(Received for publication, October 2, 1996)
From the In order to investigate the
involvement of cGMP-dependent protein kinase (cGK) type II
in cGMP-provoked intestinal Cl In intestinal epithelium a cGMP-signaling pathway can activate
cystic fibrosis transmembrane conductance regulator (CFTR)1
Cl Localization studies have suggested a key role for a recently cloned
isotype of cGMP-dependent protein kinase (cGK), designated type II, as the mediator of the cGMP-provoked intestinal
Cl For the analyses of the interactions of CFTR and cGK II or cGK I Protein A-Sepharose was from Pierce,
3-isobutyl-1-methylxanthine (IBMX), and rat atrial natriuretic peptide
(ANP) from Sigma, cGMP analogs from Biolog (Bremen,
Germany), and [32P]orthophosphate,
[ For the development of the recombinant virus Ad-cGKII, the
coding sequence of rat cGK II (6) was cloned into the multiple cloning
site between the CMV promoter and the bGH-3 Rat
intestine-derived IEC-6 cells stably expressing CFTR (IEC-CF7) were
prepared as described (14) and cultured in Dulbecco's modified
Eagle's medium supplemented with 5% fetal calf serum, 0.1 IU/ml
insulin, 0.2 mg/ml G418, 0.1 mg/ml streptomycin, and 0.04 mg/ml
penicillin. Two days after plating, confluent (subconfluent in the case
of patch clamp analysis) monolayers of cells were infected by replacing
the medium with fresh medium additionally containing the adenovirus
vectors (usually 5 × 109 particles/ml).
Two days after
infection cells were washed twice with ice-cold phosphate-buffered
saline, scraped with a rubber policeman in buffer A (150 mM
NaCl, 10 mM NaPO4 pH 7.4, 1 mM
EDTA, 100 µg/ml trypsin inhibitor, and 20 µg/ml leupeptin) and
homogenized by brief sonication (three bursts of 3 s, peak-to-peak
amplitude 15-20 µm). Cytosol and membranes were separated by
centrifugation at 150,000 × g for 60 min at 4 °C in
an Airfuge. Protein kinase activity was determined by incubation of the
samples (10 µg of protein) at 30 °C for 4 min in 40 µl of 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 5 mM Cells which
had been infected 2 days earlier were loaded with tracer for 1.5 h
under a humidified 95% air, 5% CO2 atmosphere at 37 °C
in a modified Meyler solution (108 mM NaCl, 4.7 mM KCl, 1.3 mm CaCl2, 1 mM
MgCl2, 0.8 mM NaH2PO4,
0.4 mM Na2HPO4, 20 mM NaHCO3, 20 mM HEPES, and 10 mM
glucose, pH 7.4) containing 5 µCi/ml 125I Whole-cell Cl Two days
after infection, confluent cells grown in 6-well plates (Costar; 10 cm2/well) were incubated at 37 °C for 1 h in 1 ml
of modified Meyler solution without phosphate supplemented with 0.5 mCi
of [32P]orthophosphate. Labeled cells were incubated for
an additional 20 min with membrane-permeable cGMP analogs or forskolin
in the same medium. Subsequently the medium was removed and the cells were lysed with 300 µl of 1% SDS containing 100 µg/ml leupeptin and 100 µM phenylmethylsulfonyl fluoride. The lysates
were diluted 2-fold with a solution of 50 mM Tris-HCl, pH
7.5, 400 mM NaCl, 10 mM EDTA, 200 mM NaF, 40 mM sodium pyrophosphate, and 5%
Nonidet P-40 and were homogenized by six passages through a thin needle (0.5 × 16 mm). 32P-Phosphorylated CFTR was
immunoprecipitated with a purified anti-CFTR antibody C449 as described
(15), separated by 6% SDS-polyacrylamide gel electrophoresis,
visualized by autoradiography, and quantitated with the Molecular
Imaging System GS-363.
To study
the interaction of CFTR with cGK I or II, we used rat intestinal
IEC-CF7 cells stably transfected with CFTR Cl
125I
8-pCPT-cGMP was unable to stimulate 125I
In excised membrane patches, the activation of CFTR Cl In agreement with our previous in vitro observations in
excised membrane patches (13), cGMP activation of CFTR in intact IEC-CF7 cells was specifically mediated by type II cGK, since neither
8-Br-PET-cGMP nor ANP provoked an increase in
125I As shown in Fig.
