(Received for publication, April 12, 1995; and in revised form, August 18, 1995)
From the
Type II cGMP-dependent protein kinase (cGKII) isolated from pig
intestinal brush borders and type I cGK (cGKI) purified from
bovine lung were compared for their ability to activate the cystic
fibrosis transmembrane conductance regulator (CFTR)-Cl
channel in excised, inside-out membrane patches from NIH-3T3
fibroblasts and from a rat intestinal cell line (IEC-CF7) stably
expressing recombinant CFTR. In both cell models, in the presence of
cGMP and ATP, cGKII was found to mimic the effect of the catalytic
subunit of cAMP-dependent protein kinase (cAK) on opening
CFTR-Cl
channels, albeit with different kinetics
(2-3-min lag time, reduced rate of activation). By contrast, cGKI
or a monomeric cGKI catalytic fragment was incapable of opening
CFTR-Cl
channels and also failed to potentiate cGKII
activation of the channels. The cAK activation but not the cGKII
activation was blocked by a cAK inhibitor peptide. The slow activation
by cGKII could not be ascribed to counteracting protein phosphatases,
since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A,
nor ATP
S (adenosine 5`-O-(thiotriphosphate)), producing
stable thiophosphorylation, was able to enhance the activation
kinetics. Channels preactivated by cGKII closed instantaneously upon
removal of ATP and kinase but reopened in the presence of ATP alone.
Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain
fragment of CFTR (amino acids 645-835) could be phosphorylated to
a similar extent with only minor kinetic differences by both isotypes
of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very
subtle differences in site-specificity between the cGK isoforms. These
results indicate that cGKII, in contrast to cGKI
, is a potential
activator of chloride transport in CFTR-expressing cell types.
Guanosine 3`,5`-cyclic monophosphate (cGMP) has been identified
as an important intracellular mediator of salt and water secretion in
intestinal epithelium(1, 2, 3) .
Secretagogues acting through the cGMP-signaling pathway include the
family of heat-stable enterotoxins (STs), low molecular
weight peptides secreted by enteropathogenic bacteria, and guanylin, a
recently discovered endogenous ST-like peptide
hormone(3, 4) . Binding of ST or guanylin to the
receptor domain of an intestine-specific isoform of guanylyl cyclase
(GC-C) triggers cyclase activation, cGMP accumulation, and stimulation
of net fluid secretion through the activation of apical Cl
channels in parallel with inhibition of coupled NaCl
transporters(3, 5, 6) . The cystic fibrosis
transmembrane conductance regulator (CFTR), an epithelial
Cl
channel mutated in CF
patients(7, 8) , appears to be involved in the
Cl
secretory response to ST and cGMP analogues, as
evidenced by the absence of this response in CF
intestine(9, 10) .
Several mechanisms have been
proposed to link cGMP to the CFTR-Cl channels,
including (i) cGMP cross-activation of cAMP-dependent protein kinase (11, 12, 13) followed by
multisite-phosphorylation of CFTR(14) , (ii) direct interaction
of cGMP with the CFTR protein(15) , and (iii) cGMP activation
of an intestine-specific isoform of cGMP-dependent protein kinase (type
II cGK; (16, 17, 18, 19) ). cGKII
was discovered as a cGMP-sensitive 86-kDa phosphoprotein localized in
intestinal brush border membranes (16) , which comigrated with
a cGMP receptor protein on one- and two-dimensional
gels(17, 18) . The intestinal isoform is clearly
distinct from the homodimeric type I
and I
cGK (153-156
kDa) identified in other mammalian tissues, as illustrated by
differences in subcellular localization, subunit composition,
isoelectric point, phosphopeptide maps, immunoreactivity, and affinity
for cyclic nucleotide analogues(17, 18, 19) .
Recently, molecular cloning of cGKII from mouse brain (20) and
rat intestine (21) demonstrated that cGKII is a different gene
product than cGKI
and I
(22, 23) .
In the
present study, evidence for a functional difference between cGK
isoenzymes was obtained from studies of the activation of
CFTR-Cl channels in excised membrane patches of an
intestinal cell line (IEC-CF7; (24) ) or NIH-3T3 fibroblasts
stably expressing recombinant CFTR(25) . In both models,
exposure of patches to a combination of cGMP and ATP failed to elicit
Cl
channel activity. However, the further addition of
purified cGKII, but not cGKI
, resulted in almost full activation
of CFTR-Cl
currents.
Differential activation of
the CFTR-Cl channel by cGKII is the first example of
isotype specificity in cGK regulation of cellular functions and
provides a plausible explanation for the prominent role of cGMP as a
regulator of Cl
transport in intestinal epithelium in
comparison to other CFTR-expressing cell types in which cGKII
expression is marginal or
absent(19, 20, 21) .
The
catalytic subunit of type II cAK and cGKI (characterized by antibody
analysis to be primarily the I isoform; (29) ) were
purified from bovine heart and bovine lung, respectively, as described (30, 31) . The specific activities (units/mg protein)
of the purified protein kinases as determined by the Kemptide
phosphorylation assay (32) were 4.2 (cAK), 2.0 (cGKI), and 1.6
(cGKII), respectively. A monomeric constitutively active cGKI fragment
was obtained by limited trypsinization as described(33) .
