Involvement of NH2 terminus of PKC-{delta} in binding to F-actin during activation of Calu-3 airway epithelial NKCC1

Nicole D. Smallwood, Bryan S. Hausman, Xiangyun Wang, and Carole M. Liedtke

Willard A. Bernbaum Cystic Fibrosis Research Center, Department of Pediatrics, Rainbow Babies and Children’s Hospital, and Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio

Submitted 1 October 2004 ; accepted in final form 7 December 2004


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Direct binding of nonmuscle F-actin and the C2-like domain of PKC-{delta} ({delta}C2-like domain) is involved in hormone-mediated activation of epithelial Na-K-2Cl cotransporter isoform 1 (NKCC1) in a Calu-3 airway epithelial cell line. The goal of this study was to determine the site of actin binding on the 123-amino acid {delta}C2-like domain. Truncations of the {delta}C2-like domain were made by restriction digestion and confirmed by nucleotide sequencing. His6-tagged peptides were expressed in bacteria, purified, and analyzed with a Coomassie blue stain for predicted size and either a 6xHis protein tag stain or an INDIA His6 probe for expression of the His6 tag. Truncated peptides were tested for competitive inhibition of binding of activated, recombinant PKC-{delta} with nonmuscle F-actin. Peptides from the NH2-terminal region, but not the COOH-terminal region, of the {delta}C2-like domain blocked binding of activated PKC-{delta} to F-actin. The {delta}C2-like domain and three NH2-terminal truncated peptides of 17, 83, or 108 amino acids blocked binding, with IC50 values ranging from 1.2 to 2.2 nmol (6–11 µM). NH2-terminal {delta}C2-like peptides also prevented methoxamine-stimulated NKCC1 activation and pulled down endogenous actin from Calu-3 cells. The proximal NH2 terminus of the {delta}C2-like domain encodes a {beta}1-sheet region. The amino acid sequence of the actin-binding domain is distinct from actin-binding domains in other PKC isotypes and actin-binding proteins. Our results indicate that F-actin likely binds to the {beta}1-sheet region of the {delta}C2-like domain in airway epithelial cells.

truncation; protein kinase C-{delta}; C2-like domain; slot blot assay; inhibitory constant; bumetanide; Na-K-2Cl cotransporter


THE REGULATION of the secretory form of the Na-K-2Cl cotransporter (NKCC1) in airway epithelial cells apparently involves a direct interaction between PKC-{delta}, a key effector enzyme in NKCC1 activation, and nonmuscle F-actin (15). Airway NKCC1 belongs to an extensive gene family of cation-Cl cotransporters that characteristically couple transport of Cl with K and Na (29). The NKCC1 isoform is expressed in the basolateral membrane and provides Cl for secretion. The renal-specific homolog NKCC2 is localized to the apical membrane of cells lining the thick ascending loop of Henle, where it plays a critical role in transcellular absorption of Na and Cl (20). Phosphorylation of NKCC1 is one major mechanism mediating its activation (11). Indeed, recent studies of shark rectal gland and rat brain NKCC1 suggest that levels of phosphorylation at the NH2 terminus play a major role in activation of NKCC1 (3, 4, 7, 25). Two enzymes, protein phosphatase 1 and Ste20-related proline-alanine-rich kinase, interact in vivo with the NH2 terminus at specific sites, and a third enzyme, oxidative stress response I, interacts in a yeast two-hybrid screen (3, 7, 25). Activity of the enzymes facilitates activation of NKCC1 at low intracellular Cl, apparently through a phosphorylation event. Recently, we demonstrated (16) that a Cl electrochemical gradient alone is not sufficient to stimulate NKCC1 activity. Rather, increased PKC-{delta} activity is necessary for hormone-stimulated NKCC1 activity.