4, 8-pCPT-cGMP caused an almost 3-fold increase in
32P labeling of CFTR in Ad-cGK II-infected IEC-CF7 cells.
In contrast, 8-Br-PET-cGMP had no effect on CFTR phosphorylation in
Ad-cGK I
In conclusion, our demonstration that cGK II expression rendered CFTR
sensitive to modulation by cGMP in cells which did not previously
display a cGMP-inducible Cl
Department of Biochemistry,
Department of Clinical
Genetics, Faculty of Medicine and Health Sciences, Erasmus University
Rotterdam, 3000 DR Rotterdam, The Netherlands and ¶ Laboratory of
Clinical Biochemistry, Medical University Clinic Würzburg, 97080 Würzburg, Germany
secretion,
cGMP-dependent activation and phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl
channels was analyzed after expression of cGK II or cGK I
in intact
cells. An intestinal cell line which stably expresses CFTR (IEC-CF7)
but contains no detectable endogenous cGK II was infected with a
recombinant adenoviral vector containing the cGK II coding region
(Ad-cGK II) resulting in co-expression of active cGK II. In these
cells, CFTR was activated by membrane-permeant analogs of cGMP or by
the cGMP-elevating hormone atrial natriuretic peptide as measured by
125I
efflux assays and whole-cell patch clamp
analysis. In contrast, infection with recombinant adenoviruses
expressing cGK I
or luciferase did not convey cGMP sensitivity to
CFTR in IEC-CF7 cells. Concordant with the activation of CFTR by only
cGK II, infection with Ad-cGK II but not Ad-cGK I
enabled cGMP
analogs to increase CFTR phosphorylation in intact cells. These and
other data provide evidence that endogenous cGK II is a key mediator of
cGMP-provoked activation of CFTR in cells where both proteins are
co-localized, e.g. intestinal epithelial cells.
Furthermore, they demonstrate that neither the soluble cGK I
nor
cAMP-dependent protein kinase are able to substitute for
cGK II in this cGMP-regulated function.
channels, resulting in the net
secretion of salt and water (1, 2). Guanylin and/or uroguanylin, small
peptides derived from larger precursor proteins synthesized by
intestinal epithelial cells, may function as the physiological
activator of the cGMP-mediated signaling route in intestine by
activating guanylyl cyclase C located in the apical membrane of
enterocytes (2-4). Heat-stable enterotoxins secreted by various
pathogenic strains of Escherichia coli mimic the action of
guanylin and elicit a severe secretory diarrhea by hyperactivating
guanylyl cyclase C (2-4).
secretion (5-7). Type II cGK is expressed
predominantly in epithelial cells of the intestine (5-7), although it
was also detected in kidney (6, 8, 9) and brain (6, 8, 10). In
contrast, type I cGK, consisting of
and
isoforms, and shown to
act as a key regulator of cardiovascular homeostasis (11, 12), is not
expressed in enterocytes (7). Furthermore, purified endogenous pig cGK
II, in contrast to bovine lung cGK I, was shown to activate CFTR in
excised membrane patches (13). However, the mechanism of the apparent
cGK isotype selectivity in activating CFTR was not clear, since both
cGK I and cGK II could phosphorylate immunoprecipitated CFTR in
vitro (13). An explanation for this discrepancy might be that cGK
II but not cGK I selectively phosphorylates CFTR in a native
environment. To examine this possibility we investigated activation and
phosphorylation of CFTR in intact cells expressing either cGK II or cGK
I
. Endogenous expression of cGK isoforms in intact cells also
permitted use of native enzyme in these experiments such that
alterations, particularly of cGK II, that occur during purification
could be avoided. Purification of the membrane-bound cGK II requires
the use and subsequent removal of detergents, a procedure which
potentially contributes to nonspecific (hydrophobic) interactions of
this enzyme. Furthermore, purification results in partial proteolytic
modification of cGK II and renders cGK II less sensitive to cGMP (5,
13).
in
intact cells, we established a highly efficient co-expression system in
which rat intestinal IEC-CF7 cells previously stably transfected with
CFTR (14) were infected with recombinant adenoviral vectors containing
the cDNA of cGK II or cGK I
. Here we report that co-expression
with cGK II but not with cGK I
renders CFTR sensitive to activation
by cGMP in intact cells. Furthermore, CFTR is shown to be a selective
substrate for only cGK II-mediated phosphorylation under physiological
conditions, providing a possible explanation for the present and
previously (13) observed isotype-specific activation of CFTR by cGK
II.
