane (pore size 0.45 µm, BIORAD, USA) using a transblot apparatus (BIO-RAD, USA) [23]. Membranes were blocked with 5% non-fat dry milk in TTBS [Trisbuffered saline (TBS, pH 7.5) containing 0.1% Tween-20] for overnight at 4 °C. After three washes with TTBS, membranes were incubated with anti-tubulin (dilution, 1:1000), anti-actin (dilution, 1:1000), or antiPKC-α (dilution, 1:1000) in TTBS containing 0.5% non-fat dry milk for 2 h at room temperature, followed by four washes with TTBS. Detection was accomplished using alkaline phosphatase-conjugated anti-mouse antibody for actin, anti-rabbit for tubulin (Jackson ImmunoResearch), and anti-rabbit for PKC-α (Jackson ImmunoResearch) in TBS containing 0.5% non-fat dry milk for 1 h at room temperature. Immunoreactive bands were visualized with NBT-BCIP reagent, which was used as the chromogenic substrate. Immunoreactive bands were photographed, and then images were digitized and analyzed by using Bio-Rad QUANTITY 1 software of the gel documentation system. Immunoreactive bands were quantitated and expressed as the ratio of each band density to control band density. 2.9. Assay of protein kinase C-α Cytosolic and membrane fraction, from the treated and untreated cells as described earlier, were prepared from COLO-205 cell line. Using that protein samples activity of protein kinase C-α was measured. PKCα activity in the cytosolic and membrane fractions was measured by a non-radioactive ELISA that utilizes a synthetic PKC pseudo-substrate
Fig. 1. Western blot analysis of the effect of E. coli STa (5 nM) on the actin content in Triton X-100-insoluble microfilaments fractions in COLO-205 cell line. Cells were deprived of serum overnight and stimulated with STa (5 nM) for 0–120 s. Triton X-100insoluble microfilaments fractions were prepared from the STa-treated and untreated cells according to the Materials and methods section. (A) Triton X-100-insoluble fraction. (B) Whole cell homogenate. Triton X-100-insoluble microfilaments content and total amount of microfilaments in whole cell homogenate were analyzed by immunoblotting with mouse anti-actin monoclonal antibody. Arrow indicates the 42 kDa actin-specific bands. (C) Densitometric analysis of immunoreactive bands of actin. Immunoreactive bands were photographed, and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean ± SEM (n = 3) and p b 0.05.
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and a monoclonal antibody that recognizes the phosphorylated form of the peptide, according to the manufacturer's instructions (Calbiochem, EMB Biosciences, San Diego, CA, USA). Protein concentration was determined by a Bradford method [24], with BSA as standard using manufacturer's protocol. PKC-α activity was expressed as OD/mg protein and the data normalized to control. 2.10. Immunofluorescence study For immunofluorescence study, the cells were plated to L-lysinecoated glass coverslips and kept in the incubator (5% CO2 and 37 °C) in humidified atmosphere for a minimum of 48 h to allow cell attachment and spreading. After incubation, the cells were washed with PBS. The cells were washed three times with PBS at every step in this immunofluoresence experiment. Following toxin treatment, both control and treated cells were fixed with 2% paraformaldehyde for 45 min at room temperature and permeabilized with 0.2%Triton-X 100 in PBS for 30 min at room temperature. The cells were incubated with blocking solution (5% bovine serum albumin in PBS) for 4 h at room temperature. For F-actin detection, the cells were stained with 50 µg/ ml of TRITC (Tetramethyl-rhodamine B isothiocyanate)-conjugated phalloidin in PBS at room temperature for 40 min. Cells were then mounted on microscope slides with glycerol phosphate buffer (2:1). The cells were visualized using a fluorescence microscope (Nikon) and photographed at magnification of ×100. 2.11. Statistical analysis Results were expressed as the mean ± standard error of the mean (SEM), where applicable, of at least three independent experiments. The data were analyzed by Student's t-test .The p values of b0.05 were considered as significant.
Fig. 2. Western blot analysis of the effect of E. coli STa (5 nM) on actin content the Triton X-100-soluble microfilaments fractions in COLO-205 cell line. Cells stimulated with 5nM STa for 0–120 s and Triton X-soluble fractions were prepared from control cells and the treated cells as described in the Materials and methods section. (A) Triton X-100-soluble fraction. (B) Whole cell homogenate. Triton X-100-soluble microfilaments content and total amount of microfilaments in whole cell homogenate were analyzed by immunoblotting with mouse anti-actin monoclonal antibody. Arrow indicates the 42 kDa actin-specific bands. (C) Densitometric analysis of immunoreactive bands of actin. Immunoreactive bands were photographed and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean± SEM (n = 3) and p b 0.05.
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Fig. 3. Western blot analysis of the effect of E. coli STa (5 nM) on the membrane and cytosol associated microfilaments content in COLO-205 cell line. Membrane fractions were prepared (as described in the Materials and methods section) from STa-treated cells in time course experiment (0–120 s) , resolved on an 12.5% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with mouse monoclonal anti-actin antibody. (A) Membrane fractions. (B) Cytosolic fractions. (C) Whole cell homogenate. Arrow indicates the 42 kDa actin-specific bands. (D) Densitometric analysis of immunoreactive bands of actin. Immunoreactive bands were photographed, and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean ± SEM (n = 3) and p b 0.05.
Fig. 4. Western blot analysis of the effect of E. coli STa (5 nM) on the microtubule content in the colonic carcinoma cell line. COLO-205 cells were incubated with 5nM STa for 0, 15, 30, 60, 90, or 120 s, and microtubule fractions were isolated as described in the Materials and methods section. A Western blot analysis of the membrane fraction was conducted with rabbit monoclonal anti-tubulin antibody, followed by a secondary alkaline phosphatase conjugated antibody. (A) Microtubule fraction. (B) Whole cell homogenate. Arrow indicates the 53 kDa tubulin-specific bands. (C) Densitometric analysis of immunoreactive bands of microtubule. Immunoreactive bands were photographed, and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean ± SEM (n = 3) and p b 0.05.
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3. Results 3.1. Effect of STa on the distribution of microfilaments and microtubules To evaluate dynamic changes in the levels of microfilaments and microtubules following STa treatment in COLO-205 cells, filamentous (Fig. 1A) and soluble forms of microfilaments (Fig. 2A) and microtubules (Fig. 4A) preparations were analyzed by immunoblotting for their respective contents of actin and tubulin. Densitometric analysis indicated that STa (5nM) treatment induced a timedependent increase in the level of actin in Triton X-100-insoluble microfilaments fraction (1.40 ± 0.02, 2.36 ± 0.02 and 3.72 ± 0.02 fold increase, lane 2: 15 s, lane 3: 30 s, lane 4: 60 s, respectively, compared to control in lane 1 in Fig. 1C; n = 3, p b 0.05) and decreased thereafter (i.e., 90 s and 120 s, Fig. 1C). In contrast, Fig. 2C showed that the level of actin in Triton-soluble fraction decreased with time (0.73 ± 0.03, 0.47 ± 0.04, 0.35 ± 0.025 fold, lane 2: 15 s, lane 3: 30 s, lane 4: 60 s, respectively, compared to control in lane 1, P b 0.05). In addition, it was also found that membrane-associated actin content became maximum (2.68 ± 0.02 fold increase; n = 3; p b 0.05) at 1 min of toxin treatment (Fig. 3A and D). Thereafter, membrane-associated actin content decreased but remained higher than the basal value (Fig. 3A and D). In contrast, within the same experimental period, the actin content in the cytosolic fraction became minimum (0.327 ± 0.03 fold in lane 4 compared to control, Fig. 3B and D). This dynamic association of actin with the membrane (Fig. 3) and the increase in actin content in the Triton-insoluble fraction (Fig. 1) correspond to an increase in polymerization within 1 min of STa treatment, which was not due to in new synthesis of actin, as total levels of actin in the cell lysate did not change during the experimental period with STa. Unlike membrane-associated microfilaments (Fig. 3A), we did not find any change in the level of microtubules associated with membrane in STainduced COLO-205 cell (data not shown). Similarly, it was also found that toxin treatment did not promote any polymerization of filamentous microtubules in comparison to the control (Fig. 4). Like actin, the total amount of tubulin in cell homogenates did not change during the experimental period.