We also discovered (15) colocalization of PKC-{delta} with F-actin in a Calu-3 airway epithelial cell line and avid binding of recombinant PKC-{delta} to purified nonmuscle F-actin. Binding of PKC-{delta} to actin was concentration dependent and enhanced by the presence of PKC activators. We also reported (15) for the first time that binding of PKC-{delta} to actin is a necessary step toward activation of NKCC1. Actin binds to PKC-{delta} at an NH2-terminal {delta}C2-like domain. This domain encodes a 123-amino acid segment of the NH2-terminal region of PKC-{delta} and, structurally, has antiparallel {beta}-sheets with a P-type topology similar to phospholipase-{delta} C2 domain (22). Our research revealed that the C2-like domain of PKC-{delta} presents a novel phospholipid-independent binding site for interaction with nonmuscle actin. A recombinant {delta}C2-like domain peptide blocked binding of PKC-{delta} to nonmuscle actin and activation of NKCC1 by the {alpha}1-adrenergic agonist methoxamine. Thus the {delta}C2-like domain likely expresses a specific binding site for nonmuscle F-actin that accounts for high-affinity binding of the two proteins, leading to activation of NKCC1.

The {delta}C2-like domain is analogous to the C2, or Ca2+-binding, domain of conventional PKC isotypes and may also share functional properties with the NH2 terminus of PKC-{epsilon} (15). An eight-amino acid segment embedded in the V1, or NH2-terminal, region of PKC-{epsilon} binds to receptor for activated C kinase 1 (RACK1) in Calu-3 cells and in cardiac myocytes (2, 16, 28). The purpose of our current study was to identify a site of F-actin binding on the {delta}C2-like domain of PKC-{delta}. Preliminary bioinformatic analysis indicated a unique {delta}C2-like domain amino acid sequence that lacked homology to other actin-binding PKC isotypes or actin-associated proteins. Therefore, we prepared truncations of recombinant His6-tagged {delta}C2-like domain for in vitro binding assays, in vivo functional evaluation of NKCC1 activation by methoxamine, and pulldown of actin from Calu-3 airway epithelial cells. The results of the study demonstrate inhibition of binding of PKC-{delta} to nonmuscle F-actin by a proximal 17-amino acid segment at the NH2 terminus of the {delta}C2-like domain. The truncated peptide blocked activation of NKCC1 by methoxamine and pulled down actin from total cell lysates of Calu-3 cells.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Construction of truncated PKC-{delta} C2-like domain expression vectors. Template DNA for these constructs was the rat {delta}C2-like domain coding region cloned into a pET-14b vector, kindly provided by L. Dekker (University of London), which we refer to as pET-14b-{delta}C2 (22). DNA was extracted from bacterial cell pellets, analyzed on a 0.8% TBE (45 mM Tris-borate, pH 8.0, 1 mM EDTA) agarose gel for predicted size, and stored at –20°C in 10 mM Tris·HCl, pH 8.0, 1 mM EDTA. Analysis of the cDNA sequence of pET-14b-{delta}C2 revealed three unique restriction sites within the {delta}C2-like domain coding sequence: SbfI at bp 843, BstEII at bp 638, and Bsu36I at bp 560. Six separate double digestions were set up with each of the three restriction enzymes and with either NdeI at bp 891 or XhoI at bp 515. The latter two restriction sites were the original 5' and 3' cloning sites, respectively, of pET-14b-{delta}C2. COOH-terminal truncations were prepared by sequential restriction digestions of pET-14b-{delta}C2 using 2 µl of NdeI in an overnight digestion at 37°C followed by digestion with SbfI (C1 truncation) at 37°C for 3 h, BstEII (C3 truncation) at 60°C for 3 h, or Bsu36I (C5 truncation) at 37°C for 3 h. NH2-terminal truncations were prepared by sequential restriction digestions of pET-14b-{delta}C2 using 2 µl of XhoI at 37°C overnight followed by digestion with SbfI (N2 truncation) at 37°C for 3 h, BstEII (N4 truncation) at 60°C for 3 h, or Bsu36I (N6 truncation) at 37°C for 3 h. The six DNA truncations from the double digestion were extracted from the digestion mixture with phenol-chloroform-isoamyl alcohol (25:24:1), ethanol precipitated, and resuspended in 50 µl of 10 mM Tris·HCl, pH 8.5, 0.1 mM EDTA. Recovered DNA was Klenow end-filled and religated with T4 DNA ligase (New England Biolabs, Beverly, MA) at room temperature for 30 min and transformed into TOP10 One Shot-competent cells (Invitrogen). DNA from 10 colonies of each of the six truncations was extracted and analyzed for correct construction with AvaI restriction sites at bp 515 and bp 2102. These sites are located in the pET-14b vector but not in the {delta}C2-like domain coding sequence. Digestion fragments were separated on a 0.8% TBE agarose gel for size analysis. Restriction digestion of pET-14b-{delta}C2 yields two fragments of 3,453 and 1,587 bp. Restriction digestion of pET-14b-{delta}C2 truncations produces a fragment of 3,453 bp and another of <1,587 bp, depending on the truncation. Clones of each truncation construct that matched the predicted digestion pattern were selected and verified by nucleotide sequence analysis with ABI Prism Big Dye Terminator (Applied Biosystems, Foster City, CA).

