Cystic Fibrosis Center and Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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Protein kinase C
(PKC) regulates cystic fibrosis transmembrane conductance regulator
(CFTR) channel activity but the PKC signaling mechanism is not yet
known. The goal of these studies was to identify PKC isotype(s)
required for control of CFTR function. CFTR activity was measured as
36Cl efflux in a Chinese hamster
ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a
Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased
CFTR activity induced with phorbol 12-myristate 13-acetate or with the
cAMP-generating agents ()-epinephrine or forskolin plus
8-(4-chlorophenylthio)adenosine 3',5'- cyclic
monophosphate. Immunoblot analysis of Calu-3 cells revealed that
PKC-
, -
II, -
, -
, and
-
were expressed in confluent cell cultures. Pretreatment of cell
monolayers with Lipofectin plus antisense oligonucleotide to PKC-
for 48 h prevented stimulation of CFTR with (
)-epinephrine,
reduced PKC-
activity in unstimulated cells by 52.1%, and decreased
PKC-
mass by 76.1% but did not affect hormone-activated protein
kinase A activity. Sense oligonucleotide to PKC-
and antisense
oligonucleotide to PKC-
and -
did not alter
(
)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-
.
Chinese hamster ovary cell line; chelerythrine; chloride efflux; epinephrine; protein kinase A; protein kinase C; cystic fibrosis transmembrane conductance regulator
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is a disease of electrolyte transport abnormalities that is characterized by the production of dehydrated viscous secretions in lungs, pancreatic duct, and intestinal tract. The genetic basis for CF is mutation of the CF transmembrane conductance regulator (CFTR), a secretory Cl channel and conductance regulator. CFTR is a 1,480-amino acid protein with a unique structure characterized by three cytoplasmic domains, two nucleotide binding folds, and a regulatory (R) domain that contains consensus sequences for phosphorylation by protein kinase A (PKA) and by protein kinase C (PKC). There is direct evidence for phosphorylation of serine residues in the R domain by PKA (5, 24) and by PKC (24). Firm evidence for PKA-mediated phosphorylation of CFTR as the primary regulator of CFTR activity comes from studies showing that altering or removing sites of in vivo phosphorylation in the R domain reduces, but does not eliminate, PKA stimulation of CFTR in intact cells and excised patches (4, 27, 33). However, the role of PKC in regulating the CFTR channel is less clear.
Addition of phorbol ester potentiated cAMP responses in Xenopus oocytes expressing wild-type CFTR (28) and in HT-29 colonic cells (1), T84 cells (6), C127 cells (7), and pancreatic duct cells (34). In membrane patches excised from cells expressing CFTR, addition of exogenous PKC caused a modest increase in CFTR channel activity and enhanced the rate and magnitude of subsequent PKA stimulation of open probability (29). Nevertheless, the interpretation of effects of phorbol 12-myristate 13-acetate (PMA) is not uniform and, instead, varies from direct PKC regulation of CFTR channel (6, 7, 29) to PMA-mediated increase in cell membrane area (34) and also PKC-mediated de novo insertion of channels into the plasma membrane (1). Trying to pinpoint how PKC regulates CFTR is complicated, however, by the use of PMA, which also influences CFTR expression (30) and degradation (3). Moreover, although PKC is considered to be the major receptor of PMA, its interactions with other enzymes might obscure a specific role for PKC in CFTR function.
Our studies on PKC regulation of a Cl secretory pathway in tracheal
epithelial cells (20, 22) led us to test the effects of a potent PKC
inhibitor, chelerythrine, on CFTR function. Preliminary data showed
that chelerythrine delayed efflux of
36Cl in Calu-3 lung cells
stimulated by ()-epinephrine, a cAMP-generating agent (see Fig.
1), suggesting that constitutive PKC activity in unstimulated cells is
necessary for maximal activation of CFTR. Similar findings were
reported by Jia et al. (14), from patch-clamp studies of Chinese
hamster ovary (CHO) and baby hamster kidney (BHK) cells expressing
wild-type CFTR. The question of how PKC regulates CFTR is still
unanswered. One step in understanding the regulation of CFTR by PKC is
to identify PKC isotype(s) that are required for CFTR function. That is
the goal of these studies.
