Stability of actin cytoskeleton and PKC-delta binding to actin regulate NKCC1 function in airway epithelial cells

Carole M. Liedtke, Melinda Hubbard, and Xiangyun Wang

Warren Alan Bernbaum, M.D. Center for Cystic Fibrosis Research, Department of Pediatrics, Rainbow Babies & Children Hospital, and Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4948


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Activation of airway epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity of protein kinase C (PKC)-delta , which localizes predominantly to the actin cytoskeleton. Prompted by reports of a role for actin in NKCC1 function, we studied a signaling mechanism linking NKCC1 and PKC. Stabilization of actin polymerization with jasplakinolide increased activity of NKCC1, whereas inhibition of actin polymerization with latrunculin B prevented hormonal activation of NKCC1. Protein-protein interactions among NKCC1, actin, and PKC-delta were verified by Western blot analysis of immunoprecipitated proteins. PKC-delta was detected in immunoprecipitates of NKCC1 and vice versa. Actin was also detected in immunoprecipitates of NKCC1 and PKC-delta . Pulldown of endogenous actin revealed the presence of NKCC1 and PKC-delta . Binding of recombinant PKC-delta to NKCC1 was not detected in overlay assays. Rather, activated PKC-delta bound to actin, and this interaction was prevented by a peptide encoding delta C2, a C2-like domain based on the amino acid sequence of PKC-delta . delta C2 also blocked stimulation of NKCC1 function by methoxamine. Immunofluorescence and confocal microscopy revealed PKC-delta in the cytosol and cell periphery. Merged images of cells stained for actin and PKC-delta indicated colocalization of PKC-delta and actin at the cell periphery. The results indicate that actin is critical for the activation of NKCC1 through a direct interaction with PKC-delta .

C2-like domain; coimmunoprecipitation; protein kinase C; rottlerin; jasplakinolide; latrunculin; sodium-potassium-chloride cotransport


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THE AIRWAY EPITHELIAL Na-K-2Cl (NKCC) cotransporter is a member of a large gene family of cation-Cl cotransporters, which are characterized by the coupled transport of Cl with K in the absence (KCC) or presence (NKCC1, NKCC2) of Na (34). Of the cation-Cl cotransporters, NKCC1 is distinguished by its widespread distribution in animal cells. Epithelial NKCC isoforms function during fluid and electrolyte secretion and absorption, depending on the localization of NKCC to basolateral or apical plasma membrane, respectively. NKCC1 function is highly regulated in the diverse cell types expressing the intrinsic membrane protein. Recently, we reported (23) our studies of NKCC1 and its regulation by protein kinase C (PKC)-delta and by intracellular Cl (Cli) in a Calu-3 cell line. The study demonstrated that a Cl electrochemical gradient alone is not sufficient to stimulate NKCC1 activity; rather, increased PKC-delta activity is necessary. These results further implied that NKCC1 function is modulated by intracellular electrolyte concentration, which impacts thermodynamic stability of the interaction between intracellular proteins. Thus elevated Cli may promote prolonged formation of a complex of proteins that might regulate NKCC1.

Ion transport proteins, including NKCC1, have been implicated in cytoskeletal anchoring, with subsequent effects on mechanical stability, cell shape, and assembly of transport proteins in specialized membrane domains (11). The actin cytoskeleton and actin-associated proteins participate in the regulation of NKCC1 as well as other ion transport proteins, including epithelial Na channels (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR). Disruption of actin filament polymerization alters endogenous ENaC and confers sensitivity to cAMP-dependent protein kinase A phosphorylation (29). Actin directly regulates ENaC incorporated into lipid bilayers (18) and has a regulatory effect on ENaC single-channel conductance, open probability, and downregulation by CFTR (2, 15). In addition to actin itself, the actin-associated protein alpha -actinin activates and filamin inhibits ENaC function (4). ENaC also colocalizes with ankrin and spectrin, but the functional consequences of the interaction are not clear (36, 42). Thus an organized actin network is apparently necessary for optimal ENaC function and is also required for cAMP-dependent activation of CFTR. Several studies demonstrate a direct interaction between actin and CFTR (5, 7, 30) and between actin and other ion channels, such cardiac L-type Ca channels (35).

In the intestinal T84 epithelial cell line, manipulation of actin polymerization with phalloidin and cytochalasin D alters cAMP-dependent Cl secretion and NKCC1 function (9, 26). The actin-stabilizing compound phalloidin, a mushroom-derived bicyclic heptapeptide toxin that binds to actin and prevents its depolymerization, prevents activation of NKCC1 and attenuates cAMP-dependent Cl secretion (9) and hypotonicity-induced NKCC1 activity (27). F-actin disassembly induced with the fungal metabolite cytochalasin D diminishes hypertonicity-induced NKCC1 activity. Although a role of the actin cytoskeleton in NKCC1 function has been demonstrated, the molecular nature of the involvement is not clearly understood. One study points to a role for basolateral endocytosis as a link between F-actin and NKCC1 (37). Another possibility is direct interaction between actin and NKCC1 or with another regulatory protein that is necessary or in close proximity to NKCC1.