Materials
-32P]ATP, 125I
, and the
enhanced chemiluminescence (ECL) system from Amersham Corp. Polyclonal
cGK II or cGK I antibodies raised against recombinant cGK II or cGK
I
expressed in E. coli were prepared as described (7).
The polyclonal antibody C449 against CFTR (15) was a gift from A. C. Nairn (Rockefeller University, New York, NY). The cGK substrate peptide
2A3 (RRKVSKQE) and the Walsh inhibitor peptide (PKI-(5-24)amide) were
synthesized by D. Palm (University of Würzburg, Germany).
and cGK
II
untranslated region of the
adenoviral transfer plasmid pZS2 (16). Upstream of the expression
cassette the plasmid also contains 0.5 kilobase 5
terminal sequence of
wild-type adenovirus type 5. The recombinant plasmid cGKII/pZS2 was
linearized with XbaI and ligated with the long
XbaI fragment of the DNA of RR5, an Ad5 mutant carrying a unique XbaI site and a deletion of the E1 region ranging
from nucleotide position 445-3333 (16). The ligation product was then
transfected into the E1a-transformed human embryonic kidney cell line
293, and after 10-14 days, recombinant virus was screened for by
polymerase chain reaction, recovered, and plaque-purified as described
previously (16). For the construction of the recombinant virus Ad-cGK
I
, the expression cassette from pCl (an expression vector obtained
from Promega containing the CMV promoter and the SV40 poly(A)) was
cloned into the multiple cloning site of the adenoviral transfer
plasmid p
E1sp1A (a plasmid containing N-terminal E1-deleted
sequences of Ad5, obtained from Microbix Biosystems lnc., Toronto,
Canada) to yield the recombinant plasmid pCMVI. Then the coding
sequence of human cGKI
(17, 18) was cloned into the expression
cassette of pCMVI resulting in the plasmid cGKI
/pCMVI. Recombinant
adenovirus was generated via homologous recombination between
cGKI
/pCMVI and pJM17 (Microbix), a bacterial plasmid containing
full-length Ad5 DNA, following cotransfection of both plasmids into 293 cells (19). The titer of the adenoviral preparations was approximately
1 plaque-forming unit per 500 particles.
-mercaptoethanol, 0.1 mM
3-isobutyl-1-methylxanthine, 25 mM Na-
-glycerophosphate,
200 nM protein kinase A inhibitor (PKI), 0.1 mg/ml of a cGK
substrate peptide 2A3 (RRKVSKQE; see Ref. 18), 1 µCi of
[
-32P]ATP, 300 µM unlabeled ATP and cGMP
or cGMP analogs as described (20). Immunoblotting was performed as
described earlier (21). Immunoreactive proteins were detected after
incubation with cGK II or cGK I antibody (1:3000) by the enhanced
chemiluminescence method and quantitated by the Molecular Imaging
System GS-363 (Bio-Rad) using standards of purified bovine lung cGK I,
recombinant rat intestine cGK II expressed in and purified from Sf9
cells (22), or rat intestinal brush border membranes containing an established amount of endogenous cGK II.
Efflux Studies
.
After removal of extracellular isotope with three washes of 3 ml of
modified Meyler solution, isotope efflux from the cells was determined
at 37 °C by addition and consecutive replacements of 1 ml of
modified Meyler solution at 1-3-min intervals and was expressed as
fractional efflux per minute as described (23).
currents were measured 1 or 2 days after viral infection using a
Biologic RK-300 amplifier essentially as described (24). Signals were
filtered at 1 kHz, digitized (Digidata 1200, Axon Instruments Inc.,
Foster City, CA), and analyzed using pClamp 6.0 software (Axon
Instruments). Heat-polished patch pipettes (3-6 M
) filled with an
intracellular solution containing 120 mM
N-methyl-D-glucamine, 85 mM aspartic
acid, 3 mM MgCl2, 1 mM EGTA, 1 mM MgATP, 5 mM
N-Tris-(hydroxymethyl)-methyl-2-aminoethanesulfonic acid
(TES; pH adjusted to 7.3 with HCl; [Cl
], 43 mM) were used. The extracellular solution contained 140 mM NaCl, 1.2 mM MgSO4, 1.2 mM CaCl2, 10 mM glucose, and 10 mM TES (pH 7.3). Cells were clamped at a holding potential
of 0 mV and membrane currents were measured during depolarizing and
hyperpolarizing voltage steps (+50 to
90 mV in 20-mV decrements). All
experiments were performed at 32 °C.