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MATERIALS AND METHODS |
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Materials.
36Cl (specific activity 260 MBq/g,
7.5 mCi/g) was purchased from ICN Radiochemical (Irvine, CA) and
[-32P]ATP (specific
activity 111 TBq/mmol, 3,000 Ci/mol) was purchased from Amersham Life
Science (Arlington Heights, IL). An enhanced chemiluminescence kit was
purchased from Amersham, and the PKC assay system, PKA assay system,
and Lipofectin reagent were from GIBCO BRL Life Technologies
(Gaithersburg, MD). KN-93 and chelerythrine chloride were purchased
from Calbiochem (La Jolla, CA), PMA and forskolin were obtained from
Research Biochemicals International (Natick, MA), and
8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate
(CPT-cAMP) and (
)-epinephrine were from Sigma (St. Louis, MO).
Rabbit polyclonal anti-PKC isotype-specific antibodies were purchased
from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal mouse
anti-human CFTR (epitope 729-736 of the R domain) was purchased
from Genzyme (Cambridge, MA). Tissue culture supplies were obtained
from GIBCO BRL (Grand Island, NY) and Sigma. All other chemicals were
reagent grade.
Cell isolation and culture.
The Calu-3 cell line (American Type Culture Collection HTB-55; Ref. 26)
was grown submerged in Eagle's medium with Earle's balanced salt
solution and 10% fetal bovine serum (FBS) at 37°C in a humidified
CO2 incubator. For experiments,
cells were seeded onto six-well tissue culture plastic dishes and grown
to confluence, typically 7-8 days after subculture. A CHO cell
line stably expressing wild-type CFTR (CHO-wtCFTR) was generously given
to us by Dr. J. Riordan (Mayo Clinic, Scottsdale, AZ). The CHO-wtCFTR
cell line was maintained in culture medium consisting of MEM, 8%
FBS, 200 µM methotrexate, and 1% penicillin-streptomycin. CHO cells were grown to confluence in a 5%
CO2 incubator at 37°C.
Measurement of CFTR activity as Cl efflux.
CFTR activity was assayed by measuring the rate of
36Cl efflux (32). Cell cultures
were grown to confluence in six-well tissue culture dishes and
preincubated in serum-free medium for 24 h before use. Cells were
preincubated for 1 h at 35°C with 3.5 µCi 36Cl in 10 mM HEPES-buffered
Ringer (HBR; pH 7.5) solution that contained (in mM) 136.9 NaCl, 5.4 KCl, 0.4 KH2PO4,
0.3 NaHPO4, 4.2 NaHCO3, 1.3 CaCl2, 0.5 MgCl2, 0.4 MgSO4, and 5.6 D-glucose. Medium with
radioactive tracer was removed, and cells were washed four times with
HBR to remove extracellular 36Cl.
After the wash, 0.5-ml aliquots of isotope-free HBR were added and
sequentially removed every 60 s for up to 11 min. The first three
aliquots were used to establish a stable baseline in efflux buffer
only. Agonists were added after the third aliquot was removed. Inhibitors were present in the bathing medium for the last 30 min of
the 36Cl loading period and during
the efflux period. Radioactive counts remaining in the cells were
extracted with 0.1 N NaOH. The fraction of intracellular
36Cl remaining in the cell layer
during each time point was calculated from the sample and extract
counts. Time-dependent rates of
36Cl efflux were calculated as
ln (36Clt = 1/36Clt = 2)/(t1 t2),
where 36Cl is the percent
intracellular Cl at time t and
t1 and
t2 are successive
time points.
Oligonucleotide treatment of cells.
Phosphodiester oligonucleotides were purchased from GIBCO BRL.
Antisense oligonucleotides to PKC isotypes were complementary to the
translation initiation region of mRNA specific for the animal species
delineated in Table 1. Sense
oligonucleotides were used as controls. Oligonucleotides were dissolved
in sterile deionized water to a final concentration of 1 mM, aliquoted,
and stored at 20°C until ready for use.