In this study, we examine the role of the actin cytoskeleton in the regulation of airway epithelial NKCC1, a basolateral ion transporter that is selectively activated by hyperosmotic stress or by alpha 1-adrenergic agonists (21, 22). The actin architecture was manipulated with agents that produce opposing effects on the polymerization state of cytoskeletal actin. From agents capable of affecting the actin cytoskeleton, we selected two actin inhibitors that easily penetrate cellular plasma membranes and have opposite effects on actin microfilaments: latrunculin B and jasplakinolide (38). Jasplakinolide is isolated from the marine sponge Jaspis johnstoni. It is a cyclic peptide with a 15-carbon macrocyclic ring containing three amino acids and has fungicidal and antiproliferative activity. Jasplakinolide promotes polymerization of actin under nonpolymerizing conditions and stabilizes actin filaments (3). Latrunculin B is a membrane-permeant macrolide derived from the marine sponge Latrunculia magnifica. Latrunculin B prevents actin polymerization by binding to G-actin in a nonpolymerizable 1:1 complex. In T84 cells, latrunculin B reduced transepithelial resistance with minimal effects on Cl secretion. In contrast, jasplakinolide inhibited cAMP-dependent Cl secretion and NKCC1 function (27). Our study of airway epithelial NKCC1 function demonstrates that manipulation of the actin cytoskeleton has profound effects on NKCC1 function. The results also demonstrate the interaction of actin with PKC-delta and NKCC1, suggesting that a protein complex is involved in the regulation of NKCC1. We also show that a direct interaction between actin and PKC-delta modulates NKCC1 function and that this interaction involves a C2-like domain on the regulatory arm of PKC-delta .


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Cell isolation and culture. Calu-3 cells were grown in cell culture on 100-mm2 tissue culture plastic, on 0.4-µm-pore size Transwell-Clear polyester filter inserts with a growth area of 4.4 cm2 for transport experiments (Corning Costar, Cambridge, MA), or on a filter insert with a growth area of 1.0 cm2 for immunofluorescence. Cells were maintained as described previously (23) and assessed for confluence by microscopic examination and by measurement of electrical resistance across the cell monolayers grown on filter inserts. Electrical resistance was quantitated with chopstick electrodes and an epithelial voltohmmeter (EVOM; World Precision Instruments, New Haven, CT). Values were corrected for background resistance of the filter alone bathed in medium. Filters were used for experiments when resistance exceeded 250 Omega  · cm2 6-9 days after seeding.

Measurement of NKCC1 activity. NKCC1 activity was measured as bumetanide-sensitive uptake of 86Rb, a congener of K (23). Cells were serum deprived for 24 h before experiments. After EVOM readings were taken, cells were preincubated for 30 min at 32°C after addition of vehicle, 10 µM bumetanide, or indicated inhibitor in HEPES-buffered Hanks' balanced salt solution (HPSS) consisting of (in mM) 10 HEPES, 137 NaCl, 4.2 NaHCO3, 5.8 KCl, 0.3 Na2HPO4, 1.2 CaCl2, 0.4 MgSO4, and 10 glucose, pH 7.5. To initiate radiotracer uptake, filters were transferred to a well of a six-well tissue culture dish containing 1 µCi of 86Rb in HPSS. Influx was measured for a 4-min time interval and then terminated by rapidly immersing filters four times in an ice-cold isotonic buffer consisting of 100 mM MgSO4 and 137 mM sucrose. Intracellular radioactivity was extracted by incubating cell monolayers in 0.1 N NaOH. Aliquots of extract were assayed for radioactive counts by liquid scintillation counting and for protein with a Pierce protein assay kit with bovine serum albumin as the standard. Intracellular radioisotopic content was calculated as nanomoles of potassium per milligram of protein (86Rb).

To determine whether a peptide that inhibits protein-protein interactions is also effective in preventing hormone-stimulated NKCC1 activity, peptides were delivered into Calu-3 cells with a BioPORTER protein delivery system followed by measurement of bumetanide-sensitive 86Rb uptake. BioPORTER reagent was dissolved in methanol, and a 10-µl aliquot per filter insert was transferred to a tube and dried under a stream of N2. To prepare peptide-BioPORTER complexes, the dried reagent was reconstituted in 50 µl of HPSS per filter insert containing an aliquot of peptide in phosphate-buffered saline (PBS). The solution was mixed by repeated pipetting followed by incubation at room temperature for 5 min. The total volume of the peptide-BioPORTER complex per filter insert was increased to 500 µl with serum-free culture medium. Cells were incubated on the apical surface with the peptide-BioPORTER complex or BioPORTER reagent alone for 3 h at 37°C. The apical solution was aspirated and replaced with HPSS. Cells were incubated for 1 h at 32°C before 86Rb uptake was initiated as described above.

Immunoprecipitation and immunoblot analysis. Cells were grown to confluence, serum deprived overnight, and washed with ice-cold PBS. Cells were lysed and proteins were immunoprecipitated as previously described, with modifications (23, 24). In some experiments, Calu-3 cells were treated with the alpha 1-adrenergic agonist methoxamine or, as a control, with HPSS vehicle. To halt stimulation, cells were rapidly immersed in ice-cold PBS and then harvested in 1 ml of lysis buffer consisting of 100 mM NaCl, 50 mM NaF, 50 mM Tris · HCl, pH 7.5, 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM Na vanadate, and protease inhibitor mixture. Lysates were clarified by pretreatment with agarose beads and then incubated with antibody directed against the protein of interest. Immune complexes were recovered with protein G agarose beads, washed, resuspended in Laemmli buffer, and heated for 5 min in a boiling water bath. Western blot analysis of immunoprecipitated protein was used to titrate antiserum and to select an optimal antiserum concentration (22).