Expression and isolation of recombinant peptides. Recombinant full-length {delta}C2-like domain and truncated peptides were produced with a pET-14b expression vector (Novagen), which placed a polyhistidine tag at the NH2 terminus of the construct. His6-tagged peptides were expressed in TOP10 One Shot-competent cells on induction with 0.1 mM isopropyl-{beta}-D-1-thiogalactopyranoside for 4 h at 37°C. Cells were harvested, and recombinant protein was extracted from the bacterial cell pellet with a B-PER 6xHis spin purification kit (Pierce Biotechnology, Rockford, IL) and eluted in 2 ml of elution buffer. Peptides were purified with Centricon YM-3 extraction columns (Millipore), dialyzed against PBS, pH 7.4, and stored at –80°C. Protein content was determined with a bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology) with bovine serum albumin as the standard. To verify the size of the peptide, the truncations were subjected to SDS gel electrophoresis on an 18% Tris·HCl gel under reducing conditions. Gels were stained for protein bands with GelCode Blue stain reagent (Pierce Biotechnology) and analyzed with a 6xHis protein tag stain (Pierce Biotechnology) or an INDIA His6 probe (Pierce Biotechnology) for the His6 tag.

Binding assay. Each truncation was individually analyzed in solid-phase binding studies with nonmuscle {beta}-actin (Cytoskeleton, Denver, CO). Monomeric actin (G-actin) was stored in G-actin buffer consisting of (in mM) 5 Tris·HCl, pH 8.0, 0.2 ATP, 0.2 CaCl2, and 1.0 {beta}-mercaptoethanol at a final concentration of 100 µg/ml. On the day of use, G-actin was diluted to a concentration of 0.4 µg/µl in G-actin buffer. Filamentous actin (F-actin) was polymerized from G-actin by the addition of 1/10th volume of (in mM) 500 KCl, 20 MgCl2, and 10 ATP. The mixture was incubated for 1 h at room temperature and then diluted to 0.005 µg/µl in (in mM) 50 KCl, 2 MgCl2, and 1 ATP. Polymerized F-actin was vacuumed onto polyvinylidene difluoride (PVDF) membrane paper (Schleicher & Schuell) with a slot-blot apparatus. The membrane was blocked in F-actin buffer for 1 h, incubated with preactivated PKC-{delta} with or without varying amounts of truncated peptide for 25 min, and washed extensively. PKC-{delta} binding was detected by immunoblot analysis using a polyclonal antibody to PKC-{delta}. As a positive control, membranes were reprobed for actin with a monoclonal antibody to {beta}-actin.

Measurement of NKCC1 activity. NKCC1 activity was measured as bumetanide-sensitive uptake of 86Rb, a congener of K, as previously described (16). Calu-3 cells were grown in submerged cell culture on 0.4-µm-pore Transwell-Clear polyester filter inserts and serum deprived for 24 h before experiments. His6-tagged full-length {delta}C2-like domain or truncated peptides were delivered into Calu-3 cells with a BioPORTER protein delivery system followed by measurement of bumetanide-sensitive 86Rb uptake. To initiate radiotracer uptake, filters were transferred to a well of a six-well tissue culture dish containing 1 µCi of 86Rb in HEPES-buffered HBSS (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 BCA protein assay kit (Pierce Biotechnology) using bovine serum albumin as the standard. Intracellular radioisotopic content was calculated as nanomoles of K per milligram of protein (86Rb).