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predominantly present in the membrane (0.89 ± 0.025, compared to control; Fig. 6C, lane 2 and Fig. 6D, bar 2) and decreased in the cytosol (0.152 ± 0.02, compared to control; Fig. 6A, lane 2 and Fig. 6B, bar 2). 1 h preincubation with cytochalasin D (a microfilament disrupting agent) blocked the STa-induced translocation of PKC-α (lane 3 of
3.2. Fluorescence studies Immunofluorescence studies were conducted to further confirm the redistribution of microfilaments within the cell. We have examined more than 50 cells originating from 3 independent experiments for each set of experiment. We had stained COLO-205 cells with rhodamineconjugated phalloidin for F-actin detection. Fig. 5 showed the structural distribution of F-actin in untreated (Fig. 5A) and STa-treated cells (Fig. 5B, C). In the untreated cells, F-actin filament was distributed throughout the cytosol surrounding the nucleus. Whereas in the STatreated cells for 1 min F-actin was condensed as a ring in the periphery of the cell (Fig. 5B). Fig. 5C represented the cells treated with STa for 120 s, which had a very similar F-actin filament distribution to that of control. A kinetic analysis of the effect of STa on the F-actin reorganization showed that the effect started at 15 s and lasted for 60 s of treatment (data not shown). 3.3. Effect of cytoskeleton disruptors on the translocation of PKC-α Cytosolic and membrane fractions of the COLO-205 cell line were investigated by immunoblot technique using PKC-α specific antibody to identify the effect of cytoskeleton disruptors in the translocation process of PKC-α. Immunoreactive bands of PKC-α demonstrated that in untreated COLO-205 cell, it was predominantly present at the cytosol (0.811 ± 0.012, compared to control; Fig. 6A, lane 1 and Fig. 6B, bar 1) and a little or no PKC-α present at the membrane fraction (0.21 ± 0.02, compared to control; Fig. 6C, lane 1 and Fig. 6D, bar 1). After 1-min incubation with STa (5nM), PKC-α was
Fig. 5. Effect of STa on the actin cytoskeleton in subconfluent COLO-205 colonic carcinoma cells. Laser scanning confocal imaging of actin in control and STa-treated COLO-205 cells. Cells were cultured for 3 days on L-lysine-coated coverslips and then incubated for various time periods (B: cells treated for 60 s with 5nM of STa, C: cells treated for 120 s with 5nM of STa) in serum-free medium in absence (A) or presence of 5nM of STa (B and C). After paraformaldehyde fixation and permeabilization with 0.1% triton X-100. Cells were processed for immunolabeling using Tetramethyl-rhodamine B isothiocyanate (TRITC)-conjugated phalloidin as described in the Materials and methods section. Images represent those obtained in 3 individual experiments in which a total of N 50 cells were evaluated from each experimental group. The cells were photographed at magnification of × 100.
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Fig. 7. Immunoprecipitation analysis of the effect of STa on the level of PKC-α associated with actin in colonic carcinoma cell line. COLO-205 cells were incubated in 37 °C in serum-free medium in absence (lane 1) or presence of 5nM STa for 1 min (lanes 2, 3). Membrane fractions were immunoprecipitated with anti-actin antibody as described in the Materials and methods section. Immunoblots were performed with anti-PKC-α monoclonal antibody (lanes 1 and 2) and anti-PKC-βII monoclonal antibody (lane 3). Arrow indicates the 80-kDa PKC-α-specific bands. Blots are representative illustrations of results obtained in three independent experiments.
Fig. 6A and C and bar 3of Fig. 6B and D). Whereas pretreatment of COLO-205 cells with nocodazole (a microtubules disrupting agent) for 1 h did not block the STa-induced translocation of PKC-α significantly (lane 4 of Fig. 6A and C and bar 4 of Fig. 6B and D). PMA was used as a positive control for PKC-α activation. These results indicate that intact actin cytoskeleton is required for the translocation of PKC-α. 3.4. Association of PKC-alpha with cytoskeleton The level of actin associated with membrane was also found to increase after 1 min (3.37 ± 0.02 compared to the control; Fig. 3). In addition, 1-min incubation with STa strongly increased the amount of PKC-alpha associated with the membrane (Fig. 6C, lane 3). Densitometric analysis indicated that the level of PKC-alpha associated with the membrane was increased by 3.9 ± 0.7 fold (n = 3; p b 0.05 compared to the control; Fig. 6D, bar 2) after 1 min. This transient increase in PKC-alpha in the membrane preparation might be associated with the membrane-associated actin .To confirm the association of PKCalpha with actin, membrane fractions were immunoprecipitated with anti-actin antibody and then processed for immunoblotting analysis with anti-PKC-alpha antibody. The results presented in Fig. 7 confirm that PKC-alpha was associated with actin in the membrane fraction after STa stimulation (lane 2). To exclude unspecific binding to actin, membrane fraction was subjected to immunoblotting by using antiPKC-βII antibody (Fig. 7, lane 3).
Fig. 6. Western blot analysis of protein kinase C (PKC)-α translocation in the presence of microfilament disruptor (cytochalasin D) and microtubules disruptor (nocodazole) in the colonic carcinoma cell line. COLO-205 cells were treated for 0 s (Control) or 60 s with 5 nM STa. Cells were also treated with STa for 60 s after 1-h exposure to cytochalasin D (1 mM) or nocodazole (1 mM). Then cytosolic and membrane fraction were prepared as described in the Materials and methods section. In this study, cytosolic and membrane fractions were loaded in the respective lanes. PMA, a known activator of PKC isoenymes that translocate PKC-α from cytosol to membrane, was used as a positive control for PKC-α translocation. (A) Representative Western blot of cytosolic fraction of PKC-α. Lane P: cytosolic fraction of the PMA-treated cells (positive control). Lane 1: cytosolic fraction of untreated cells (control). Lane 2: cytosolic fraction of STa treated cells. Lane 3: cytosolic fraction of cytochalasin-D-treated cells prior to STa treatment. Lane 4: cytosolic fraction of nocodazole treated cells prior to STa treatment. Arrow indicates the 80 kDa PKC-α-specific bands. (B) Desitometric analysis of the Western blot data of the cytosolic fraction of PKC-α expressed as the ratio of each band density to positive control (PKC-α) band density. Bar 1: cytosolic fraction of untreated cells (Control). Bar 2: cytosolic fraction of STa-treated cells. Bar 3: cytosolic fraction of cytochalasin-D-treated cells prior to STa treatment. Bar 4: cytosolic fraction of nocodazole treated cells prior to STa treatment. Data represent the mean ± SEM (n = 3) and p b 0.05. (C) Representative Western blot of membrane fraction of PKC-α. Lane P: membrane fraction of the PMA-treated cells (positive control). Lane 1: membrane fraction of untreated cells (Control). Lane 2: membrane fraction of STa-treated cells. Lane 3: membrane fraction of cytochalasin-D-treated cells prior to STa treatment. Lane 4: membrane fraction of nocodazole-treated cells prior to STa treatment. Arrow indicates the 80 kDa PKC-α-specific bands. (D) Desitometric analysis of the Western blot data membrane fraction of PKC-α expressed as the ratio of each band density to positive control (PKC-α) band density. Bar 1: membrane fraction of untreated cells (Control). Bar 2: membrane fraction of STa-treated cells. Bar 3: membrane fraction of cytochalasin-Dtreated cells prior to STa treatment. Bar 4: membrane fraction of nocodazole-treated cells prior to STa treatment. Data represent the mean± SEM (n = 3) and p b 0.05.