NKCC1 was immunoprecipitated from Calu-3 cells as described previously (23). Briefly, cells were lysed in 1 ml of ice-cold 10 mM HEPES (pH 7.4) buffer supplemented with 3.5 mM MgCl2, 150 mM NaCl, 0.3% Triton X-100, 1 mM benzamide, and protease inhibitor mixture. Lysates were precleared by treatment with agarose beads, as described above. A 0.5-ml aliquot of lysate was incubated with 1.1% sodium dodecyl sulfate (SDS) for 1 h at room temperature. The SDS-solubilized lysates were combined with 1.4 ml of 3.0% Triton X-100 in lysis buffer and incubated for 1 h on ice. NKCC1 was immunoprecipitated by overnight incubation at 4°C with 1:2,500 dilution of T4 monoclonal antibody. Immune complexes were incubated with prewashed protein G agarose beads, recovered by centrifugation, washed five times, and subjected to gel electrophoresis on 4-15% gradient slab gels. NKCC1 and coimmunoprecipitated proteins were detected by Western blot analysis with specific antibodies and enhanced chemiluminescence.

G- and F-actin solutions. Monomeric actin (G-actin) was stored in G-actin buffer consisting of (in mM) 2 Tris · HCl, pH 8.0, 0.5 ATP, 0.5 CaCl2, and 0.5 beta -mercaptoethanol at a final concentration of 100 µg/ml. Filamentous actin (F-actin) was polymerized from G-actin by the addition of 2 mM MgCl2 and 50 mM KCl to G-actin in G-actin buffer. The mixture was incubated for 1 h at room temperature.

Expression of recombinant proteins. Recombinant native delta C2 domain was produced with a pET14b expression vector, which placed a polyhistidine tag at the NH2 terminus of the construct. The expression system was kindly provided by Dr. Lodewijk V. Dekker (University College London). For protein production, 400 ml of LB medium containing 50 µg/ml ampicillin was inoculated with an overnight culture of DH5alpha cells transformed with the pET14b-delta C2 construct and grown for 3 h at 37°C. Isopropyl-beta -D-thiogalactopyranoside was then added to a final concentration of 0.1 mM. Cells were incubated for 3-5 h at 37°C and harvested, and recombinant protein was isolated with a B-PER 6XHIS Spin Purification Kit (Pierce, Rockford, IL), according to the manufacturer's instructions. Isolation of fusion protein was monitored by immunoblot analysis with a polyclonal antibody to the HIS6 tag or India HisProbe (data not shown).

Binding assays. In vitro binding of PKC-delta to G-actin or F-actin was studied by two methods. In the first method, 1 µg of G-actin or F-actin was immobilized on polyvinylidene difluoride (PVDF) membrane paper in a slot blot apparatus (GIBCO), blocked, incubated with recombinant PKC-delta in the absence or presence of PKC activators, and washed extensively. rPKC-delta binding was detected by immunoblot analysis. In a second method, a solution binding assay was performed. rPKC-delta was incubated with phosphatidylserine (PtdSer) and dioctanylglycerol (DOG) for 15 min at 30°C to activate PKC-delta . G-actin or F-actin was added, and the incubation continued for 30 min at room temperature. Complexes of PKC-delta and actin were pulled down with anti-actin antibody coupled to agarose beads. Beads were washed five times to remove unbound material and analyzed by immunoblot analysis for PKC-delta .

Immunofluorescence and confocal microscopy. Cell monolayers were washed three times with HPSS and fixed in fresh 4% paraformaldehyde for 15 min at room temperature. Cells were washed three times with PBS and permeabilized with 0.2% Triton X-100 in 10% normal goat serum in PBS. The fixed, permeabilized cells were stained for 1 h at room temperature with a polyclonal antibody directed to PKC-delta (1:100 dilution) or Texas red-phalloidin (1:80 dilution). Cell monolayers were washed three times with PBS. Secondary Oregon green-conjugated anti-rabbit antibody for PKC-delta was applied at 1:200 dilution for 1 h at room temperature. After three final washes with PBS, the polyester filters were carefully cut from their support inserts and mounted in Slow Fade mounting medium on glass microscope slides. Fluorescence was analyzed with a LSM410 confocal scanning microscope (Carl Zeiss) equipped with an external argon-krypton laser. Optical sections of 512 × 512 pixels were digitally recorded in 16× line-averaging mode, and z-sectioning was done in 1-µm increments. Images were processed for reproduction with Photoshop software (Adobe Systems, Mountainview, CA).

Data analysis. Immunoreactive protein bands detected by chemiluminescence were quantitated by laser densitometry. Values are representative of three or more experiments, unless otherwise stated, and treatment effects were evaluated with a two-sided Student's t-test. Values are reported as means ± SE.