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Immunoblot analysis of CFTR and PKC isotypes. Culture medium was replaced with Hanks' balanced salt solution supplemented with 10 mM HEPES (pH 7.5). Cells were treated with vehicle or drugs of interest at 35°C for times indicated. Cell cultures were immediately washed twice with ice-cold PBS and then harvested in 1 ml 100 mM NaCl, 50 mM NaF, 50 mM Tris · HCl (pH 7.55), 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 100 µM leupeptin, 1 µg/ml aprotinin, and 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride. Immunoblot analysis of cell proteins for PKC isotypes was performed as described previously (22). Protein bands immunoreactive to specific antibodies were detected using enhanced chemiluminescence and analyzed by laser densitometry in a Sciscan 5000 (United States Biochemical), using the OS-Scan image analysis system software package (Oberlin Scientific).
To confirm the expression of wtCFTR in transfected cells and CFTR in Calu-3 cells, immunoblot analysis for CFTR was performed on lysates of cells. Lysis buffer was supplemented with 0.1% SDS. Lysates were incubated at 30°C for 30 min in 50 mM Tris (pH 6.8), 100 mM dithiothreitol, 5% glycerol, 4% SDS (wt/vol), and 0.1Measurement of PKC and PKA activity.
Cell cultures grown on 60-mm tissue culture plastic dishes were treated
with vehicle or the drug of interest and then harvested in 0.5 ml of
lysis buffer. Lysates were clarified and incubated with antiserum
against a specific PKC isotype, as previously described (22). Kinase
activity of immune complexes of PKC isotypes was measured using histone
III as the substrate for PKC-,
-
II, -
, and -
and a
peptide derived from the pseudosubstrate region of PKC-
as the
substrate for PKC-
(25). PKA activity of clarified lysates was
measured using a commercially available assay system (GIBCO BRL Life
Technologies).
Data analysis. Protein levels were determined with a Bradford assay kit (Bio-Rad, Hercules, CA) using BSA as the standard. Data were analyzed by ANOVA followed by Bonferroni multiple comparison tests or by Student's t-tests for unpaired samples. Data are reported as means ± SE for the number of cell monolayers tested (n).
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RESULTS |
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Effect of PKC inhibitor on cAMP-stimulated efflux.
CFTR function has been assessed in CHO-wtCFTR and Calu-3 cells using
iodide efflux and whole cell and patch-clamp recordings of
cell-attached and cell-excised patches (13, 29). These methods detected
a cAMP-regulated Cl permeability that was indistinguishable from CFTR.
For the studies reported here,
36Cl efflux was used as an
indicator of CFTR function. Figure
1 illustrates the effect of
chelerythrine, a general PKC inhibitor, on CFTR function in CHO-wtCFTR
and Calu-3 cells. Addition of the combination of forskolin and CPT-cAMP
to CHO-wtCFTR cells rapidly increased the rate of
36Cl efflux, with peak rates at 2 min after addition of stimulatory agents (Fig.
1A). Efflux rates subsequently
declined to the steady-state level observed just before addition of
stimulatory agents. Calu-3 cells gave a similar response to 3 µM
()-epinephrine, an endogenous hormone that occupies
- and
-adrenergic receptors and increases cAMP levels (Fig.
1B). Maximal rates of
36Cl efflux occurred 1-2 min
after addition of hormone and declined afterward to prestimulatory
steady-state levels. Figure 2 summarizes the sensitivity of CFTR activity at peak rates to cAMP-generating agents. The combination of forskolin and CPT-cAMP significantly increased the rate of efflux in CHO-wtCFTR to 0.78 ± 0.11 min
1
(n = 9; Fig.
2A) and in Calu-3 cells to 0.80 ± 0.16 min
1
(n = 5; Fig.
2B). As seen in Fig.