Adenovirus-mediated Transfer of cGK I and cGK II
channels
(14), which contain no detectable endogenous cGK I or cGK II by
immunoblotting (data not shown), as well as extremely little soluble
and no measurable membrane-associated cGMP-stimulated phosphotransferase activity (Fig. 1). Infection of
IEC-CF7 cells with 5 × 109 particles of replication
deficient adenovirus containing the cDNA of rat cGK II (Ad-cGK
II) resulted in the expression of 0.5 ± 0.2 µg of cGK II/mg
protein as assessed by immunoblotting (n = 4; Fig. 1),
which is in the range of the endogenous cGK II content of isolated rat
enterocytes determined by immunoblotting (0.2-0.4 µg/mg protein;
data not shown). Furthermore, only the full-length 86-kDa form of cGK
II was observed in homogenates of Ad-cGK II-infected IEC-CF7 cells
(Fig. 1), which is similar to cGK II in native rat intestinal brush
border membranes (Fig. 1) (7). In contrast, a mixture of cGK II forms
(86 kDa intact and 70 and 75 kDa proteolyzed forms) was present in the
purified preparations of pig cGK II used in previous experiments
demonstrating cGK II-mediated activation of CFTR in excised membrane
patches (5, 12). Infection of IEC-CF7 cells with a similar dose of
adenovirus vector containing the coding region of human cGK I
(Ad-cGK I
) produced a relatively high expression level of cGK I
(2.4 ± 1 µg/mg protein; detected by immunoblotting,
n = 4) corresponding to a large increase in phosphotransferase activity exceeding the activity measured for cGK II
by 5-fold (Fig. 1). In contrast to cGK II, which was associated primarily with the membrane fraction, cGK I
was cytosolic (Fig. 1).
The observed subcellular localization of the recombinant cGKs after
adenovirus-mediated transfer are in agreement with the membrane localization of endogenous cGK II (5, 7) and the soluble character of
endogenous cGK I
(25). The selectivities of the recombinant cGK II
and cGK I
expressed in the IEC-CF7 cells for membrane permeant cGMP
analogs were similar to those previously determined for endogenous or
recombinant forms of cGK II and I
(18, 22, 26,
27),)2 i.e. cGK II was
preferentially activated by 8-pCPT-cGMP over 8-Br-PET-cGMP
(Ka = 0.1 and 1.7 µM, respectively;
data not shown), and cGK I
was more readily activated by
8-Br-PET-cGMP than by 8-pCPT-cGMP (Ka = 0.04 and 0.9 µM, respectively; data not shown). Taken together, these
results show that both the amount and subcellular distribution of cGK
isotype expression produced by adenovirus-mediated gene transfer in
IEC-CF7 cells reflects that of endogenous cGK I and cGK II, allowing a
meaningful comparison of the effects of recombinant cGK isotypes on
CFTR in IEC-CF7 cells.
Fig. 1.
Adenovirus-mediated transfer of
cGMP-dependent protein kinases (cGK) in IEC-CF7 cells.
Rat intestinal IEC-CF7 cells stably transfected with CFTR
Cl channels were infected with 5 × 109
particles/ml (approximately 107 plaque-forming units/ml) of
a replication-deficient adenovirus containing the cDNA of
luciferase (mock; solid bar), cGK II (hatched bar), or cGK I
(open bar). Two days after infection,
cells were harvested, homogenized, and separated into cytosol and
membrane fractions. Phosphotransferase activity was determined with a
cGK-selective substrate (2A3) in the presence or absence of 10 µM cGMP and was expressed per milligram of homogenate
protein after correction for basal activity in the absence of cGMP.
Inset, immunoblots of the homogenate (5 µg protein;
hom), cytosol (cyt), and membrane (mem) fractions of cells infected with adenovirus containing
cGK I
(top) or cGK II (bottom). The blots were
labeled with the respective specific antibodies against cGK I and II.