Materials. Polyclonal anti-PKC isotype antibodies, horseradish peroxidase-coupled secondary antibodies, anti-actin antibody coupled to agarose beads, and protein G-agarose were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Baculovirus-expressed rPKC isotypes were obtained from Pan Vera (Madison, WI), monoclonal antibodies to beta -actin and to HIS6 tag from Abcam (Cambridge, UK), and monoclonal antibody to the COOH terminus of NKCC1 from the Developmental Studies Hybridoma Bank (Iowa City, IA). The BioPORTER protein delivery system was obtained from Gene Therapy Systems (San Diego, CA). India HisProbe-HRP was purchased from Pierce (Rockford, IL), methoxamine HCl, bumetanide, and chelerythrine from Sigma, rottlerin from Research Products International (Natick, MA), nonmuscle actin (85% beta -actin, 15% alpha -actin) from Cytoskeleton (Denver, CO), and protease inhibitor cocktail set III and latrunculin B from Calbiochem-Novabiochem (San Diego, CA). An enhanced chemiluminescence reagent was purchased from Amersham (Piscataway, NJ), jasplakinolide from Molecular Probes (Eugene, OR), and Transwell Clear filter inserts from Fisher Scientific (Hanover Park, IL). Agarose beads, LB broth base, ampicillin, and tissue culture supplies were purchased from Invitrogen-GIBCO (Gaithersburg, MD). Precast 4-15% gradient slab gels and silver stain kits were obtained from Bio-Rad (Hercules, CA). All other chemicals were reagent grade.


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Antiactin drugs alter activity of NKCC1 cotransporter and its regulation. In the absence of actin inhibitor, the basal activity of NKCC1, measured as bumetanide-sensitive 86Rb uptake across the basolateral membrane, was 37.7 ± 10.6 nmol K/mg protein (n = 6), which represented 28.2% of the total 86Rb uptake (Fig. 1A). The alpha 1-adrenergic agent methoxamine caused a 3.0-fold increase in NKCC1 activity to 111.91 ± 16.8 nmol K/mg protein (n = 8). Methoxamine also increased the bumetanide-sensitive component of total K uptake from 28.2% to 61.1%, a 2.2-fold increase that, together with stimulation of K uptake, indicates activation of NKCC1. Latrunculin B did not significantly affect basal NKCC1 activity but did block methoxamine-stimulated flux. In contrast, jasplakinolide induced a twofold increase in NKCC1 activity from 37.6 ± 10 (n = 6) to 80.9 ± 14 (n = 7) nmol K/mg protein (P < 0.02) in the absence of methoxamine. In these experiments, jasplakinolide increased the percent bumetanide-sensitive flux 2.1-fold from 28.2% to 54.4% total flux, indicating activation of NKCC1. To determine whether PKC-delta is necessary for jasplakinolide-stimulated NKCC1 activity, cells were pretreated with rottlerin, an inhibitor of PKC-delta . Jasplakinolide stimulated a rottlerin-sensitive uptake of 58.6 ± 6 nmol K/mg protein (n = 6), which was not significantly different from the bumetanide-sensitive K uptake or a combined rottlerin-, bumetanide-sensitive uptake of 69.6 ± 11 nmol K/mg protein (n = 6). When cells were treated with jasplakinolide and then with methoxamine, NKCC1 activity increased to 108.5 ± 11 nmol K/mg protein (n = 8). The results indicate that jasplakinolide stimulates NKCC1 activity as robustly as methoxamine.


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Fig. 1.   Activity of Na-K-2Cl cotransporter (NKCC)1 in the presence of actin inhibitors. Calu-3 cells were grown to confluence on filter inserts and serum-deprived overnight. A: cells were preincubated with 10 µM latrunculin B for 1 h or 50 nM jasplakinolide for 2 h or left untreated. Thirty minutes before the end of the preincubation period, bumetanide was added to the basolateral medium to a final concentration of 10 µM. Uptake of 86Rb was measured for 4 min and then terminated as described in METHODS. Bumetanide-sensitive flux, which represents NKCC1 activity, was determined by subtracting bumetanide-insensitive flux from total flux. Dose-dependent effect of latrunculin-B (B) and jasplakinolide (C) on NKCC1 activity was measured in cells stimulated with methoxamine (filled bars) or HEPES-buffered Hanks' balanced salt solution (HPSS) vehicle (open bars). Data are means ± SE for 3-9 independent cell cultures. Levels of significance: #P < 0.05, ##P < 0.02, ###P < 0.001 compared with methoxamine-stimulated uptake in the absence of latrunculin B; *P < 0.03 compared with uptake in cells not treated with jasplakinolide.

The effect of the antiactin drugs showed dose dependence, as shown in Fig. 1, B and C. Latrunculin B did not significantly alter baseline NKCC1 activity but did cause a dose-dependent decrease in the stimulatory response to methoxamine (Fig. 1B). The EC50 value for the inhibitory effect of latrunculin B on methoxamine-sensitive NKCC1 activity was 3.6 µM. In contrast, jasplakinolide increased NKCC1 activity in the absence of alpha 1-adrenergic hormone in a dose-dependent manner, with an EC50 of 27.5 nM (Fig. 1C). At jasplakinolide concentrations ranging from 1 to 30 nM, treatment with methoxamine induced a significant increase in NKCC1 activity. At higher concentrations of actin inhibitor, methoxamine failed to induce a secondary increase in NKCC1 activity.

Coimmunoprecipitation of PKC-delta , actin, and NKCC1. A recent study from this laboratory (23) demonstrated an association between NKCC1 and PKC-delta by coimmunoprecipitation of the two proteins from the same lysis buffer. The results of Fig. 1 suggest that F-actin may also be important in hormone-stimulated NKCC1 activity. We now find that PKC-delta and NKCC1 coimmunoprecipitate with actin (Fig. 2). Lysates from Calu-3 cells were treated with antibodies to PKC-delta or with antibodies to actin coupled to agarose beads. PKC-delta and actin were recovered and resolved on SDS-PAGE. Protein bands were transferred to PVDF membrane paper and probed by immunoblot analysis for the other protein. Actin was detected in immunoprecipitates of PKC-delta and in total cell lysates (Fig. 2A, left). Likewise, PKC-delta was detected in pulldowns of actin and in total cell lysates (Fig. 2A, right). The results demonstrate coimmunoprecipitation of actin and PKC-delta . NKCC1 and actin also coimmunoprecipitate, as shown in Fig. 2B. Actin was detected in immune complexes of NKCC1 (Fig. 2B, left), and NKCC1 was found in pulldowns of actin (Fig. 2B, right).