2B, forskolin and CPT-cAMP mimicked
the effect of (
)-epinephrine in Calu-3 cells. A signaling mechanism for the effects of forskolin plus CPT-cAMP focuses on the
elevation of cAMP levels by bypassing membrane receptors, which leads
to activation of PKA and subsequent phosphorylation of CFTR and
increased channel activity. However, the data of Fig. 1 suggest a role
for PKC in cAMP-dependent activation of CFTR. Figure 1 shows that
pretreatment with 10 µM chelerythrine, a general PKC inhibitor,
abolished the stimulatory effects of forskolin and CPT-cAMP on
CHO-wtCFTR cells and of (
)-epinephrine on Calu-3 cells,
suggesting that PKC activity in unstimulated cells regulates CFTR
function. As seen in Fig. 2A,
chelerythrine significantly reduced peak rates elicited with
cAMP-generating agents and, more importantly, with the phorbol ester
PMA, an activator of PKC, in CHO-wtCFTR cells. The sensitivity of
PMA-induced CFTR activity to chelerythrine indicates that inhibition of
PKC blocks PKC-induced CFTR activity.
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Effect of antisense oligonucleotides on cAMP-dependent CFTR
activation.
The approach of antisense technology to reduce PKC isotype mass and
activity was used by this laboratory to identify a critical role for
PKC- in the regulation of Na-K-2Cl cotransport in human tracheal
epithelial cells (20) and in CF/T43 cells (21). Calu-3 cells were used in the next series of experiments, in which an antisense approach was used to mimic the effect of inhibition of PKC by
chelerythrine on CFTR function. First, PKC isotypes expressed by Calu-3
cells were identified by immunoblot analysis. Calu-3 cells were
immunoreactive with polyclonal antibodies to PKC-
,
-
II, -
, -
, and -
(Fig.
3A).
PKC-
I, -
, and -
were not
detected. Proteins immunoreactive to PKC isotypes corresponded closely
in apparent molecular weight to recombinant PKC isotypes and to
calculated PKC isotype molecular weights. Immunoreactive protein bands
to PKC isotypes that were found in Calu-3 cells were also found in
normal human tracheal epithelial cells (22) and in CF/T43 cells (21).
The relative distribution of PKC isotypes to cytosol and a particulate
fraction in Calu-3 cells (Fig. 3A) shared similarities with normal human tracheal epithelial cells (22)
and with CF/T43 cells (21), with PKC-
and -
localized predominantly in cytosol and
PKC-
II distributed
approximately evenly between cytosol and a particulate fraction.
PKC-
and -
were distributed predominantly to cytosol.
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Effect of antisense oligonucleotide on PKC-
expression and activity.
Antisense oligonucleotide to PKC-
could block activation of CFTR by
cAMP by diminishing PKC-
activity in unstimulated cells, decreasing
the amount of PKC-
, or both. The first possibility was investigated
by measuring kinase activity of immune complexes of PKC isotypes in
untreated cells or in cells preincubated for 24 h with 1 µg/ml
Lipofectin or with Lipofectin plus antisense oligonucleotide to
PKC-
. Incubation of Calu-3 cells with antisense oligonucleotide
reduced total PKC-
activity by 52.1% (Table
3). Moreover, PKC-
activity per unit
protein also significantly decreased, indicating a loss of PKC-
activity. Treatment of cells with Lipofectin did not significantly
alter baseline PKC-
activity (Table 3). PKC-
expression was next
evaluated in cells treated with antisense oligonucleotide to PKC-
and, as a control, in untransfected cells (Fig.
3B). Antisense oligonucleotide
reduced PKC-
by 76.1 ± 4.5% (n = 6) and did not affect PKC-
,
-
II, -
, or -
mass (Fig.
3B). These results indicate that
antisense oligonucleotide to PKC-
blocks CFTR function by
decreasing PKC-
mass and activity in Calu-3 cells.
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Effect of antisense oligonucleotide on PKA activity.
Regulation of CFTR function by PKC- could be due to modulation of
PKA to alter PKA-dependent phosphorylation of CFTR. To test this
possibility, PKA activity was quantitated in untransfected cells,
Lipofectin-treated cells, and cells transfected with antisense oligonucleotide to PKC-
. As seen in Table
4, antisense oligonucleotide to PKC-
did not affect (
)-epinephrine-stimulated PKA
activity, indicating the independence of PKA from PKC-
. Therefore,
PKC-
regulates CFTR function at a different site.