Fig. 8. Effect of STa, cytochalasin D, and nocodazole on PKC-α activity COLO-205 cell. COLO-205 cells were treated with and without STa or with cytochalasin D (1 mM) and nocodazole (1 mM) separately for 1 h before stimulation with 5nM STa for 1 min and PKC activity in the cytosol and membrane particulate fraction was determined by using non-radioactive ELISA. Data are normalized to cytosolic control, and values are expressed as mean ± SEM (p b 0.05; n = 6).
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3.5. Effect of cytoskeleton disruptors on the activity of PKC-α We tested the effect of cytoskeleton disruptors on the PKC-α activity upon STa exposure in COLO-205 cells by using a non-radioactive ELISA. Under control conditions, PKC-α activity was found mainly in the cytosolic compartment and translocated to the membrane compartment upon activation, but total PKC-α activity in the whole cell homogenate remained unchanged. Moreover, it is also known that the increase in the ratio of membrane to cytosol activity accurately reflects activation of PKC-α [25–27]. PKC-α activity in cytosolic and particulate membrane fractions was quantified after STa treatment to COLO-205 cells for 1 min. STa exposure for 1 min significantly increased PKC-α activity in the particulate fraction (1.11 ± 0.06 per mg of protein; n = 6; p b 0.05), whereas activity was decreased in the cytosol (0.44 ± 0.03 per mg of protein; n = 6; p b 0.05) (Fig. 8). This increase in the ratio of membrane to cytosol activity clearly indicates activation of PKC-α. PKCα activity was not increased in the membrane fraction upon 1 min STa induction in COLO-205 cells preincubated with cytochalasin D (1 mM) for 1 h (0.37 ± 0.03 per mg of protein; n = 6; p b 0.05). In contrast PKC-α activity was significantly increased in the membrane fraction upon 1 min STa induction in COLO-205 cells preincubated with nocodaloze (1 mM) for 1 h (1.07± 0.06 per mg of protein; n = 6; p b 0.05) (Fig. 8). These findings suggest that microtubule filaments are not involved in the translocation PKC-α. 4. Discussion STa is an 18–19 amino acid [28–30] containing methanol soluble [31] heat-stable toxin, which triggers a cascade of reaction including increase of intracellular calcium levels [Ca2+], and activation and translocation of PKC-α [6–8] in the colonic carcinoma cell line. But the exact mechanisms involved in the STa-mediated translocation of PKCα have not yet been clarified. With regard to the molecular mechanism behind PKC translocation, it is tempting to implicate the cytoskeleton, based on the previous works where only an association between PKC isoforms and cytoskeletal component have been reported [32–34]. Therefore, in the present study, we have evaluated the involvement of cytoskeletal proteins in the E. coli heat-stable enterotoxin (STa) mediated translocation of PKC-α in COLO-205 cell line. The cytoskeletal proteins are organized as the filamentous networks distributed throughout the entire cell extending from the plasma membrane to the interior of the nucleus. These proteins account for a large fraction of total cellular protein and are involved in all structural and dynamic aspects of the living cells, including maintenance of the cell morphology, cell adhesion, motility, replication, apoptosis, differentiation, and cell signaling [35]. Cytoskeleton exists in dynamic form in response to extracellular signals [36]. Here we have investigated the effect of cytoskeleton upon E. coli heat-stable enterotoxin (STa) treatment in the colonic carcinoma cell line COLO205. Densitometric analysis of the membrane fraction reveals that 1min incubation of the cells with STa is sufficient to induce a huge increase in membrane association of actin compared to control, that is, actin is redistributed upon STa activation. This rapid increase in membrane-associated actin during STa stimulation signifies the importance of the role of microfilaments in the early events of STa action. Furthermore, actin content of cytosolic fraction decreases after STa stimulation. This redistribution study of microfilaments within the cell is further confirmed by the immunofluorescence studies. Whereas time-dependent study of the Triton X-100-insoluble fraction of microfilaments content reveals that STa increases Triton X-100insoluble F-actin content, which has been found to increase by more than 3-fold within 1 min of toxin treatment in comparison to the control. In contrast, time-dependent study of the Triton X-100-soluble fraction of microfilaments content demonstrates that STa decreases Triton X-100-soluble G-actin content within 1 min of toxin treatment
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compared to the control which indicates that STa not only induces actin redistribution within 1 min of incubation but also promoted polymerization. These results suggest a dynamic redistribution and reorganization of actin cytoskeleton. This study corroborates with the report of Jennings et al., which demonstrate that a 15-s stimulation of platelets with thrombin increases the amount of F-actin by 65%. [37]. In addition to this, in the present study, it has been found that the microtubular network is not significantly modified by STa induction. We have reported earlier that 1-min stimulation with STa strongly increased the amounts of PKC-α association with membrane [8]. In the present study, we have shown that within the same period of time STa stimulation strongly increased the amounts of microfilaments association with membrane. From these data, it may be assumed that STa triggers the association of PKC-α and microfilaments in the membrane. To confirm this in the STa-stimulated membrane fraction, actin is coimmunoprecipitated with PKC-α by using anti PKC-α antibody. It has been found that PKC-α is not only present in the membrane but also closely associated with microfilaments in the membrane fraction. This result corroborates with findings of Cote et. al. (1996) [20]. It was already reported that PKC-α translocation requires an intact cytoskeleton [38]. To identify the specific cytoskeleton proteins such as actin or microtubule involved in PKC-α translocation, the effects of cytochalasin D and nocodazole have been examined. Our study reveals that, prior to toxin treatment, incubation of cells with cytochalasin D inhibits the translocation of PKC-α from cytosol to membrane fraction. Whereas incubation of cells with nocodazole prior to toxin treatment does not have any effect on the translocation of PKC-α from cytosol to membrane. These findings suggest that microfilaments are involved in the translocation process not the microtubule filaments. In the present study, we have shown that even in absence of microtubules, PKC-α translocates from cytosol to membrane. This observation suggests that the blockage of PKC-α translocation by actin depolymarization is not the result of common cytoskeleton derangement, because it is not the case of tubulin depolymerization. Rather it is due to the requirement of highly selective interaction of PKC-α with the actin cytoskeleton, which in turn required for the translocation of the enzyme. This contrast with another report showing that the translocation of PKC-α is maintained in cytochalasin-D-treated GH3B6 cells [39]. In conclusion, the present study demonstrates that the organization of the actin cytoskeleton is rapidly rearranged following induction of E. coli STa in human colonic carcinoma cell line COLO205. It is in dynamic equilibrium between Triton X-100-insoluble and Triton X-100-soluble microfilaments. We do not observe any changes in total actin content under STa stimulation, indicating that STa promotes the polymerization of actin either directly or indirectly without modification of the total actin content. In addition, we have shown for the first time the involvement of intact actin-based cytoskeleton in STa-mediated PKC-alpha translocation. Therefore, we have proposed that in colonic carcinoma cells, the actin cytoskeleton network allows PKC-α to reach the appropriate position at the membrane, where it is likely to interact and phosphorylate its target substrates.