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Fig. 2.   Coimmunoprecipitation of actin and Calu-3 cellular proteins. A: endogenous protein kinase C (PKC)-delta was immunoprecipitated from total cell lysate (TCL), and actin pulldown was accomplished with anti-actin antibodies conjugated to agarose beads. Recovered proteins were subjected to SDS-PAGE gel electrophoresis on 4-15% gradient slab gels and analyzed by immunoblot analysis for actin or PKC-delta . Left, actin is detected in immunoprecipitates of PKC-delta as a 45-kDa protein band. Right, PKC-delta is detected in pulldowns of actin as a 75-kDa protein band. B: NKCC1 was immunoprecipitated from lysates of Calu-3 cells and actin recovered by pulldown as described in A. Immunoprecipitates were subjected to SDS-PAGE and analyzed by immunoblot analysis for actin or NKCC1. Left, the presence of actin in immunoprecipitates of NKCC1. Right, NKCC1 is detected in immunoprecipitates of endogenous actin. Results are representative of 4 separate experiments. IP, immunoprecipitate; PD, pulldown; WB, Western blot.

Stimulation of NKCC1 requires increased activity of PKC-delta ; however, association of PKC-delta and NKCC1 might involve active or inactive protein. To determine whether active enzyme is necessary for association with NKCC1, PKC-delta activity was manipulated by treating Calu-3 cells with methoxamine in the absence or presence of rottlerin for various time intervals. NKCC1 was immunoprecipitated from cell lysates, subjected to SDS-PAGE, transferred to PVDF membrane paper, and probed for PKC-delta with a polyclonal antibody. As seen in Fig. 3, manipulation of the activity of PKC-delta with alpha 1-adrenergic agonist in the absence or presence of the PKC-delta inhibitor rottlerin did not affect coimmunoprecipitation of PKC-delta with NKCC1.


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Fig. 3.   Association of NKCC1 and PKC-delta is independent of PKC-delta activity. Calu-3 cells were serum deprived overnight, treated with methoxamine (Mox), a selective NKCC1 stimulant, in the absence or presence of rottlerin (Rott), a specific blocker of PKC-delta activity for 1-10 min at 35°C. Cell monolayers were washed with ice-cold PBS and then lysed, and NKCC1 was immunoprecipitated from cell lysates. Immunoprecipitates were electrophoresed on 4-15% gradient slab gels and immunoblotted with antibodies to PKC-delta . Blots were stripped and reprobed for NKCC1. PKC-delta (A) and NKCC1 (B) were detected in TCL of Calu-3 cells (lane B) and, as a positive control for the antibody, in TCL of T84 cells (lane A). Western blot analysis for PKC-delta and laser densitometry (data not shown) demonstrate that stimulation with Mox with or without the PKC-delta inhibitor Rott did not alter coimmunoprecipitation of PKC-delta with NKCC1, indicating that activity of PKC-delta is not a prerequisite for coimmunoprecipitation. Results are representative of 4 separate experiments.

In vitro binding of PKC-delta to nonmuscle actin. Binding properties of actin and PKC-delta were examined with recombinant proteins. Binding studies were performed in a solution binding assay (Fig. 4A) or in a slot blot apparatus with immobilized F- or G- nonmuscle actin (Fig. 4, B and C). In the first experiment, nonmuscle F-actin was incubated together with inactive or preactivated rPKC-delta . F-actin was recovered by pulldown with anti-actin antibody coupled to agarose beads. To preactivate rPKC-delta , enzyme was preincubated with the PKC activators PtdSer and DOG. Figure 4A shows that PKC-delta binds to nonmuscle F-actin and that binding is enhanced threefold by preactivation of enzyme. To directly investigate binding of PKC-delta to nonmuscle G-and F-actin, we used a slot blot assay. In the slot blot assay, nonmuscle G- or F-actin was immobilized on PVDF membrane paper. As illustrated in Fig. 4B, PKC-delta binds to both nonmuscle F- and G-actin; however, there is a 3.9-fold greater binding to F-actin than G-actin, suggesting preferential binding to F-actin. This was confirmed by using antiactin drugs to manipulate actin polymerization. Nonmuscle G-actin was incubated with jasplakinolide to promote polymerization of G-actin, and nonmuscle F-actin was incubated with latrunculin B, which mimics the activity of monomer sequestering proteins. Jasplakinolide increased binding of PKC-delta by 84%, and latrunculin B decreased PKC-delta binding by 28% (Fig. 4B). Binding of PKC-delta to F-actin is dose dependent, with an EC50 of 72 ng (Fig. 4C). The results indicate that the polymerization state of nonmuscle actin is an important determinant of PKC-delta binding.


