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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ABSTRACT |
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Activation of airway
epithelial Na-K-2Cl cotransporter (NKCC)1 requires increased activity
of protein kinase C (PKC)-, 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-
were verified by Western
blot analysis of immunoprecipitated proteins. PKC-
was detected in
immunoprecipitates of NKCC1 and vice versa. Actin was also detected in
immunoprecipitates of NKCC1 and PKC-
. Pulldown of endogenous actin
revealed the presence of NKCC1 and PKC-
. Binding of recombinant
PKC-
to NKCC1 was not detected in overlay assays. Rather, activated
PKC-
bound to actin, and this interaction was prevented by a peptide
encoding
C2, a C2-like domain based on the amino acid sequence of
PKC-
.
C2 also blocked stimulation of NKCC1 function by
methoxamine. Immunofluorescence and confocal microscopy revealed
PKC-
in the cytosol and cell periphery. Merged images of cells
stained for actin and PKC-
indicated colocalization of PKC-
and
actin at the cell periphery. The results indicate that actin is
critical for the activation of NKCC1 through a direct interaction with PKC-
.
C2-like domain; coimmunoprecipitation; protein kinase C; rottlerin; jasplakinolide; latrunculin; sodium-potassium-chloride cotransport
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INTRODUCTION |
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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)- 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-
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 -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
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-
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-
modulates
NKCC1 function and that this interaction involves a C2-like domain on
the regulatory arm of PKC-
.
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METHODS |
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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 · 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 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).
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 -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 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 DH5
cells transformed with
the pET14b-
C2 construct and grown for 3 h at 37°C.
Isopropyl-
-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- 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-
in the absence or presence of PKC activators, and washed extensively. rPKC-
binding was detected by immunoblot analysis. In a
second method, a solution binding assay was performed. rPKC-
was
incubated with phosphatidylserine (PtdSer) and dioctanylglycerol (DOG)
for 15 min at 30°C to activate PKC-
. G-actin or F-actin was added,
and the incubation continued for 30 min at room temperature. Complexes
of PKC-
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-
.
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- (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-
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 -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%
-actin,
15%
-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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RESULTS |
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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
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-
is necessary for jasplakinolide-stimulated NKCC1 activity, cells were pretreated with rottlerin, an inhibitor of
PKC-
. 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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Coimmunoprecipitation of PKC-, actin, and NKCC1.
A recent study from this laboratory (23) demonstrated an
association between NKCC1 and PKC-
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-
and NKCC1 coimmunoprecipitate with
actin (Fig. 2). Lysates from Calu-3 cells
were treated with antibodies to PKC-
or with antibodies to actin
coupled to agarose beads. PKC-
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-
and in total cell lysates (Fig. 2A, left). Likewise, PKC-
was detected in
pulldowns of actin and in total cell lysates (Fig. 2A,
right). The results demonstrate coimmunoprecipitation of
actin and PKC-
. 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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In vitro binding of PKC- to nonmuscle actin.
Binding properties of actin and PKC-
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-
.
F-actin was recovered by pulldown with anti-actin antibody coupled to
agarose beads. To preactivate rPKC-
, enzyme was preincubated with
the PKC activators PtdSer and DOG. Figure 4A shows that
PKC-
binds to nonmuscle F-actin and that binding is enhanced
threefold by preactivation of enzyme. To directly investigate binding
of PKC-
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-
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-
by 84%, and latrunculin B decreased PKC-
binding by 28%
(Fig. 4B). Binding of PKC-
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-
binding.
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Intracellular localization of actin and PKC-.
The new findings on PKC-
-actin interaction in Calu-3 cells imply
that PKC-
and actin might colocalize to the same region of the cell.
The intracellular localization of PKC-
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-
and actin. Actin was detected at the cell periphery. PKC-
,
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-
and actin at discrete sites in the
cell periphery but not the cytosol.