In the lanes at right, 10 ng of pure cGK I (top)
or rat intestinal brush borders containing 10 ng of cGK II
(bottom) were loaded as standards (st). Shown are
results of a typical experiment which was performed three times.
[View Larger Version of this Image (34K GIF file)]
efflux measurements provide
a simple assay for monitoring the activation of CFTR Cl
channels (14, 23). The cAMP-elevating agent forskolin caused a large
increase in 125I
efflux from IEC-CF7 cells
(Fig. 2) but not from CFTR-deficient wild-type or
mock-transfected IEC-6 cells (14). In contrast, both the
membrane-permeable cGMP analog 8-pCPT-cGMP and the cGMP-elevating hormone atrial natriuretic peptide (ANP) (28) were unable to mimic the
forskolin-provoked increase in 125I
efflux in
either IEC-CF7 cells infected with control adenovirus containing
luciferase cDNA (Fig. 2B, mock), or in
noninfected IEC-CF7 cells (data not shown). These results indicate that
cGMP was unable to provoke activation of CFTR via endogenous
cAMP-dependent protein kinase (cAK) in cGK-deficient
IEC-CF7 cells under the conditions tested. However, after infection of
IEC-CF7 cells with 5 × 109 particles/ml Ad-cGK II,
addition of 8-pCPT-cGMP caused a gradual increase in the
125I
efflux rate, reaching a maximum after 4 min (14 ± 4%/min above basal, n = 6; Fig. 2). A
5-fold lower dose of Ad-cGK II (109 particles/ml) resulted
in an approximately 5-fold lower expression of cGK II, and a 3-4-fold
lower increment in 125I
efflux rate (3.8 ± 1.3%/min; n = 3) in response to 8-pCPT-cGMP (data
not shown). Relatively high doses of Ad-cGK II (>2 × 1010 particles/ml) were more effective than the standard
dose of 5 × 109 particles/ml in facilitating the
8-pCPT-cGMP-provoked increase in 125I
efflux
but were also toxic for the IEC-CF7 cells as judged from their
morphology and an increased rate of basal
125I
efflux (data not shown).
Fig. 2.
cGMP increases iodide efflux in IEC-CF7 cells
expressing cGK II but not cGK I. Rat intestinal IEC-CF7 cells
stably transfected with CFTR Cl
channels were infected
with replication deficient adenovirus (Ad) containing the cDNA of
either luciferase (mock), cGK II, or cGK I
. Two days
after infection, CFTR activity was monitored by measurements of
fractional 125I
efflux. A, time
course of 125I
efflux in Ad-cGK II-infected
IEC-CF7 cells. At 5 min (arrow) 50 µM
8-pCPT-cGMP (
), 0.1 µM ANP (
), 10 µM
forskolin (
), or vehicle (control;
) was added to the
efflux medium. B, maximal increment in
125I
efflux as determined 2 min after
addition of forskolin (fors; open bars) and ANP
(hatched bars) or 4 min after addition of cGMP analogs
(cGMP; solid bars) to IEC-CF7 cells infected as shown. 125I
efflux was corrected for basal efflux at
the same time points determined in the absence of the agonists.
Concentration of the agonists added was as in A, except that
20 µM 8-Br-PET-cGMP was used to activate cGK I
. Data
are means ± S.E. of 3-6 experiments.
[View Larger Version of this Image (21K GIF file)]
efflux in CFTR-deficient IEC-6 cells infected with Ad-cGK II (data not
shown), further strengthening the concept that the CFTR
Cl
channel is the mediator of cGMP/cGK II-enhanced
125I
efflux in IEC-CF7 cells. Accordingly,
whole-cell patch clamp analysis of Ad-cGK II-infected IEC-CF7 cells
stimulated with 8-pCPT-cGMP revealed rapid induction of a linear
Cl
current that was indistinguishable from
forskolin-provoked currents (Fig. 3) (24). This
8-pCPT-cGMP-triggered anion current was observed in 8 of 9 IEC-CF7
cells infected with Ad-cGK II. In contrast, only in 1 of 6 mock-infected cells was a small increase observed, indicating that cGK
II expression was a prerequisite for CFTR Cl
channel
activation.