Acknowledgments We wish to thank Dr. G.B. Nair, Director, National Institute of Cholera and enteric Diseases, Kolkata, for his encouragement throughout the study. For confocal microscopic study, we are grateful to the Head, Department of Biotechnology, Ballygunge Science College, Kolkata and Prof. Gouri Sankar Sa, Bose Institute Kolkata. Funding supported by the Indian Council of Medical Research (New Delhi, India) in the form of Senior Research Fellowship to the authors N.M. and D.P. is also highly acknowledged.
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Biochimica et Biophysica Acta j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / b b a g e n
Reorganization of cytoskeletal proteins by Escherichia coli heat-stable enterotoxin (STa)-mediated signaling cascade Nibedita Mahata, Debasis Pore, Amit Pal, Manoj K. Chakrabarti ⁎ Division of Pathophysiology, National Institute of Cholera and Enteric Diseases, P-33, C.I.T. Road, Scheme-XM, Beliaghata, Kolkata-700010, West Bengal, India
a r t i c l e
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Article history: Received 21 October 2009 Received in revised form 8 March 2010 Accepted 10 March 2010 Available online 23 March 2010 Keywords: Escherichia coli heat-stable enterotoxin Cytoskeleton Cytochalasin D PKC-α COLO-205 cell line
a b s t r a c t Background: IP3-mediated calcium mobilization from intracellular stores activates and translocates PKC-α from cytosol to membrane fraction in response to STa in COLO-205 cell line. The present study was undertaken to determine the involvement of cytoskeleton proteins in translocation of PKC-α to membrane from cytosol in the Escherichia coli STa-mediated signaling cascade in a human colonic carcinoma cell line COLO-205. Methods: Western blots and consequent densitometric analysis were used to assess time-dependent redistribution of cytoskeletal proteins. This redistribution was further confirmed by using confocal microscopy. Pharmacological reagents were applied to colonic carcinoma cells to disrupt the microfilaments (cytochalasin D) and microtubules (nocodazole). Results: STa treatment in COLO-205 cells showed dynamic redistribution and an increase in actin content in the Triton-insoluble fraction, which corresponds to an increase in polymerization within 1 min. Moreover, pharmacological disruption of actin-based cytoskeleton greatly disturbed PKC-α translocation to the membrane. Conclusions: These results suggested that the organization of actin cytoskeleton is rapidly rearranged following E. coli STa treatment and the integrity of the actin cytoskeleton played a crucial role in PKC-α movement in colonic cells. Depolymerization of tubulin had no effect on the ability of the kinase to be translocated to the membrane. General significance: In the present study, we have shown for the first time that in colonic carcinoma cells, STa-mediated rapid changes of actin cytoskeleton arrangement might be involved in the translocation of PKC-α to membrane. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Heat-stable enterotoxin (STa), a major cause of watery diarrhea in human, is secreted by enterotoxigenic Escherichia coli (ETEC) [1]. Two hundred eighty million episodes of diarrhea due to ETEC are estimated annually in children less than 5 years old in developing countries [2]. STa binds with cell surface receptor guanylyl cyclase C (GC-C) and leads to the rise of intracellular cGMP level. cGMP stimulates the cGMP-dependent protein kinases that regulate the activation of CFTR channels and Cl− secretion [3,4]. But the specific signaling pathway behind E. coli STa induced rise in cGMP activity and its secretory response is still not clear [5]. Previous reports from our laboratory [6–8] and others [9,10] suggested that besides cGMP, other signaling molecules have been anticipated in the STa-mediated
Abbreviations: cGMP, cyclic GMP; STa, E. coli heat-stable enterotoxin; PKC-α, protein kinase C-α; PMA, phorbol 12-myristate-13-acetate; BSA, bovine serum albumin; Ca2+, calcium ⁎ Corresponding author. Tel.: +91 33 2370-5533; fax: +91 33 2350 5066. E-mail address: [email protected] (M.K. Chakrabarti). 0304-4165/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagen.2010.03.006
intestinal secretion. It has been reported earlier by our laboratory that STa causes an inositol-triphosphate (IP3)-mediated release of Ca2+ from intracellular store [6,7]. It is also reported that by perturbing the Ca2+ homeostasis, toxin may also affect the cytoskeletal architecture and alter the barrier function of epithelial cells, because of the critical role of Ca2+ in the regulation of cytoskeleton dynamics induced by bacteria [11–13]. However, the basis of this dynamic regulation of cytoskeleton remains to be defined [11]. Furthermore, it has also been reported by us that STa induces an increase in [Ca2+]i that leads to the activation and translocation of the protein kinase C-α (PKC-α) from cytosol to membrane, which might be required for the stimulation of a membrane-bound guanylyl cyclase [8]. However, the molecular mechanism behind the translocation of PKC-α is still not clear [14]. The present study has been aimed to investigate the reorganization of cytoskeletal proteins and their role in translocation of PKC-α to membrane in the E. coli STa-mediated signaling mechanism in a human colonic carcinoma cell line COLO-205. Our results clearly demonstrate that actin network is significantly rearranged in STatreated COLO-205 colonic carcinoma cell line and this rearrangement leads to the translocation of PKC-α.