Pulldown of endogenous actin. Pulldowns were performed with His6-tagged full-length {delta}C2-like domain or truncated peptides. Total cell lysates from Calu-3 cells were prepared and incubated at room temperature for 30 min with 20 µg of recombinant protein. Talon beads were added to pull down His6-tagged peptide. Beads were recovered by centrifugation, washed extensively with PBS, and resuspended in Laemmli buffer. Samples were heated for 5 min in a boiling water bath, cooled, and then subjected to 4–15% SDS-PAGE and immunoblot analysis for F-actin with a monoclonal antibody to actin. Endogenous actin was detected as a 45-kDa protein band.

Data analysis. Immunoreactive protein bands were quantitated with a VersaDoc Imaging System (Bio-Rad, Hercules, CA) or laser densitometry. Data are representative of three or more experiments and are reported as means ± SE. Treatment effects were evaluated with a two-sided Student's t-test. An inhibitory constant or IC50 was calculated from Hill plots of data from dose-response curves with a GraphPad Prism 4.0 software program.

Materials. 86Rb (specific activity 154 Bq/g Rb, 4,200 Ci/g Rb) was purchased from Amersham Life Science. Restriction enzymes, Klenow polymerase, and T4 DNA ligase were obtained from New England Biolabs. Polyclonal anti-PKC-{delta} antibody and horseradish peroxidase-coupled secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Baculovirus-expressed recombinant PKC-{delta} was obtained from Pan Vera (Madison, WI) and monoclonal antibody to {beta}-actin and nonmuscle actin (85% {beta}-actin, 15% {alpha}-actin) were from Cytoskeleton. The BioPORTER protein delivery system was obtained from Gene Therapy Systems (San Diego, CA). Methoxamine HCl and bumetanide were purchased from Sigma (St. Louis, MO), 18% precast slab gels from Bio-Rad, and protease inhibitor cocktail set III from Calbiochem-Novabiochem (San Diego, CA). An enhanced chemiluminescence reagent was purchased from Amersham (Piscataway, NJ) and Transwell Clear filter inserts from Fisher Scientific (Hanover Park, IL). INDIA His6 probe and Supersignal West Pico Chemiluminescent substrate were purchased from Pierce Biotechnology. Agarose beads and tissue culture supplies were purchased from Invitrogen-GIBCO (Gaithersburg, MD). All other chemicals were reagent grade.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Preparation and expression of truncated {delta}C2 peptides. The NH2-terminal regulatory domain of PKC-{delta}, referred to as the {delta}C2-like domain, has been found to prevent binding of PKC-{delta} to F-actin and to prevent hormone-stimulated activation of NKCC1 in a Calu-3 airway epithelial cell line (15). To clarify which region of the {delta}C2-like domain is essential for these functions, a series of constructs encoding truncated {delta}C2 peptides was prepared with restriction enzyme digestions. A map of the pET-14b vector with {delta}C2-like domain cDNA cloned into the NdeI and XhoI restriction sites of the multiple cloning site is shown in Fig. 1. Also shown are restriction sites selected for creating and verifying truncations. Truncations were prepared with Bsu36I, BstEII, and SbfI restriction sites. The AvaI restriction site was used to verify truncations for correct size. Three truncated peptides retained the NH2 terminus plus varying lengths of {delta}C2-like domain sequence, and three peptides retained the COOH terminus with varying lengths of peptide (Fig. 2). The DNA sequence of each truncation-encoding plasmid was sequenced to confirm the predicted nucleotide sequence. Expressed and purified truncated peptides were detected with an INDIA His6 probe, confirming the presence of the His6 tag.



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Fig. 1. Circular vector diagram for pET-14b-{delta}C2 construct. The construct of the {delta}C2-like domain of PKC-{delta} in the circular pET-14b vector is shown with restriction sites selected for making and verifying truncations. Full-length {delta}C2-like domain was cloned into NdeI and XhoI restriction sites of the multiple cloning site. Truncations were prepared with Bsu36I, BstEII, and SbfI restriction sites. The AvaI restriction site was used to verify truncations for correct size and T7 regions to prime for DNA sequence verification.