Fig. 3.
cGMP increases whole-cell chloride current in
IEC-CF7 cells expressing cGK II. Rat intestinal IEC-CF7 cells
stably transfected with CFTR Cl channels were infected
with replication-deficient adenovirus containing the cDNA of either
cGK II or luciferase (mock). One or two days after
infection, whole-cell Cl
currents were measured. Cells
were clamped at a holding potential of 0 mV, and membrane currents were
recorded during depolarizing and hyperpolarizing voltage steps (+50 to
90 mV in 20-mV decrements; see inset above first trace).
A, current traces from cGK II or mock-infected single cells
stimulated with 8-pCPT-cGMP (50 µM) and subsequently
forskolin (10 µM). Control traces represent basal currents prior to stimulation. B, current-to-voltage
relationship of basal (
) membrane current and the currents provoked
by 8-pCPT-cGMP (
) and forskolin (
) in the cGK II-infected cell
shown in A.
[View Larger Version of this Image (16K GIF file)]
channels by purified pig cGK II was relatively slow in comparison to their activation by cAK (13). In Ad-cGK II-infected IEC-CF7 cells
8-pCPT-cGMP caused a similar sluggish activation of CFTR (Fig.
2A). As ANP was shown previously to induce a prompt rise in
intracellular cGMP in IEC cells by activating endogenous guanylyl cyclases (28), the time course of ANP activation of
125I
efflux was compared with the response to
the cGMP analog. As shown in Fig. 2A, the rate of
ANP-stimulated 125I
efflux was similar to
that observed with the cAMP-elevating agonist forskolin, suggesting
that cGK II and cAK are able to activate CFTR in intact cells with
similar kinetics and that the lag phase in activation by 8-pCPT-cGMP is
due to a relatively slow permeation of the analog across the IEC-CF7
cell membrane. These results also imply that the delayed CFTR channel
opening in response to purified cGK II observed in excised patches (13)
is apparently an in vitro artifact and may represent the lag
time needed for re-anchoring of the solubilized enzyme to the
membrane.
efflux in Ad-cGK I
-infected cells
(Fig. 2B). However, these cells showed a normal forskolin
response, excluding any deleterious effect of cGK I
expression on
CFTR activation (Fig. 2B). Furthermore, the ANP-provoked
increase in cGMP did not differ between Ad-cGK II and Ad-cGK
I
-infected IEC-CF7 cells (data not shown). Since infection of
IEC-CF7 cells with equivalent doses of adenovirus caused a 5-fold
higher expression of cGK I
than cGK II, and this dose of Ad-cGK I
did not stimulate 125I
efflux, whereas even a
5-fold lower dose of Ad-cGK II still did, we conclude that cGK II is at
least 25-fold more effective than cGK I
in activating CFTR
Cl
channels. However, since both cGK II and cGK I were
found to phosphorylate immunopurified CFTR and a cloned regulatory
domain fragment of CFTR (CF-2) in vitro with similar
kinetics (13), we next investigated the in situ
phosphorylation of CFTR in cGK II and cGK I
-expressing IEC-CF7
cells.
-infected cells, despite the fact that CFTR phosphorylation in response to forskolin was similar in both cGK I
and cGK
II-expressing cells. Therefore, the increase in CFTR phosphoryl content
paralleled the activation of CFTR-mediated
125I
efflux observed upon cGMP application.
This suggests that selective phosphorylation of CFTR, rather than
subtle differences in the phosphorylation of individual sites within
the CFTR molecule, may be the molecular basis for the present and
previously (13) observed cGK II isotype-selective activation of CFTR.
The cGK I-mediated phosphorylation of immunoprecipitated CFTR (13) may be another example of promiscuous substrate utilization inherent in
in vitro phosphorylation studies. We might speculate that
the different capabilities of the two cGK isotypes for CFTR
phosphorylation in intact cells may be due to: (i) a potential
difference in the structure of the regulatory domain of native CFTR in
comparison to that of immunoprecipitated CFTR or (ii) a kinetic
advantage of cGK II in phosphorylating native CFTR due to their
co-localization in the same subcellular (membrane) compartment (20).
Even a small kinetic advantage in phosphorylation may be important for tipping the balance between phosphorylation and dephosphorylation of
CFTR in a native environment that may contain other regulatory components like phosphatases, which are absent in isolated preparations of CFTR or CF-2. Finally, since the in vivo phosphorylation
data strictly do not distinguish whether CFTR is directly
phosphorylated by cGK II or by another kinase, the remote possibility
that the cGK II isotype-specific activation of CFTR is at the level of a regulator/cofactor endogenously expressed in IEC-CF7 cells cannot as
yet be ruled out.