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2. Materials and methods 2.1. Materials The chemicals used in the present study were obtained from the following sources: RPMI-1640 medium, fetal bovine serum (FBS), trypsin-EDTA, Hank's balanced salt solution, sodium bicarbonate, glucose and sodium pyruvate from Gibco BRL, USA; Dimethyl sulfoxide (DMSO), Phenylmethylsulphonylfluoride (PMSF), Dithiothreitol (DTT), Leupeptin, Aprotinin, E. coli heat-stable enterotoxin (STa), phorbol 12-myristate-13-acetate (PMA), penicillin, streptomycin sulfate, cytochalasin D, Nocodazole, taxol, PIPES, TRITC (tetramethylrhodamine B isothiocyanate)-conjugated phalloidin , and isoformspecific antibody against PKC-α from Sigma, USA; Nonidet P-40 from Pierce; anti-actin antibody from Santa Cruz Biotechnology; anti-tubulin antibody from Cell Signaling and secondary antibodies from Jackson ImmunoResearch. All other reagents were of analytical grade, and deionized water was used throughout the study. 2.2. Cell culture and preparation of viable cells and cell treatment COLO-205 cells, which were procured from National Centre for Cell Sciences (NCCS), Pune, India, were routinely cultured in tissue culture flasks and grown up to monolayer in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 2.4 g/l sodium bicarbonate, 100 U/ml penicillin, and 100 μg/ml streptomycin sulfate in humidified 5% CO2 atmosphere at 37 °C.Confluent monolayer were passaged using trypsin-EDTA. Viability of the cells was routinely checked by trypan blue exclusion. After reaching 70% confluence, cells were washed with Hank's balanced salt solution (HBSS) and cultured in serum-free RPMI 1640 medium for 24 h and then used for experimental purpose. For the dynamics study of the cytoskeleton, proteins COLO-205 cells were treated with or without 5nM STa in serum-free medium for different time periods. COLO-205 cells were exposed to different drugs at the following concentrations: 1 mM cytochalasin D, 1 mM nocodazole in serum free medium for 1 h at 37 °C. 2.3. Preparation of microtubules Microtubules were prepared from COLO-205 cells (2 ×106 cells/ml) according to Solomon et al. (1986) [15] with some modification. The cells were treated with 1 mM taxol for 2 h before extraction of microtubules, which in turn stabilizes microtubules without promoting polymerization. The culture medium was then aspirated and replaced with 0.1 M PIPES buffer(PIPES buffer containing 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, 0.04 TIU/ml aprotinin, 2 mM phenylmethylsulfonylfluoride, and 1 mM benzamidine, pH 6.9) containing 1 mM taxol. Cells were scraped from the substratum with a cell scraper and centrifuged at 1000×g for 5 min at room temperature. The cell pellet was then resuspended with PIPES buffer (0.1 M PIPES buffer containing 2 M glycerol, 5 mM MgCl2, 2 mM EGTA, 0.04 TIU/ml aprotinin, 2 mM phenylmethylsulfonylfluoride, and 1 mM benzamidine, pH 6.9) containing 1% Nonidet P-40 and 1 mM taxol and incubated for 15 min at 37 º C. After the incubation, the suspension was centrifuged at 1000 ×g for 5 min at 37 °C. The pellet consisting of the microtubules was then solubilized in 125 mM Tris buffer, pH 6.8, containing 4% SDS, 20% glycerol, and 10% 2-mercaptoethanol and heated to 100 °C for 5 min. After centrifugation at 10,000 ×g for 5 min, the supernatant was stored at −20 º C for subsequent Western blot analysis. For total cell extracts, same number of cells was taken and washed twice with HBSS buffer and solubilized as described above. 2.4. Preparation of microfilaments Microfilament-enriched preparations were extracted from COLO205 cells (2×106 cells/ml) as described by Phillips et al. (1980) [16,17]. Culture medium was aspirated and changed to HBSS buffer. Cells were scraped from the tissue culture flasks with a cell scraper. Cells were
centrifuged at 100 ×g for 5 min at room temperature. The pellet was dissolved in 500 µl of Triton solution (1% Triton X-100, 10 mM EGTA, and 0.1 M Tris–HCl, pH 7.4) and kept on ice for 10 min. The preparation was then centrifuged at 8000 ×g for 5 min at room temperature. Tritonsoluble G-actin fraction was contained in the supernatant. The pellet, which corresponds to the Triton-insoluble fraction, was solubilized in 2% SDS-2% 2-mercaptoethanol (v/v) by boiling at 100 °C for 10 min. 2.5. Preparation of cytosolic and membrane-bound PKC-α Cytosolic and membrane-bound PKC-α were prepared according to Datta Gupta et al. (2005) [8] with little modification. Confluent monolayers of COLO-205 cells (2× 106 cells/ml) were treated in the presence and absence of STa or with disruptors for 1 h before stimulation of STa for 1 min. The reactions were terminated using the ice-cold Hank's balanced salt solution (HBSS). Cells were washed twice in 20 mM Tris–HCl (pH 7.5), suspended in the same buffer containing PMSF, and leupeptin and then homogenized with a motor driven Teflon homogenizer (REMI Udyog, India). The homogenate was centrifuged for 60 min at 30,000 rpm at 4 °C. The Supernatant was separated from pellet and used for measuring the cytosolic PKC-α [18]. The pellet was then resuspended in 20 mM Tris–HCl (PH 7.5) containing PMSF and leupeptin, homogenized briefly in the presence of 1% Triton X-100 to solubilize the membrane-bound proteins and recentrifuged as before (for 60 min at 30,000 rpm at 4 °C). The resulting supernatant served as the source of membrane-bound PKC-α [19]. 2.6. Isolation of cytosolic and membrane fraction Membrane and cytosolic fractions were obtained by using the method of Cote et. al. (1996) [20]. Briefly, the confluent monolayer of COLO-205 cells (2×106 cells/ml) were treated with STa (5nM) and incubated at 37 °C for 0–120 s, cells were washed twice with HBSS buffer and then with 10 mM ice-cold Tris–HCl buffer (containing 0.5 mM EDTA, 1 mM MgCl2, 1 µg/ml leupeptin, 0.21 µg/ml aprotinin1mM PMSF, and10 µM benzimidine, pH 8.0). The cells were then scraped and homogenized in the same buffer. Cell extracts were centrifuged at 800 ×g for 5 min, and the supernatant was then centrifuged at 15,000 ×g for 30 min. After centrifugation, the resulting supernatant was used as cytosolic fraction. The pellet was then resuspended in 50 mM Tris–HCl buffer (containing 2 mM EDTA, 5 mM MgCl2, and 250 mM sucrose) to obtain the membrane fractions. 2.7. Immunoprecipitation Immunoprecipitation was performed according to Ron et al. (1999) [21] with little modification. Each membrane fraction (prepared as described above) was pre-cleaned by incubation with 50 μl of protein A/G plus agarose (Santa Cruz Biotechnology) for 2 h at 4 °C. The samples were centrifuged at 200×g for 1 min, to collect the supernatant, and protein content was measured. Immunoprecipitation was performed overnight at 4 °C with 5 μg of actin antibody per 500 μg protein diluted with an equal volume of immunoprecipitation buffer (2% Triton X-100, 300 mM NaCl, 20 mM Tris–HCl (pH 7.4), 2 mM EDTA, 2 mM EGTA, 0.4 mM sodium vanadate, 1 mM PMSF, leupeptin and aprotinin). Fifty microliters of protein A/G plus agarose was then added, and the samples were incubated at 4 °C for 2 h. Finally, the samples were centrifuged at 14,000×g for 10 min, the supernatant was discarded, and the pellet was resuspended in SDS protein gel loading solution. It was then subjected to immunoblotting by using anti-PKC-alpha antibody. It was also subjected to immunoblotting by using anti-PKC-βII antibody to exclude unspecific binding to actin. 2.8. Immunoblotting and densitometric analysis Western blot analysis was done to detect the actin, tubulin, and PKC-α by using anti-actin, anti-tubulin, and anti-PKC-α antibodies.
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Samples from a same number of cells were compared in each experiment. Samples were added to SDS-PAGE buffer and kept at boiling bath for 5 min to solubilize the proteins [22]. Then the samples were subjected to 12.5% SDS-polyacrylamide gels for separation and then transferred to nitrocellulose membrane (pore size 0.45 µm, BIORAD, USA) using a transblot apparatus (BIO-RAD, USA) [23]. Membranes were blocked with 5% non-fat dry milk in TTBS [Trisbuffered saline (TBS, pH 7.5) containing 0.1% Tween-20] for overnight at 4 °C. After three washes with TTBS, membranes were incubated with anti-tubulin (dilution, 1:1000), anti-actin (dilution, 1:1000), or antiPKC-α (dilution, 1:1000) in TTBS containing 0.5% non-fat dry milk for 2 h at room temperature, followed by four washes with TTBS. Detection was accomplished using alkaline phosphatase-conjugated anti-mouse antibody for actin, anti-rabbit for tubulin (Jackson ImmunoResearch), and anti-rabbit for PKC-α (Jackson ImmunoResearch) in TBS containing 0.5% non-fat dry milk for 1 h at room temperature. Immunoreactive bands were visualized with NBT-BCIP reagent, which was used as the chromogenic substrate. Immunoreactive bands were photographed, and then images were digitized and analyzed by using Bio-Rad QUANTITY 1 software of the gel documentation system. Immunoreactive bands were quantitated and expressed as the ratio of each band density to control band density. 2.9. Assay of protein kinase C-α Cytosolic and membrane fraction, from the treated and untreated cells as described earlier, were prepared from COLO-205 cell line. Using that protein samples activity of protein kinase C-α was measured. PKCα activity in the cytosolic and membrane fractions was measured by a non-radioactive ELISA that utilizes a synthetic PKC pseudo-substrate
Fig. 1. Western blot analysis of the effect of E. coli STa (5 nM) on the actin content in Triton X-100-insoluble microfilaments fractions in COLO-205 cell line. Cells were deprived of serum overnight and stimulated with STa (5 nM) for 0–120 s. Triton X-100insoluble microfilaments fractions were prepared from the STa-treated and untreated cells according to the Materials and methods section. (A) Triton X-100-insoluble fraction. (B) Whole cell homogenate. Triton X-100-insoluble microfilaments content and total amount of microfilaments in whole cell homogenate were analyzed by immunoblotting with mouse anti-actin monoclonal antibody. Arrow indicates the 42 kDa actin-specific bands. (C) Densitometric analysis of immunoreactive bands of actin. Immunoreactive bands were photographed, and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean ± SEM (n = 3) and p b 0.05.