 


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Fig. 2. Schematic diagram of truncated peptides used in this study. Notation for truncated peptides is listed at left. FL, full-length {delta}C2-like domain; N2, N4, N6, peptides derived after restriction digestion to remove varying lengths of the COOH terminus; C1, C3, C6, peptides derived after restriction digestion to remove varying lengths of the NH2 terminus. Restriction enzymes XhoI and NdeI are the original cloning sites of the {delta}C2-like domain into the pET-14b vector. SbfI, BstEII, and Bsu36I are unique restriction sites at bp 843, bp 638, and bp 560, respectively, within the {delta}C2-like domain portion of the pET-14b-{delta}C2 construct. The sizes of the {delta}C2-like domain and each truncated fragment are given in number of amino acids (aa). Peptides were expressed in TOP10 One Shot-competent cells and purified with a B-PER 6xHis spin purification kit as described in METHODS. Protein content was measured with a Pierce protein assay kit. Detection of expressed His6-tag peptides with a His6 probe (Pierce) is illustrated at right.

 
Inhibition of PKC-{delta} binding to F-actin. The {delta}C2-like domain comprises the NH2 terminus of PKC-{delta}, a region that is analogous to the C2, or Ca2+-binding, domain of conventional PKC isotypes and is thought to be important for unique protein-protein interactions (2, 6). Reports from this laboratory (15) detail experiments establishing the binding of recombinant {delta}C2-like domain to nonmuscle F-actin in in vitro binding assays. The recombinant {delta}C2-like domain also blocked binding of PKC-{delta} to F-actin. In this series of experiments, we tested each truncated {delta}C2 peptide for inhibition of the interaction between PKC-{delta} and F-actin with a solid-phase slot-blot assay. The results illustrated in Fig. 3A demonstrate that 20-µg full-length {delta}C2-like domain blocks 55.8% of the binding of PKC-{delta} to F-actin (P < 0.0001). An IC50, or concentration of full-length {delta}C2-like domain that inhibits 50% of the binding of PKC-{delta} to F-actin, was calculated from Hill plots of dose-response binding curves as 1.2 nmol, or 6.0 µM. Addition of COOH-terminal truncated peptides did not significantly affect binding of PKC-{delta} to F-actin. However, binding was inhibited by NH2-terminal truncated peptides. A dose-response curve for each NH2-terminal truncated peptide was performed, and an IC50 was calculated from a Hill plot of the resulting data (Fig. 3B). IC50 values were 2.2 nmol (11 µM) for N2, 1.2 nmol (6 µM) for N4, and 1.3 nmol (6.5 µM) for N6 truncated peptides. The similar IC50 values suggest that each truncated protein expressed the same critical binding site for interaction with F-actin.



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Fig. 3. Inhibition of in vitro binding of PKC-{delta} to nonmuscle F-actin. Aliquots containing 0.5 µg of F-actin were immobilized on polyvinylidene difluoride paper and overlaid with 25 ng of PKC-{delta} in the absence (Veh) or presence of 20 µg of full-length {delta}C2-like domain or varying amounts of one of the truncated {delta}C2 peptides. After 25 min at room temperature, unbound material was removed by washing, and bound PKC-{delta} was detected by immunoblot analysis. Exposed bands were quantitated with a VersaDoc Imaging System (Bio-Rad) or by laser densitometry. A: COOH-terminal truncated peptides. Binding of PKC-{delta} to immobilized F-actin is reported as total ± SE optical density (OD) units for 5–8 independent experiments for each peptide studied. Percentages refer to mean % inhibition of PKC-{delta} binding to F-actin by the indicated peptide. Full-length {delta}C2-like domain blocks binding of PKC-{delta} to F-actin with an IC50 of 1.2 nmol, as determined from dose-response curves and calculated with a GraphPad Prism 4.0 software program. COOH-terminal truncated peptides C1, C3, and C5 did not affect binding. *P < 0.0001 compared with no peptide addition. B: NH2-terminal truncated peptides. Percent maximum binding is calculated from laser densitometry units. NH2-terminal truncated peptides N2, N4, and N6 block binding of PKC-{delta} to F-actin in a dose-dependent manner at the indicated IC50 values. The IC50 values are not significantly different.