Fig. 4.
cGMP promotes phosphorylation of CFTR in
IEC-CF7 cells expressing cGK II but not cGK I. Rat intestinal
IEC-CF7 cells stably transfected with CFTR Cl
channels
were infected with replication-deficient adenovirus containing the
cDNA of either cGK II or I
(5 × 109
particles/ml). Two days after infection, cells were metabolically labeled with inorganic 32P for 1 h and subsequently
incubated for 20 min with vehicle (
, none), 10 µM forskolin (forsk), or either 50 µM 8-pCPT-cGMP in the case of cGK II (cGMP) or
20 µM 8-Br-PET-cGMP in the case of cGK I
(cGMP). Subsequently CFTR was immunoprecipitated and
separated by 6% SDS-polyacrylamide gel electrophoresis. A,
autoradiograph showing 32P-phosphorylated CFTR (30-day
exposure). The position of CFTR as determined with in vitro
32P-phosphorylated CFTR (see Refs. 13 and 14) is
indicated with an arrowhead. The 220-kDa protein in
lane 5 is nonspecific, as it does not comigrate with CFTR
and was not observed in other experiments. B, amount of
32P incorporated into CFTR was quantitated by a
phospho-imager and expressed relative to the basal 32P
incorporation into CFTR in Ad-cGK II-infected IEC-CF7 cells (cGK
II, none). Data are means ± S.E. of three experiments.
[View Larger Version of this Image (32K GIF file)]
conductance indicates that
cGK II is a key mediator of cGMP-provoked activation of CFTR in,
e.g., intestinal epithelial cells where both proteins are
co-localized (7). This conclusion is also corroborated by the
previously observed correlation between the presence of cGK II and
detection of cGMP-induced CFTR-mediated Cl
secretion in
different intestinal segments (7). Furthermore, the role of cGK II in
mediating cGMP-provoked activation of CFTR activation cannot be
mimicked by cGK I or cAK in our cell system. In particular, the
ascribed role of cAK in the cGMP-induced activation of CFTR in several
cell lines (29-33) may therefore not be widely valid but restricted to
cells either expressing a type III cGMP-inhibited phosphodiesterase to
elevate cAMP (29) or cells in which very high levels of cGMP can be
attained to cross-activate cAK (30-32). The intestinal cell lines T84
and Caco-2 used to demonstrate the latter mechanism appear nevertheless
to be unsuitable models for studying physiological mechanisms, since
they do not contain detectable levels of cGK II or cGK I (7).
Furthermore, we have shown (7) that the major localization of cGK I in
smooth muscle cells of the villus lamina propria, in contrast to the
epithelial brush border where cGK II is located, makes cGK I an
unlikely endogenous mediator of cGMP effects on CFTR. The particularly
intriguing aspect of our present results is the observation that even
when present cGK I cannot substitute for cGK II in phosphorylating CFTR
in intact cells or in stimulating CFTR-mediated Cl
conductance. This not only reinforces the caveat regarding in vitro phosphorylation studies but, more importantly, may be a clue
to mechanisms of subcellular compartmentalization of functions.
*
This work was supported by the Netherlands Organization for
Scientific Research (NWO) and the Deutsche Forschungsgemeinschaft (SFB
355). 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.
§
To whom correspondence should be addressed: Dept.
Biochemistry, Medical Faculty, Erasmus University Rotterdam, P. O. Box
1738, 3000 DR Rotterdam, The Netherlands. Tel.: 31-10-408-1111; Fax: 31-10-436-0615.
1
The abbreviations used are: CFTR, cystic
fibrosis transmembrane conductance regulator; cGK,
cGMP-dependent protein kinase, cAK,
cAMP-dependent protein kinase; 8-pCPT-cGMP,
8-(4-chlorophenylthio)-cGMP; 8-Br-PET-cGMP,
-phenyl-1,N2-etheno-8-bromo-cGMP; ANP,
atrial natriuretic peptide.
2
A. B. Vaandrager, unpublished
observations.
©1997 by The American Society for Biochemistry and Molecular Biology, Inc.