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and a monoclonal antibody that recognizes the phosphorylated form of the peptide, according to the manufacturer's instructions (Calbiochem, EMB Biosciences, San Diego, CA, USA). Protein concentration was determined by a Bradford method [24], with BSA as standard using manufacturer's protocol. PKC-α activity was expressed as OD/mg protein and the data normalized to control. 2.10. Immunofluorescence study For immunofluorescence study, the cells were plated to L-lysinecoated glass coverslips and kept in the incubator (5% CO2 and 37 °C) in humidified atmosphere for a minimum of 48 h to allow cell attachment and spreading. After incubation, the cells were washed with PBS. The cells were washed three times with PBS at every step in this immunofluoresence experiment. Following toxin treatment, both control and treated cells were fixed with 2% paraformaldehyde for 45 min at room temperature and permeabilized with 0.2%Triton-X 100 in PBS for 30 min at room temperature. The cells were incubated with blocking solution (5% bovine serum albumin in PBS) for 4 h at room temperature. For F-actin detection, the cells were stained with 50 µg/ ml of TRITC (Tetramethyl-rhodamine B isothiocyanate)-conjugated phalloidin in PBS at room temperature for 40 min. Cells were then mounted on microscope slides with glycerol phosphate buffer (2:1). The cells were visualized using a fluorescence microscope (Nikon) and photographed at magnification of ×100. 2.11. Statistical analysis Results were expressed as the mean ± standard error of the mean (SEM), where applicable, of at least three independent experiments. The data were analyzed by Student's t-test .The p values of b0.05 were considered as significant.
Fig. 2. Western blot analysis of the effect of E. coli STa (5 nM) on actin content the Triton X-100-soluble microfilaments fractions in COLO-205 cell line. Cells stimulated with 5nM STa for 0–120 s and Triton X-soluble fractions were prepared from control cells and the treated cells as described in the Materials and methods section. (A) Triton X-100-soluble fraction. (B) Whole cell homogenate. Triton X-100-soluble microfilaments content and total amount of microfilaments in whole cell homogenate were analyzed by immunoblotting with mouse anti-actin monoclonal antibody. Arrow indicates the 42 kDa actin-specific bands. (C) Densitometric analysis of immunoreactive bands of actin. Immunoreactive bands were photographed and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean± SEM (n = 3) and p b 0.05.
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Fig. 3. Western blot analysis of the effect of E. coli STa (5 nM) on the membrane and cytosol associated microfilaments content in COLO-205 cell line. Membrane fractions were prepared (as described in the Materials and methods section) from STa-treated cells in time course experiment (0–120 s) , resolved on an 12.5% SDS-polyacrylamide gel, transferred to nitrocellulose membrane and probed with mouse monoclonal anti-actin antibody. (A) Membrane fractions. (B) Cytosolic fractions. (C) Whole cell homogenate. Arrow indicates the 42 kDa actin-specific bands. (D) Densitometric analysis of immunoreactive bands of actin. Immunoreactive bands were photographed, and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean ± SEM (n = 3) and p b 0.05.
Fig. 4. Western blot analysis of the effect of E. coli STa (5 nM) on the microtubule content in the colonic carcinoma cell line. COLO-205 cells were incubated with 5nM STa for 0, 15, 30, 60, 90, or 120 s, and microtubule fractions were isolated as described in the Materials and methods section. A Western blot analysis of the membrane fraction was conducted with rabbit monoclonal anti-tubulin antibody, followed by a secondary alkaline phosphatase conjugated antibody. (A) Microtubule fraction. (B) Whole cell homogenate. Arrow indicates the 53 kDa tubulin-specific bands. (C) Densitometric analysis of immunoreactive bands of microtubule. Immunoreactive bands were photographed, and then images were digitized and analyzed. Immunoreactive bands were quantitated and compared with the whole cell lysate band density. Data represent the mean ± SEM (n = 3) and p b 0.05.
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3. Results 3.1. Effect of STa on the distribution of microfilaments and microtubules To evaluate dynamic changes in the levels of microfilaments and microtubules following STa treatment in COLO-205 cells, filamentous (Fig. 1A) and soluble forms of microfilaments (Fig. 2A) and microtubules (Fig. 4A) preparations were analyzed by immunoblotting for their respective contents of actin and tubulin. Densitometric analysis indicated that STa (5nM) treatment induced a timedependent increase in the level of actin in Triton X-100-insoluble microfilaments fraction (1.40 ± 0.02, 2.36 ± 0.02 and 3.72 ± 0.02 fold increase, lane 2: 15 s, lane 3: 30 s, lane 4: 60 s, respectively, compared to control in lane 1 in Fig. 1C; n = 3, p b 0.05) and decreased thereafter (i.e., 90 s and 120 s, Fig. 1C). In contrast, Fig. 2C showed that the level of actin in Triton-soluble fraction decreased with time (0.73 ± 0.03, 0.47 ± 0.04, 0.35 ± 0.025 fold, lane 2: 15 s, lane 3: 30 s, lane 4: 60 s, respectively, compared to control in lane 1, P b 0.05). In addition, it was also found that membrane-associated actin content became maximum (2.68 ± 0.02 fold increase; n = 3; p b 0.05) at 1 min of toxin treatment (Fig. 3A and D). Thereafter, membrane-associated actin content decreased but remained higher than the basal value (Fig. 3A and D). In contrast, within the same experimental period, the actin content in the cytosolic fraction became minimum (0.327 ± 0.03 fold in lane 4 compared to control, Fig. 3B and D). This dynamic association of actin with the membrane (Fig. 3) and the increase in actin content in the Triton-insoluble fraction (Fig. 1) correspond to an increase in polymerization within 1 min of STa treatment, which was not due to in new synthesis of actin, as total levels of actin in the cell lysate did not change during the experimental period with STa. Unlike membrane-associated microfilaments (Fig. 3A), we did not find any change in the level of microtubules associated with membrane in STainduced COLO-205 cell (data not shown). Similarly, it was also found that toxin treatment did not promote any polymerization of filamentous microtubules in comparison to the control (Fig. 4). Like actin, the total amount of tubulin in cell homogenates did not change during the experimental period.