 
Inhibition of methoxamine-stimulated NKCC1 activity. In a previous study (15), we demonstrated that activation of NKCC1 by methoxamine was inhibited by recombinant {delta}C2-like domain in a dose-dependent manner. Inhibition of PKC-{delta} binding to F-actin by specific truncated {delta}C2-like peptides led us to predict that NH2-terminal truncated peptides might also prevent hormone-dependent activation of NKCC1. To test this hypothesis, we introduced 20-µg {delta}C2-like truncated peptide into Calu-3 cells grown on filter inserts, using a BioPORTER delivery system. In the absence of peptide, the baseline activity of NKCC1, measured as bumetanide-sensitive 86Rb uptake across the basolateral membrane, was 28.5 ± 3.6 nmol K/mg protein (n = 6), which represented 21.8% of the total 86Rb uptake (Fig. 4). Treatment with methoxamine, an {alpha}1-adrenergic agent, increased NKCC1 activity 3.6-fold to 102.9 ± 15.8 nmol K/mg protein (n = 8). Methoxamine also increased the proportion of total K uptake sensitive to bumetanide from 21.8% to 50.6%, a 2.3-fold increase that, together with stimulation of bumetanide-sensitive K uptake, indicates activation of NKCC1.



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Fig. 4. Inhibition of Na-K-2Cl cotransporter isoform 1 (NKCC1) activation by truncated {delta}C2 peptides. Calu-3 cells were grown to confluence on filter inserts, serum deprived overnight, and incubated with BioPORTER reagent alone, with 20 µg of full-length {delta}C2-like domain, or with 20 µg of truncated {delta}C2 peptide, as described in METHODS. Uptake of 86Rb was measured for a 4-min time period. Line represents baseline bumetanide-sensitive 86Rb uptake of 28.5 ± 3.6 (n = 8) nmol/mg protein in cells treated with vehicle (Veh) of HEPES-buffered HBSS. In cells treated with 10 µM methoxamine, bumetanide-sensitive 86Rb uptake increased to 102.9 ± 15.8 (n = 8) nmol/mg protein. Baseline NKCC1 activity was not affected by treatment with recombinant peptide in a BioPORTER complex. Delivery of full-length {delta}C2-like domain and NH2-terminal truncated peptides into Calu-3 cells prevented or significantly reduced methoxamine-stimulated NKCC1 activation. Truncations at the COOH terminus did not alter the effect of methoxamine on NKCC1 activity. *P < 0.01, compared with cells treated with vehicle.

 
Activity of NKCC1 after delivery of full-length {delta}C2-like domain was 36.1 ± 4.7 nmol K/mg protein (n = 4), a value not significantly different from baseline NKCC1 activity. However, addition of methoxamine to the basolateral bathing solution did not increase NKCC1 activity. Furthermore, delivery of NH2-terminal truncated peptides reduced activation of NKCC1 by methoxamine by at least 52.8% (N6) and maintained percent bumetanide-sensitive 86Rb uptake at 17.1% for N2, 26.1% for N4, and 23.1% for N6. These values were not significantly different from baseline values in cells not treated with methoxamine. In contrast, COOH-terminal truncated peptides did not prevent activation of NKCC1 by methoxamine. The apparent higher stimulation with C3 truncated peptide represents a 2.3-fold increase in NKCC1 activity and 2.4-fold increase in percent total K uptake over baseline after stimulation with methoxamine. These data are comparable to the response of cells not treated with peptide, as detailed above. Overall, the functional studies provide evidence for inhibition of NKCC1 activation by an NH2-terminal truncated {delta}C2-like domain peptide.

Pulldown of endogenous actin by His6-tagged peptides. If the {delta}C2-like domain encodes a binding site for F-actin, we predict that the peptide will bind to endogenous F-actin. To examine this possibility, we used His6-tagged full-length {delta}C2-like domain or truncated peptides in a pulldown assay using total cell lysate derived from Calu-3 cells. The results are shown in Fig. 5. Full-length {delta}C2-like domain pulled down 27.8% of total actin detected in Calu-3 cell lysates. N2, N4, and N6 truncated peptides pulled down 21.5%, 25.2%, and 24.6% of total actin, respectively. C1, C3, and C5 truncated peptides pulled down 5.7%, 6.2% and 6.2% of total actin, respectively. The fourfold greater recovery of actin with the NH2-terminal peptides indicates preferential association of endogenous F-actin with the NH2 terminus of PKC-{delta}.