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Fig. 4.   In vitro binding of recombinant human PKC-delta (rPKC-delta ) to nonmuscle actin. A: for in vitro binding, 25 µg of F-actin and 100 ng of preactivated rPKC-delta were mixed and incubated at 30°C for 20 min. rPKC-delta was preactivated by incubation for 15 min at 30°C with mixed micelles (MM) consisting of 30 µg/ml phosphatidylserine (PtdSer) and 2 µg/ml dioctanylglycerol (DOG) (+). Pulldown of actin was accomplished with anti-actin antibody conjugated to agarose beads. Beads were recovered by centrifugation, washed extensively with ice-cold PBS, and resuspended in Laemmli buffer. Soluble proteins were separated by gel electrophoresis on 4-15% SDS-PAGE gradient slab gels and probed by immunoblot analysis for PKC-delta . Protein bands were analyzed by laser densitometry (LD), as shown in the summary graph (bottom). The results show preferential binding of activated PKC-delta to nonmuscle F-actin. Negative control for the omission of PKC-delta shows no immunoreactive protein band. Results are representative of 2 independent experiments in which conditions were replicated in triplicate. B: effect of actin inhibitors on binding of PKC-delta to actin. Jasplakinolide was added to an aliquot of G-actin in G-actin buffer to a final concentration of 50 nM, and latrunculin B was added to an aliquot of F-actin in F-actin buffer to a final concentration of 0.2 µM. The mixtures were incubated for 15 min at room temperature. Aliquots containing 0.5 µg of actin were immobilized on polyvinylidene difluoride (PVDF) paper and overlaid with 50 ng of inactive or preactivated PKC-delta . Bound PKC was detected by immunoblot analysis, and exposed bands were quantitated by LD. Summary graphs of LD values are shown. Underlined numbers represent net binding and are calculated as the difference in LD units between paired samples, one with (+) MM and the other without (-) MM. In the absence of actin inhibitor, PKC-delta binds to G- and F-actin with greater binding to F-actin. For both forms of actin, activation of PKC-delta with MM of PtdSer and DOG promotes binding. Jasplakinolide (Jasp), an actin stabilizer, increased PKC-delta binding to G-actin by 84%. Latrunculin B (Latr), an actin destabilizer, reduced binding of PKC-delta to F-actin by 28%. C: concentration-dependent binding of PKC-delta to F-actin. F-actin (0.5 µg) was immobilized to PVDF membrane paper and overlaid with the indicated amounts of PKC-delta . Bound PKC-delta was detected by immunoblot analysis, and exposed bands were quantitated by LD. A summary graph of LD values is shown at bottom. Results are representative of 3 independent experiments.

Intracellular localization of actin and PKC-delta . The new findings on PKC-delta -actin interaction in Calu-3 cells imply that PKC-delta and actin might colocalize to the same region of the cell. The intracellular localization of PKC-delta relative to actin was determined with double-label immunofluorescence of confluent cell cultures grown on filter inserts. Figure 5 shows en face and orthogonal images of PKC-delta and actin. Actin was detected at the cell periphery. PKC-delta , on the other hand, was present in the cytosol and at the cell periphery. Merged images showed focal spots of yellow-orange, indicating colocalization of PKC-delta and actin at discrete sites in the cell periphery but not the cytosol.


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Fig. 5.   Localization of actin and PKC-delta in Calu-3 cells. En face and orthogonal computer-generated images of a midsection plane through epithelial cell layer are shown. Serum-deprived Calu-3 cells were fixed in 4% paraformaldehyde, permeabilized in 0.2% Triton X-100, and incubated for 60 min with primary antibody directed against PKC-delta and with Texas red conjugated to phalloidin for detection of actin. For PKC-delta , cells were incubated with secondary antibody of Oregon green-conjugated anti-rabbit antibody for 60 min. En face images of actin (A) and PKC-delta (B) and a merged image (C) were visualized by confocal microscopy. Actin was detected at the cell periphery. PKC-delta , on the other hand, was found in the cytosol and at the cell periphery. Merged images indicate colocalization of actin and PKC-delta at selective sites at the cell periphery but not in the cytosol. Cells incubated with secondary antibody alone displayed no image (data not shown).

Competitive inhibition of binding of PKC-delta to actin by a PKC-delta C2-like domain (delta C2). The C2 domain expressed in novel PKC isotypes is thought to have, instead of a Ca2+ binding function, a function of protein interaction (8, 17, 25). We used a recombinant His6-tagged delta C2 to investigate its binding to nonmuscle F-actin in a slot blot binding assay. Figure 6A shows that delta C2 binds to nonmuscle F-actin in a dose-dependent manner. Binding of PKC-delta to muscle actin was negligible (data not shown). We next tested inhibition of the binding of PKC-delta to F-actin by delta C2. The results, illustrated in Fig. 6B, demonstrate that delta C2 blocks binding of rPKC-delta to F-actin in a dose-dependent manner with an IC50 (inhibitory concentration) of 2.26 µg.


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Fig. 6.   In vitro binding of delta C2 to nonmuscle F-actin. A: binding of delta C2 and actin. Aliquots containing 0.5 µg of F-actin were immobilized on PVDF paper and overlaid with varying amounts of delta C2 for 25 min at room temperature. Unbound material was removed by washing, and bound delta C2 was detected by immunoblot analysis with India HisProbe-HRP and quantitated by LD (bottom). The result shown is representative of 4-6 independent experiments. B: effect of delta C2 on binding of PKC-delta to actin. Equal aliquots with 0.5 µg of F-actin were immobilized on PVDF membrane paper and overlaid with 25 ng of rPKC-delta and indicated amounts of delta C2 for 25 min at room temperature. Bound PKC-delta was detected by immunoblot analysis, and exposed bands were quantitated by LD. A summary of LD values as arbitrary units is shown at bottom. The analysis indicates dose-dependent inhibition of binding of PKC-delta to nonmuscle actin with an apparent Ki of 2.26 µg. Results are representative of 4 independent experiments.