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Competitive inhibition of binding of PKC- to actin by a PKC-
C2-like domain (
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
C2 to investigate its binding to nonmuscle
F-actin in a slot blot binding assay. Figure 6A shows that
C2 binds to
nonmuscle F-actin in a dose-dependent manner. Binding of PKC-
to
muscle actin was negligible (data not shown). We next tested inhibition
of the binding of PKC-
to F-actin by
C2. The results, illustrated
in Fig. 6B, demonstrate that
C2 blocks binding of
rPKC-
to F-actin in a dose-dependent manner with an IC50
(inhibitory concentration) of 2.26 µg.
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DISCUSSION |
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Regulation of NKCC1 function in airway epithelial cells is a
complex process, involving a signaling cascade in which PKC- 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-
(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-
with actin (Fig. 5) and avid binding of
PKC-
to nonmuscle actin (Figs. 4 and 6). Binding of PKC-
to actin
was concentration dependent and enhanced by the presence of PKC
activators. A functional role for actin-PKC-
binding was studied
with
C2, a peptide encoding the C2-like domain of PKC-
, as an
inhibitory peptide in binding and transport experiments, respectively
(Figs. 6 and 7). Binding of PKC-
to nonmuscle actin was blocked by
C2 (Fig. 6), which also prevented stimulation of NKCC1 activity by
methoxamine (Fig. 7). We showed previously that methoxamine increases
activity of PKC-
and that this is necessary for activation of NKCC1
(20). Now we learn that binding of PKC-
to
actin is also a necessary step toward activation of NKCC1.
The C2 domain encodes a 123-amino acid segment of the
NH2-terminal region of PKC-
and, structurally, has an
antiparallel
-sandwich with a P-type topology similar to
phospholipase-
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-
presents a novel phospholipid-independent binding site interacting with
nonmuscle actin.
C2 also interacts with other cellular proteins.
GAP-43, also known as neuromodulin, forms a complex with PKC-
in
intact cells through direct binding of GAP-43 with the regulatory
domain of PKC-
in the absence of phospholipid (10). The
interaction between GAP-43 and PKC-
was narrowed to the V0/C2 region
comprising amino acids 1-121, which is referred to as the
C2
domain in our study. These results and our new finding of binding of
C2 to nonmuscle actin indicate that the
C2 domain is not just a
target region for a PKC cofactor but serves as a protein-protein
interaction domain. The binding of PKC-
to nonmuscle actin via the
C2-like domain is reminiscent of the binding of the novel PKC
isotype PKC-
to receptor for activated C kinase 1 (RACK1) in Calu-3
cells (24). An eight-amino acid sequence in the
regulatory domain of PKC-
, designated
V1-2, localizes to a
region analogous to
C2 of PKC-
and is necessary for binding of
activated PKC-
to RACK1. As with
V1-2,
C2 prevented
binding of PKC-
to nonmuscle actin (Fig. 6B). The absence
of a requirement for phospholipid for binding indicates that the
C2
domain lacks a recognition site for phosphatidylserine. The C2 region
of the conventional PKC isotype PKC-
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-
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.
C2 appears to block activation of NKCC1 by binding to actin
and thus preventing binding of PKC-
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- 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-
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-
by phorbol
12-myristate 13-acetate (PMA) was reported to disrupt the actin
cytoskeleton in lymphocytes (32). The Ca-dependent PKC
isotype PKC-
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 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-
activity are hormone receptor-mediated activation and hyperosmotic
stress. A common kinase critical for activation of PKC-
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- 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-
binding in Calu-3 cells is not yet clear.
Binding may bring PKC-
near its substrate or near protein kinases
and phosphatases, which regulate its activity. Another possibility is
that PKC-
is sequestered to specific subcellular compartment(s).
Because the overall result of activation of PKC-
is rapid
stimulation of NKCC1, the positioning of PKC-
near its substrate
would be advantageous for fidelity and specificity in the regulation of
NKCC1 function.
How the interaction between PKC- and actin leads to activated NKCC1
remains to be clarified. Anchoring of activated PKC-
close to its
target substrate might enhance substrate phosphorylation. NKCC1 is one
of several likely target substrates for activated PKC-
, as are
actin, actin-binding proteins, and other PKC binding proteins (6,
16). Another possibility is that activated PKC-
stabilizes
the F-actin cytoskeleton, thus promoting activation of NKCC1
(19). The relationship between PKC-
, 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 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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