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predominantly present in the membrane (0.89 ± 0.025, compared to control; Fig. 6C, lane 2 and Fig. 6D, bar 2) and decreased in the cytosol (0.152 ± 0.02, compared to control; Fig. 6A, lane 2 and Fig. 6B, bar 2). 1 h preincubation with cytochalasin D (a microfilament disrupting agent) blocked the STa-induced translocation of PKC-α (lane 3 of
3.2. Fluorescence studies Immunofluorescence studies were conducted to further confirm the redistribution of microfilaments within the cell. We have examined more than 50 cells originating from 3 independent experiments for each set of experiment. We had stained COLO-205 cells with rhodamineconjugated phalloidin for F-actin detection. Fig. 5 showed the structural distribution of F-actin in untreated (Fig. 5A) and STa-treated cells (Fig. 5B, C). In the untreated cells, F-actin filament was distributed throughout the cytosol surrounding the nucleus. Whereas in the STatreated cells for 1 min F-actin was condensed as a ring in the periphery of the cell (Fig. 5B). Fig. 5C represented the cells treated with STa for 120 s, which had a very similar F-actin filament distribution to that of control. A kinetic analysis of the effect of STa on the F-actin reorganization showed that the effect started at 15 s and lasted for 60 s of treatment (data not shown). 3.3. Effect of cytoskeleton disruptors on the translocation of PKC-α Cytosolic and membrane fractions of the COLO-205 cell line were investigated by immunoblot technique using PKC-α specific antibody to identify the effect of cytoskeleton disruptors in the translocation process of PKC-α. Immunoreactive bands of PKC-α demonstrated that in untreated COLO-205 cell, it was predominantly present at the cytosol (0.811 ± 0.012, compared to control; Fig. 6A, lane 1 and Fig. 6B, bar 1) and a little or no PKC-α present at the membrane fraction (0.21 ± 0.02, compared to control; Fig. 6C, lane 1 and Fig. 6D, bar 1). After 1-min incubation with STa (5nM), PKC-α was
Fig. 5. Effect of STa on the actin cytoskeleton in subconfluent COLO-205 colonic carcinoma cells. Laser scanning confocal imaging of actin in control and STa-treated COLO-205 cells. Cells were cultured for 3 days on L-lysine-coated coverslips and then incubated for various time periods (B: cells treated for 60 s with 5nM of STa, C: cells treated for 120 s with 5nM of STa) in serum-free medium in absence (A) or presence of 5nM of STa (B and C). After paraformaldehyde fixation and permeabilization with 0.1% triton X-100. Cells were processed for immunolabeling using Tetramethyl-rhodamine B isothiocyanate (TRITC)-conjugated phalloidin as described in the Materials and methods section. Images represent those obtained in 3 individual experiments in which a total of N 50 cells were evaluated from each experimental group. The cells were photographed at magnification of × 100.
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Fig. 7. Immunoprecipitation analysis of the effect of STa on the level of PKC-α associated with actin in colonic carcinoma cell line. COLO-205 cells were incubated in 37 °C in serum-free medium in absence (lane 1) or presence of 5nM STa for 1 min (lanes 2, 3). Membrane fractions were immunoprecipitated with anti-actin antibody as described in the Materials and methods section. Immunoblots were performed with anti-PKC-α monoclonal antibody (lanes 1 and 2) and anti-PKC-βII monoclonal antibody (lane 3). Arrow indicates the 80-kDa PKC-α-specific bands. Blots are representative illustrations of results obtained in three independent experiments.
Fig. 6A and C and bar 3of Fig. 6B and D). Whereas pretreatment of COLO-205 cells with nocodazole (a microtubules disrupting agent) for 1 h did not block the STa-induced translocation of PKC-α significantly (lane 4 of Fig. 6A and C and bar 4 of Fig. 6B and D). PMA was used as a positive control for PKC-α activation. These results indicate that intact actin cytoskeleton is required for the translocation of PKC-α. 3.4. Association of PKC-alpha with cytoskeleton The level of actin associated with membrane was also found to increase after 1 min (3.37 ± 0.02 compared to the control; Fig. 3). In addition, 1-min incubation with STa strongly increased the amount of PKC-alpha associated with the membrane (Fig. 6C, lane 3). Densitometric analysis indicated that the level of PKC-alpha associated with the membrane was increased by 3.9 ± 0.7 fold (n = 3; p b 0.05 compared to the control; Fig. 6D, bar 2) after 1 min. This transient increase in PKC-alpha in the membrane preparation might be associated with the membrane-associated actin .To confirm the association of PKCalpha with actin, membrane fractions were immunoprecipitated with anti-actin antibody and then processed for immunoblotting analysis with anti-PKC-alpha antibody. The results presented in Fig. 7 confirm that PKC-alpha was associated with actin in the membrane fraction after STa stimulation (lane 2). To exclude unspecific binding to actin, membrane fraction was subjected to immunoblotting by using antiPKC-βII antibody (Fig. 7, lane 3).
Fig. 6. Western blot analysis of protein kinase C (PKC)-α translocation in the presence of microfilament disruptor (cytochalasin D) and microtubules disruptor (nocodazole) in the colonic carcinoma cell line. COLO-205 cells were treated for 0 s (Control) or 60 s with 5 nM STa. Cells were also treated with STa for 60 s after 1-h exposure to cytochalasin D (1 mM) or nocodazole (1 mM). Then cytosolic and membrane fraction were prepared as described in the Materials and methods section. In this study, cytosolic and membrane fractions were loaded in the respective lanes. PMA, a known activator of PKC isoenymes that translocate PKC-α from cytosol to membrane, was used as a positive control for PKC-α translocation. (A) Representative Western blot of cytosolic fraction of PKC-α. Lane P: cytosolic fraction of the PMA-treated cells (positive control). Lane 1: cytosolic fraction of untreated cells (control). Lane 2: cytosolic fraction of STa treated cells. Lane 3: cytosolic fraction of cytochalasin-D-treated cells prior to STa treatment. Lane 4: cytosolic fraction of nocodazole treated cells prior to STa treatment. Arrow indicates the 80 kDa PKC-α-specific bands. (B) Desitometric analysis of the Western blot data of the cytosolic fraction of PKC-α expressed as the ratio of each band density to positive control (PKC-α) band density. Bar 1: cytosolic fraction of untreated cells (Control). Bar 2: cytosolic fraction of STa-treated cells. Bar 3: cytosolic fraction of cytochalasin-D-treated cells prior to STa treatment. Bar 4: cytosolic fraction of nocodazole treated cells prior to STa treatment. Data represent the mean ± SEM (n = 3) and p b 0.05. (C) Representative Western blot of membrane fraction of PKC-α. Lane P: membrane fraction of the PMA-treated cells (positive control). Lane 1: membrane fraction of untreated cells (Control). Lane 2: membrane fraction of STa-treated cells. Lane 3: membrane fraction of cytochalasin-D-treated cells prior to STa treatment. Lane 4: membrane fraction of nocodazole-treated cells prior to STa treatment. Arrow indicates the 80 kDa PKC-α-specific bands. (D) Desitometric analysis of the Western blot data membrane fraction of PKC-α expressed as the ratio of each band density to positive control (PKC-α) band density. Bar 1: membrane fraction of untreated cells (Control). Bar 2: membrane fraction of STa-treated cells. Bar 3: membrane fraction of cytochalasin-Dtreated cells prior to STa treatment. Bar 4: membrane fraction of nocodazole-treated cells prior to STa treatment. Data represent the mean± SEM (n = 3) and p b 0.05.