Inhibition of PKC-delta binding to F-actin by delta C2 indicates that a binding site for actin is localized on the delta C2 peptide. We predicted that if activation of NKCC1 requires PKC-delta binding to F-actin, then elevated intracellular delta C2 peptide should prevent hormone-dependent activation of NKCC1. To test this hypothesis, we introduced delta C2 into Calu-3 cells with a BioPORTER delivery system. BioPORTER reagent alone did not affect baseline activity of NKCC1 (data not shown) and did not significantly alter methoxamine-dependent activation of NKCC1 (Fig. 7). delta C2 decreased methoxamine-stimulated NKCC1 activity in a dose-dependent manner. An IC50 of 0.36 µg was calculated from Hill plots of uptake data. The results demonstrate that binding of PKC-delta to the actin cytoskeleton is necessary for activation of NKCC1.


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Fig. 7.   Effect of delta C2 on activation of NKCC1 by methoxamine. Calu-3 cells were grown to confluence on filter inserts, serum deprived overnight, and incubated with BioPORTER reagent alone or with varying concentrations of delta C2, as described in METHODS. Uptake of 86Rb was measured for 4 min. As a control for cells treated with BioPORTER reagent, uptake was also measured in untreated cells (UT). BioPORTER reagent alone did not significantly alter methoxamine-stimulated NKCC1 activity or NKCC1 activity in cells treated with vehicle (data not shown). delta C2 decreased methoxamine-stimulated NKCC1 activity with a maximal effect at 10 µg of delta C2 (nominal 1 µM concentration) and an IC50 of 0.36 µg. *P < 0.01, **P < 0.001 compared with cells treated with 0 µg delta C2.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Regulation of NKCC1 function in airway epithelial cells is a complex process, involving a signaling cascade in which PKC-delta is a major effector enzyme (23). A more recent study led us to conclude that protein-protein interactions were critically involved in the regulation of NKCC1. In this study, we show that inhibitors of actin integrity have marked effects on NKCC1 function. We also identify an association of actin with NKCC1 and PKC-delta (Figs. 2 and 3), a serine-threonine protein kinase that is necessary for activation of airway epithelial NKCC1 (20). In addition, we found colocalization of PKC-delta with actin (Fig. 5) and avid binding of PKC-delta to nonmuscle actin (Figs. 4 and 6). Binding of PKC-delta to actin was concentration dependent and enhanced by the presence of PKC activators. A functional role for actin-PKC-delta binding was studied with delta C2, a peptide encoding the C2-like domain of PKC-delta , as an inhibitory peptide in binding and transport experiments, respectively (Figs. 6 and 7). Binding of PKC-delta to nonmuscle actin was blocked by delta C2 (Fig. 6), which also prevented stimulation of NKCC1 activity by methoxamine (Fig. 7). We showed previously that methoxamine increases activity of PKC-delta and that this is necessary for activation of NKCC1 (20). Now we learn that binding of PKC-delta to actin is also a necessary step toward activation of NKCC1.

The delta C2 domain encodes a 123-amino acid segment of the NH2-terminal region of PKC-delta and, structurally, has an antiparallel beta -sandwich with a P-type topology similar to phospholipase-delta C2 (28). As with its counterparts in other novel PKC isotypes, it lacks the Ca-coordinating sequences characteristic of C2 domains in conventional Ca-dependent PKC isotypes and also adopts a radically different conformation, thus resulting in three nonfunctional Ca-binding loops. The C2 domain in conventional PKC isotypes functions as a Ca-regulated membrane anchor that promotes translocation to subcellular compartments through the binding of Ca and lipids. Our data indicate that the C2-like domain of PKC-delta presents a novel phospholipid-independent binding site interacting with nonmuscle actin. delta C2 also interacts with other cellular proteins. GAP-43, also known as neuromodulin, forms a complex with PKC-delta in intact cells through direct binding of GAP-43 with the regulatory domain of PKC-delta in the absence of phospholipid (10). The interaction between GAP-43 and PKC-delta was narrowed to the V0/C2 region comprising amino acids 1-121, which is referred to as the delta C2 domain in our study. These results and our new finding of binding of delta C2 to nonmuscle actin indicate that the delta C2 domain is not just a target region for a PKC cofactor but serves as a protein-protein interaction domain. The binding of PKC-delta to nonmuscle actin via the delta C2-like domain is reminiscent of the binding of the novel PKC isotype PKC-epsilon to receptor for activated C kinase 1 (RACK1) in Calu-3 cells (24). An eight-amino acid sequence in the regulatory domain of PKC-epsilon , designated epsilon V1-2, localizes to a region analogous to delta C2 of PKC-delta and is necessary for binding of activated PKC-epsilon to RACK1. As with epsilon V1-2, delta C2 prevented binding of PKC-delta to nonmuscle actin (Fig. 6B). The absence of a requirement for phospholipid for binding indicates that the delta C2 domain lacks a recognition site for phosphatidylserine. The C2 region of the conventional PKC isotype PKC-beta II contains at least part of the RACK1 binding site on PKC (33). C2-derived peptides, encoding segments of the C2 domain, inhibit translocation of PKC-beta and prevent insulin-induced regulation of Xenopus oocyte maturation. It is likely that the C2-derived peptides inhibit PKC function by binding to RACK1, a PKC binding partner native to Xenopus oocyte, and blocking subsequent binding of the enzyme. delta C2 appears to block activation of NKCC1 by binding to actin and thus preventing binding of PKC-delta to actin.