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Fig. 5. Pulldown (PD) of endogenous actin by His6-tagged full-length {delta}C2-like domain or truncated peptides. Calu-3 cells were grown to confluence and serum deprived overnight. Total cell lysate (TCL) was prepared and incubated with 20 µg of peptide for 40 min at room temperature. Talon beads (40 µl) were added and incubated for 1 h at 4°C to pull down the His6-tagged peptide and associated proteins. Unbound material was removed by washing with PBS. Immunoblot analysis for actin was performed with a monoclonal antibody to actin and, as a control, 20 µg of TCL. Results are illustrative of 3 separate experiments. Actin was detected as a 45-kDa band in the PD. Exposed bands were analyzed by laser densitometry. Recovery of actin from each PD was compared with actin detected in TCL and calculated as % actin recovered.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we have identified a binding domain for nonmuscle F-actin on PKC-{delta} that encompasses the first 17 amino acids of the NH2 terminus. In earlier studies (15), we demonstrated avid binding of PKC-{delta} to actin and linked binding to the {delta}C2-like domain of PKC-{delta}. The {delta}C2-like domain encodes a 123-amino acid peptide segment of the NH2 terminus of PKC-{delta} and, when delivered into Calu-3 airway epithelial cells, prevents hormonal activation of NKCC1 by methoxamine (15). The {delta}C2-like domain also acts as an inhibitory peptide to competitively inhibit binding of activated PKC-{delta} to F-actin. To determine which part of the {delta}C2-like domain is essential for the inhibition of binding, we truncated the {delta}C2-like domain to produce a series of six different peptides, each encoding a different segment of the {delta}C2-like domain (Fig. 2). Constructs were sequenced and then expressed as His6-tagged proteins and used in competitive inhibition experiments to test their effectiveness in blocking binding of activated PKC-{delta} to F-actin. Three constructs that retained the COOH terminus but not the NH2 terminus failed to block binding (Fig. 3) and to prevent methoxamine-induced activation of NKCC1 (Fig. 4). Three NH2 terminus constructs effectively inhibited binding of PKC-{delta} and F-actin and activation of NKCC1 and, in addition, pulled down endogenous actin from Calu-3 cell lysates (Fig. 5). The smallest truncated peptide, N2, encodes the first 17 amino acids of the {delta}C2-like domain.

F-actin is a recognized binding partner of PKC isotypes and, as such, is thought to selectively position a PKC isotype in an appropriate intracellular location to respond to specific receptor-mediated activation signals. Disruption of methoxamine-stimulated NKCC1 activation by the {delta}C2-like domain (15) and by NH2-terminal truncated peptides (Fig. 4) indicates that binding of PKC-{delta} to F-actin is necessary to maximize the hormonal response. Two other PKC isotypes, PKC-{beta}II and PKC-{epsilon}, bind directly to F-actin via specific domains (Table 1). PKC-{beta}II interacts with F-actin in in vitro assays and coimmunoprecipitates actin through a specific actin binding site on the COOH terminus (1). The COOH terminus of PKC-{beta}II shares homology with a consensus actin-binding site in troponin (Table 1) and effectively blocks binding of PKC-{beta}II and F-actin. PKC-{epsilon} directly localizes with and binds F-actin at a binding site mapped to amino acids 223–228 in the C1 domain of the NH2 terminus regulatory domain of PKC-{epsilon} (26). This binding motif has also been identified in several other actin-binding proteins, such as {alpha}-actinin, actobindin, fimbrin, myosin, plastin, and thymosin (Table 1). A hexapeptide (LKKQET) based on the PKC-{epsilon} actin-binding motif competes in a dose-dependent manner with binding of PKC-{epsilon} to F-actin (26). A computer analysis of the actin-binding domains shows that the actin-binding domain on the {delta}C2-like region of PKC-{delta} is distinct from actin binding sites on PKC-{beta}II and PKC-{epsilon} and also from actin binding sites of other recognized actin-binding proteins, such as annexin and ezrin (Table 1).