Fig. 8. Effect of STa, cytochalasin D, and nocodazole on PKC-α activity COLO-205 cell. COLO-205 cells were treated with and without STa or with cytochalasin D (1 mM) and nocodazole (1 mM) separately for 1 h before stimulation with 5nM STa for 1 min and PKC activity in the cytosol and membrane particulate fraction was determined by using non-radioactive ELISA. Data are normalized to cytosolic control, and values are expressed as mean ± SEM (p b 0.05; n = 6).
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3.5. Effect of cytoskeleton disruptors on the activity of PKC-α We tested the effect of cytoskeleton disruptors on the PKC-α activity upon STa exposure in COLO-205 cells by using a non-radioactive ELISA. Under control conditions, PKC-α activity was found mainly in the cytosolic compartment and translocated to the membrane compartment upon activation, but total PKC-α activity in the whole cell homogenate remained unchanged. Moreover, it is also known that the increase in the ratio of membrane to cytosol activity accurately reflects activation of PKC-α [25–27]. PKC-α activity in cytosolic and particulate membrane fractions was quantified after STa treatment to COLO-205 cells for 1 min. STa exposure for 1 min significantly increased PKC-α activity in the particulate fraction (1.11 ± 0.06 per mg of protein; n = 6; p b 0.05), whereas activity was decreased in the cytosol (0.44 ± 0.03 per mg of protein; n = 6; p b 0.05) (Fig. 8). This increase in the ratio of membrane to cytosol activity clearly indicates activation of PKC-α. PKCα activity was not increased in the membrane fraction upon 1 min STa induction in COLO-205 cells preincubated with cytochalasin D (1 mM) for 1 h (0.37 ± 0.03 per mg of protein; n = 6; p b 0.05). In contrast PKC-α activity was significantly increased in the membrane fraction upon 1 min STa induction in COLO-205 cells preincubated with nocodaloze (1 mM) for 1 h (1.07± 0.06 per mg of protein; n = 6; p b 0.05) (Fig. 8). These findings suggest that microtubule filaments are not involved in the translocation PKC-α. 4. Discussion STa is an 18–19 amino acid [28–30] containing methanol soluble [31] heat-stable toxin, which triggers a cascade of reaction including increase of intracellular calcium levels [Ca2+], and activation and translocation of PKC-α [6–8] in the colonic carcinoma cell line. But the exact mechanisms involved in the STa-mediated translocation of PKCα have not yet been clarified. With regard to the molecular mechanism behind PKC translocation, it is tempting to implicate the cytoskeleton, based on the previous works where only an association between PKC isoforms and cytoskeletal component have been reported [32–34]. Therefore, in the present study, we have evaluated the involvement of cytoskeletal proteins in the E. coli heat-stable enterotoxin (STa) mediated translocation of PKC-α in COLO-205 cell line. The cytoskeletal proteins are organized as the filamentous networks distributed throughout the entire cell extending from the plasma membrane to the interior of the nucleus. These proteins account for a large fraction of total cellular protein and are involved in all structural and dynamic aspects of the living cells, including maintenance of the cell morphology, cell adhesion, motility, replication, apoptosis, differentiation, and cell signaling [35]. Cytoskeleton exists in dynamic form in response to extracellular signals [36]. Here we have investigated the effect of cytoskeleton upon E. coli heat-stable enterotoxin (STa) treatment in the colonic carcinoma cell line COLO205. Densitometric analysis of the membrane fraction reveals that 1min incubation of the cells with STa is sufficient to induce a huge increase in membrane association of actin compared to control, that is, actin is redistributed upon STa activation. This rapid increase in membrane-associated actin during STa stimulation signifies the importance of the role of microfilaments in the early events of STa action. Furthermore, actin content of cytosolic fraction decreases after STa stimulation. This redistribution study of microfilaments within the cell is further confirmed by the immunofluorescence studies. Whereas time-dependent study of the Triton X-100-insoluble fraction of microfilaments content reveals that STa increases Triton X-100insoluble F-actin content, which has been found to increase by more than 3-fold within 1 min of toxin treatment in comparison to the control. In contrast, time-dependent study of the Triton X-100-soluble fraction of microfilaments content demonstrates that STa decreases Triton X-100-soluble G-actin content within 1 min of toxin treatment
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compared to the control which indicates that STa not only induces actin redistribution within 1 min of incubation but also promoted polymerization. These results suggest a dynamic redistribution and reorganization of actin cytoskeleton. This study corroborates with the report of Jennings et al., which demonstrate that a 15-s stimulation of platelets with thrombin increases the amount of F-actin by 65%. [37]. In addition to this, in the present study, it has been found that the microtubular network is not significantly modified by STa induction. We have reported earlier that 1-min stimulation with STa strongly increased the amounts of PKC-α association with membrane [8]. In the present study, we have shown that within the same period of time STa stimulation strongly increased the amounts of microfilaments association with membrane. From these data, it may be assumed that STa triggers the association of PKC-α and microfilaments in the membrane. To confirm this in the STa-stimulated membrane fraction, actin is coimmunoprecipitated with PKC-α by using anti PKC-α antibody. It has been found that PKC-α is not only present in the membrane but also closely associated with microfilaments in the membrane fraction. This result corroborates with findings of Cote et. al. (1996) [20]. It was already reported that PKC-α translocation requires an intact cytoskeleton [38]. To identify the specific cytoskeleton proteins such as actin or microtubule involved in PKC-α translocation, the effects of cytochalasin D and nocodazole have been examined. Our study reveals that, prior to toxin treatment, incubation of cells with cytochalasin D inhibits the translocation of PKC-α from cytosol to membrane fraction. Whereas incubation of cells with nocodazole prior to toxin treatment does not have any effect on the translocation of PKC-α from cytosol to membrane. These findings suggest that microfilaments are involved in the translocation process not the microtubule filaments. In the present study, we have shown that even in absence of microtubules, PKC-α translocates from cytosol to membrane. This observation suggests that the blockage of PKC-α translocation by actin depolymarization is not the result of common cytoskeleton derangement, because it is not the case of tubulin depolymerization. Rather it is due to the requirement of highly selective interaction of PKC-α with the actin cytoskeleton, which in turn required for the translocation of the enzyme. This contrast with another report showing that the translocation of PKC-α is maintained in cytochalasin-D-treated GH3B6 cells [39]. In conclusion, the present study demonstrates that the organization of the actin cytoskeleton is rapidly rearranged following induction of E. coli STa in human colonic carcinoma cell line COLO205. It is in dynamic equilibrium between Triton X-100-insoluble and Triton X-100-soluble microfilaments. We do not observe any changes in total actin content under STa stimulation, indicating that STa promotes the polymerization of actin either directly or indirectly without modification of the total actin content. In addition, we have shown for the first time the involvement of intact actin-based cytoskeleton in STa-mediated PKC-alpha translocation. Therefore, we have proposed that in colonic carcinoma cells, the actin cytoskeleton network allows PKC-α to reach the appropriate position at the membrane, where it is likely to interact and phosphorylate its target substrates.
Acknowledgments We wish to thank Dr. G.B. Nair, Director, National Institute of Cholera and enteric Diseases, Kolkata, for his encouragement throughout the study. For confocal microscopic study, we are grateful to the Head, Department of Biotechnology, Ballygunge Science College, Kolkata and Prof. Gouri Sankar Sa, Bose Institute Kolkata. Funding supported by the Indian Council of Medical Research (New Delhi, India) in the form of Senior Research Fellowship to the authors N.M. and D.P. is also highly acknowledged.
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