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DISCUSSION |
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Regulation of CFTR Cl channel by PKC has been inferred
from studies of CFTR expressed in a variety of epithelial cell lines and in heterologous expression systems (1, 6, 7, 14, 28, 34), yet few
studies provide detailed information about PKC regulation of CFTR. The
current study provides the first report of regulation of CFTR channel
function by a Ca-independent PKC isotype, PKC-. The cAMP-generating
agent forskolin in combination with CPT-cAMP increased CFTR Cl channel
activity, measured as 36Cl efflux,
from 0.23 ± 0.02 min
1
(n = 10) to 0.78 ± 0.11 min
1
(n = 9) in CHO-wtCFTR cells and from
0.12 ± 0.01 min
1
(n = 25) to 0.80 ± 0.16 min
1
(n = 5) in Calu-3 cells. Pretreatment
of both cell lines with the general PKC inhibitor chelerythrine blocked
cAMP-induced CFTR Cl channel activity (Figs. 1 and 2), suggesting that
baseline, constitutive PKC activity is necessary for acute stimulation
of CFTR. Jia et al. (14) came to a similar conclusion from a study of
two mammalian expression systems, CHO-wtCFTR and BHK-wtCFTR, using an
electrophysiological approach. The studies reported here also show that
elevated CFTR function induced by PMA in CHO-wtCFTR cells (Fig.
2A) was blocked by pretreatment with
chelerythrine, indicating that chelerythrine blocks the effects of PMA
by preventing an increase in PKC activity.
Previous studies by this laboratory on basolateral Na-K-2Cl cotransport
showed that, in the absence of maximal Cl secretion, activation of
cotransport by 1-adrenergic
agents requires a PKC signaling mechanism and that increased PKC-
activity in a cytosolic fraction is necessary for activation (20). The
identity of a PKC isotype necessary for cAMP-dependent CFTR Cl channel
activity was investigated first by establishing which PKC isotypes were expressed in Calu-3 cells and then by mimicking the effects of inhibition of PKC enzyme activity by individually downregulating PKC
isotypes. Calu-3 cells express five PKC isotypes representative of
three major types of PKC isotypes (Fig. 3). Conventional PKC isotypes
and
II, novel PKC isotypes
and
, and atypical PKC isotype
were identified by immunoblot
analysis. The same PKC isotypes were also found in human tracheal
epithelial cell cultures and in CF/T43 cells, indicating that this
panel of PKC isotypes is a signature of tracheal epithelial cells and
cell lines derived from primary tracheal epithelial cell cultures (20,
21).
Downregulation of PKC isotypes was accomplished using an antisense
approach. Using this approach, this laboratory obtained convincing
evidence for a role for PKC- in the activation of Na-K-2Cl
cotransport. A major reason for using this approach is that long-term
treatment with the phorbol ester PMA for 18 h did not deplete PKC
isotype activity or abundance in CF/T43 cells (21) or in primary
cultures of human tracheal epithelial cells (22). Treatment of Calu-3
cells with antisense oligonucleotide to PKC-
or -
for 48 h
reduced PKC-
by 73.7% and PKC-
by 81.1%, respectively, but
failed to prevent cAMP-dependent CFTR Cl channel function (Table 2) and
did not affect Cl loading. Previous studies established that antisense
oligonucleotide to PKC-
prevents
1-adrenergic activation of
Na-K-2Cl cotransport but not baseline cotransport activity in
unstimulated cells. Thus there may be sufficient cotransport activity
in cells treated with antisense oligonucleotide to PKC-
to support
Cl loading. Alternatively, activity of other Cl transport pathways
(e.g., Cl/HCO3 exchange) might
mediate Cl loading. A major finding of this study is that antisense
oligonucleotide to PKC-
potently blocked cAMP-dependent activation
of CFTR Cl channel function (Table 3 and Fig. 4). Antisense
oligonucleotide to PKC-
also reduced PKC-
by 76.1%, a finding
consistent with the half-life of ~24 h for PKC in vitro, and
significantly reduced baseline activity of PKC-
(Table 3). The
latter finding provides evidence for constitutive PKC-
activity that
was also suggested by Jia et al. (14) to explain their results.