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Table 1. Actin binding sites on interacting protein binding partners

 
The actin binding sites of PKC-{delta} and PKC-{epsilon} are located on the regulatory domain of each PKC isotype. Actin preferentially binds PKC-{delta} or PKC-{epsilon} after enzyme activation by the PKC activators phosphatidylserine and dioctanylglycerol. The latter property indicates that the actin binding site may be hidden when the protein kinase is in an inactive configuration. Our findings and reports from others indicate an actin-binding domain in different regions of the regulatory domains. An actin-binding domain on PKC-{delta} apparently encompasses amino acids 1–17, which represents a C2-like domain in a region designated V0 (22). An actin-binding domain on PKC-{epsilon} lies embedded within a C1 domain. Interestingly, PKC-{epsilon} also expresses a specific binding motif at amino acid residues 14–20 for direct interaction with the WD6 repeat of RACK1, a receptor for activated C kinase (2). Thus, barring steric hindrance due to its tertiary structure, PKC-{epsilon} could bind two scaffold proteins, RACK1 and F-actin. RACK1 may also play a role in localizing PKC-{epsilon} to cardiac myofibrils and microfilaments (12, 23) and with {alpha}-actinin, a marker of the Z line of the sarcomere (27). PKC-{delta}, on the other hand, does not coimmunoprecipitate with RACK1 in Calu-3 airway epithelial cells, indicating lack of a physiologically relevant interaction (17). Hence, in Calu-3 cells, F-actin likely plays a prominent role as an anchor protein immobilizing PKC-{delta}.

Structurally, the NH2-terminal sequence defining the {delta}C2-like domain resembles the C2 domains of PLC{delta} and synaptotagmin I (9, 22). The crystal structure of {delta}C2-like domain reveals an antiparallel {beta}-sandwich structure with a P-type topology similar to PLC{delta} C2 domain. Two {beta}-sheets are formed by the {beta}1-, {beta}4-, {beta}7-, and {beta}8-strands and the {beta}2-, {beta}5-, and {beta}6-strands. The first 17 amino acids of the {delta}C2-like domain comprise a {beta}1-strand plus 4 amino acids of the 13-residue {beta}1–{beta}2 loop. Our data indicate direct binding of the {beta}1-strand to nonmuscle F-actin with functional consequences on the activation of NKCC1. The remainder of the {delta}C2-like domain is thought to express COOH-terminal loops equivalent to the Ca2+-binding region of a C2 domain and two potential lipid binding sites (22). The C2 domain of conventional PKC isotypes facilitates Ca2+-dependent phospholipid binding with the lipid phosphatidylserine through electrostatic binding of Ca2+ (8, 13, 24, 34, 33). The {delta}C2-like domain, on the other hand, has analogous Ca2+-binding loops that lack all but one of five conserved Ca2+-coordinating side chains and hence are unable to bind Ca2+.

The function of the C2-like domain, although not fully understood, differs markedly from that of the C2 domain in Ca2+-dependent conventional PKC isotypes. C2-like domains are thought to be important for translocation of PKC isotypes from cytosol to membrane-bound anchor or scaffold proteins, thus playing a critical role in localizing the activated PKC enzyme near its target substrate. In neutrophils, PKC-{delta} colocalizes with F-actin in a PMA-sensitive manner; colocalization is blocked by the PKC-{delta} inhibitor rottlerin (19). Exposure of neutrophils to Staphylococcus aureus causes a rapid and dramatic redistribution of PKC-{delta} and F-actin. In the same study, microinjected {delta}C2-like domains colocalized with F-actin and prevented its redistribution. These findings indicate an interaction of PKC-{delta} with an active pool of actin that is necessary for F-actin redistribution. However, the observation that not all cellular PKC-{delta} interacts with cellular actin and not all F-actin colocalizes with PKC-{delta} mirrors our findings (15) from immunofluorescence and confocal microscopy of PKC-{delta} interaction with F-actin in Calu-3 airway epithelial cells. Indeed, the interaction of PKC-{delta} and F-actin appears to sequester PKC-{delta} to specific intracellular regions and, in Calu-3 cells, is necessary for activation of NKCC1.

How the interaction between PKC-{delta} and nonmuscle actin regulates NKCC1 function remains to be clarified. Anchoring of activated PKC-{delta} to F-actin might align PKC-{delta} with a target substrate for phosphorylation or sequester PKC-{delta} in a proteome that regulates NKCC1 activity. Understanding the relationship among PKC-{delta}, the actin cytoskeleton, and NKCC1 will clearly enhance our understanding of NKCC1 function and hence will be the focus of future research.


    FOOTNOTES
 

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: carole.liedtke{at}case.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.


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