These new findings indicate a unique signaling mechanism for activation of airway epithelial NKCC1. Although the actin cytoskeleton has been implicated in the regulation of NKCC1 (9, 26, 27), the mechanism that explains the regulatory role of actin is poorly understood. Differential interaction of the F-actin cytoskeleton with specific PKC isotypes has been associated with numerous cell functions (19). Activation of PKC in different cell types leads to changes in the F-actin cytoskeleton, including lymphocyte surface capping, smooth muscle contraction, and actin rearrangement in T cells, neutrophils, and epithelial cells. PKC isotypes colocalize with a range of cytoskeletal proteins and components of the actin filaments (16, 19). For example, PKC-zeta stabilizes the actin cytoskeletal structure in IL-2-mediated proliferation of T cells (13) yet disassembles F-actin stress fibers in mesangial cells from diabetic rodents (40) and in HA-Ras L61 expressed in NIH/3T3 fibroblasts (39). Association of PKC-epsilon with actin has been implicated in glutamate endocytosis in nerve terminals (31), regulation of basolateral endocytosis in human T84 colonic cells (37), neurite outgrowth (41), and cell spreading in fibroblasts (12, 14). Likewise, activation of PKC-delta by phorbol 12-myristate 13-acetate (PMA) was reported to disrupt the actin cytoskeleton in lymphocytes (32). The Ca-dependent PKC isotype PKC-beta 1, on the other hand, stabilizes the F-actin cytoskeleton during oxidant injury in a human colonic Caco-2 cell line (1).

The data from these studies point out unique differences in the role of actin in colonic T84 cells and airway epithelial cells. In colonic cells, inhibition of actin polymerization with cytochalasin D, which causes actin filament severing and thus increases short actin filaments, increased NKCC1 activity (26, 27). However, latrunculin A, which sequesters G-actin monomers and prevents polymerization of actin, neither activated nor inhibited NKCC1. In Calu-3 cells, latrunculin B abolished alpha 1-adrenergic stimulation of NKCC1 (Fig. 1). Inducing actin polymerization with jasplakinolide blocked cAMP-elicited Cl secretion, inhibited NKCC1, prevented PMA-stimulated endocytosis, and attenuated an increase in surface NKCC1 protein expression in T84 cells (26, 27, 37) but increased baseline activity of NKCC1 in Calu-3 cells (Fig. 1). One conclusion from these experiments is that, unlike T84 colonic cells, short actin fibers do not regulate airway epithelial NKCC1. Despite this difference in response to actin inhibitors, NKCC1 in both cell types depends on an intact and dynamic actin cytoskeleton for proper regulation. Two predominant stimuli of NKCC1 function and PKC-delta activity are hormone receptor-mediated activation and hyperosmotic stress. A common kinase critical for activation of PKC-delta is likely involved in the intracellular signaling for both stimuli but has yet to be identified.

Our findings suggest that in Calu-3 cells at least part of the cellular PKC-delta is tethered to a specific site, the actin cytoskeleton (Fig. 5). Other PKC isotypes have been shown to have specific subcellular localization before activation. It is thought that the locations place inactive PKC isotypes near their target substrates to ensure preferential and rapid phosphorylation on activation. The primary function of actin-PKC-delta binding in Calu-3 cells is not yet clear. Binding may bring PKC-delta near its substrate or near protein kinases and phosphatases, which regulate its activity. Another possibility is that PKC-delta is sequestered to specific subcellular compartment(s). Because the overall result of activation of PKC-delta is rapid stimulation of NKCC1, the positioning of PKC-delta near its substrate would be advantageous for fidelity and specificity in the regulation of NKCC1 function.

How the interaction between PKC-delta and actin leads to activated NKCC1 remains to be clarified. Anchoring of activated PKC-delta close to its target substrate might enhance substrate phosphorylation. NKCC1 is one of several likely target substrates for activated PKC-delta , as are actin, actin-binding proteins, and other PKC binding proteins (6, 16). Another possibility is that activated PKC-delta stabilizes the F-actin cytoskeleton, thus promoting activation of NKCC1 (19). The relationship between PKC-delta , the actin cytoskeleton, and NKCC1 appears important for understanding NKCC1 function and hence fertile ground for future research.


    ACKNOWLEDGEMENTS

We thank Dr. L. V. Dekker (University College London) for providing constructs of the delta C2 domain. We also appreciate the assistance provided for this study from Dr. Douglas Eaton for helpful discussions and Ms. Denise Hatalya for microscopy.


    FOOTNOTES

This work was supported by National Institutes of Health Grants HL-58598 and DK-27651 and by a Cystic Fibrosis Foundation Research Development Program.

Address for reprint requests and other correspondence: C. M. Liedtke, Pediatric Pulmonology, Case Western Reserve Univ., BRB, Rm. 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948 (E-mail: cxl7{at}po.cwru.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

First published October 16, 2002;10.1152/ajpcell.00357.2002

Received 1 August 2002; accepted in final form 8 October 2002.


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