A PKC- signaling mechanism involved in regulation of CFTR function
was next investigated by asking whether PKC-
modulated activity of
PKA, a protein kinase that is specifically activated by cAMP and is
necessary for increased CFTR Cl channel activity. Antisense
oligonucleotide to PKC-
did not affect a
(
)-epinephrine-mediated increase in PKA activity (Table 4)
despite blocking (
)-epinephrine-induced CFTR Cl channel activity
(Fig. 4). Hence, PKC-
regulates CFTR Cl channel function at a site
other than PKA. The phorbol ester PMA stimulates CFTR Cl channel
function, apparently by increasing PKC phosphorylation of CFTR;
however, more definitive studies are needed to determine whether
phosphorylation of CFTR by PKC-
in unstimulated cells is necessary
to achieve maximal CFTR channel activity when cAMP levels are elevated.
Downregulation of PKC- by antisense oligonucleotides or by long-term
phorbol ester treatment has implicated PKC-
in a number of cellular
functions, including regulation of electrolyte and nonelectrolyte
transporters. In a human liver epithelial BC1 cell line, long-term PMA
treatment reduced CFTR mRNA, with a concomitant inhibition of CFTR Cl
channel activity, and induced a cytosol-to-membrane translocation of
PKC-
and -
(16), indicating a role for these PKC isotypes in
previously observed PMA-sensitive CFTR expression (30). PKC-
was
also found to be necessary for inhibition of vasopressin-stimulated
Na transport in rabbit cortical collecting duct cells,
suggesting cAMP-mediated activation of PKC-
(8), and for amino acid
transport in cultured human fibroblasts (11). In rat cardiac muscle,
PKC-
translocation to cross-striated structures after stimulation
with PMA or with
1-adrenergic
agonists (10) can be blocked with chelerythrine or the V1 fragment of
PKC-
(10), resulting in loss of contractility (9, 15) and protection from ischemic injury (12). PKC isotypes, either alone or in combination
with other PKC isotypes, play major roles in cross talk among signal
transduction pathways, as demonstrated in recent studies. For example,
mechanosensitive signal transduction in endothelial cells involves
PKC-
and extracellular signal-regulated kinase-1/2 (31), and tumor
necrosis factor-
-regulated insulin signaling in HEK-293 cells
involves PKC-
(17). However, selective activation of PKC-
or -
in rat adrenal glomerulosa cells is stimulus dependent (23), and the
level of expression of human endothelial nitric oxide synthase
increases with PMA-stimulated PKC-
and -
(18).
Identification of a Ca-independent PKC isotype in the regulation of CFTR agrees with the phosphorylation studies of Picciotto et al. (24), who report that PKC phosphorylated CFTR in a Ca-independent manner. Phosphopeptide mapping localized PKC phosphorylation sites at serines 686 and 790 of the R domain; however, in vivo PKC phosphorylation using PMA as a PKC activator revealed additional sites outside the R domain. The role of PKC phosphorylation sites is still not clear, although recent studies by Wilkinson et al. (33) suggest an interaction between phosphorylated amino acid residues to explain roles for inhibitory and stimulatory phosphorylation sites (16).
The finding that PKC- regulates CFTR function is novel and clearly
differentiates PKC-
regulation of CFTR from PKC-
modulation of
Na-K-2Cl cotransport. Although both PKC isotypes are diacylglycerol dependent and Ca2+ independent,
cotransport activation requires increased PKC-
activity as opposed
to an apparently constitutive PKC-
activity linked to CFTR. These
results indicate specific roles for selective PKC isotypes in airway
epithelial cells and, furthermore, suggest that a critical component of
a PKC signaling mechanism is the targeting of a PKC isotype to its
substrate protein, which can be localized to apical or basolateral
membranes.
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ACKNOWLEDGEMENTS |
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We acknowledge the excellent technical assistance of Qiuping Shu and Peter Wung.
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FOOTNOTES |
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This research was supported by National Heart, Lung, and Blood Institute Grant HL-50160 and by the Cystic Fibrosis Foundation.
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. §1734 solely to indicate this fact.
Address for reprint requests: C. M. Liedtke, Pediatric Pulmonology, Case Western Reserve University, BRB, Room 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948.
Received 19 May 1998; accepted in final form 12 August 1998.
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