Figure 1:
cGKII but not cGKI
activates CFTR-Cl channel current in excised,
inside-out membrane patches from 3T3-CFTR fibroblasts. ATP (2
mM), cGMP (50 µM), cGKII (10 milliunits/ml), and
catalytic subunit of cAK (2 milliunits/ml) were present in the
cytosolic (bath) solution during the times indicated by the bars. Panel A, comparison of the effects of cGKI,
cGKII, and cAK. The amount of current activated by cGKII was 78
± 24% (n = 5) of the current measured after
subsequent addition of cAK. Panel B, a representative example
of an experiment showing that the cGK-activated currents rapidly
returned to near base-line values upon removal of the kinase and ATP
from the bath (also shown for the cAK-activated currents in panel
A), but could be restored almost instantaneously by the readdition
of ATP alone. Panel C, time course of activation of
CFTR-Cl
current by cGKII alone (
; 10
milliunits/ml) or a combination of cGKI (10 milliunits/ml) and cGKII
(
); n = 6. Current levels are expressed as a
percentage of the maximal CFTR-Cl
current observed
upon subsequent addition of 2 milliunits/ml
cAK.
The cGKII isoform,
but not cGKI, was capable of activating CFTR-Cl channels also in excised patches from IEC-CF7 cells (Fig. 2). In this low expression model, single-channel events
could be monitored (Fig. 2A), which had a linear I-V
relation in symmetrical Cl
concentrations, a channel
conductance of 8 pS, and a rightward shift in current reversal
potential upon lowering of the Cl
concentration in
the pipette (Fig. 2B). The mean open probability (P
) of CFTR-Cl
channels measured
at the plateau phase of activation by saturating concentrations of
cGKII (0.22 ± 0.09; n = 9) did not differ
significantly from the P
of cAK-activated channels
(0.23 ± 0.12; n = 8).
Figure 2:
Biophysical characteristics of the
cGKII-activated channel in excised, inside-out membrane patches from
IEC-CF7 cells. Panel A, current tracings of cGKII-activated
channels. Left, symmetrical 147/147 mM chloride
solutions. Right, pipette buffer was replaced by a low (49
mM) chloride buffer. Tracings were obtained at the indicated
voltages. C, all channels closed; dotted line,
single-channel current levels. Panel B, I-V characteristics of
the channel. The channel conductance was 8.0 ± 0.6 pS (n = 5). , symmetrical Cl
solution
(147/147 mM);
, reduction of Cl
in
the pipette to 49 mM by replacement with aspartic
acid.
Additional proof of the
identity of the cGKII-activated channel as CFTR came from the
observation that cGKII could not further enhance channel activity in
excised patches from 3T3-CFTR cells following their pre-phosphorylation
by cAK (2 milliunits/ml) and ATP (2 mM) (results not shown).
Another similarity between CFTR-Cl channel regulation
by cAK and cGKII was the observation that the currents rapidly returned
to near base-line values upon the removal of either kinase and ATP from
the bath, but could be restored almost instantaneously by the
readdition of 2 mM ATP alone, confirming the crucial role of
ATP in CFTR-Cl
channel functioning (Fig. 1B; cf. Refs. 25, 38, and 40).
To
eliminate the possibility that cGKII activation of CFTR resulted from a
contamination of the cGKII preparation with cAK, a specific peptide
inhibitor of cAK, PKI (0.1 µM) was added to the bath.
Under this condition CFTR-Cl channel activation in
3T3 membrane patches by cAK (2 milliunits/ml) was completely abolished,
whereas channel activation by cGKII (2 milliunits/ml) was not
significantly affected (42 ± 3% of the maximal channel activity
evoked by cAK in the same patch following removal of PKI from the bath,
as compared to 37 ± 5% in the absence of PKI; n = 5). Moreover, PKI was unable to inhibit phosphorylation
of Kemptide or CF-2 by cGKII (not shown).
Finally, the possibility
was considered that cGKI, in spite of its failure to open
CFTR-Cl channels by itself, could interfere with the
activation of CFTR-Cl
channels by the other cGK
isoform. However, neither preincubation of 3T3 patches with cGKI (10
milliunits/ml, 15 min; results not shown) nor the simultaneous addition
of cGKI (10 milliunits/ml) and cGKII (10 milliunits/ml) had any effect
on the rate or extent of CFTR-Cl
channel activation
as compared to cGKII alone (Fig. 1C).
Figure 3:
Phosphorylation of CFTR (panel A)
and CF-2 (panel B) by purified protein kinases. Panel
A, CFTR was immunoprecipitated from T84 cells and phosphorylated
as described under ``Experimental Procedures.'' The reactions
also contained: catalytic subunit of cAK (2 milliunits/ml; lanes 1 and 2), cGKI (7.5 milliunits/ml; lanes 3 and 4), and cGKII (9.4 milliunits/ml; lanes 5 and 6). Lanes 2, 4, and 6, control
experiments in which CFTR was omitted. The P-labeled
proteins were separated by 6% SDS-PAGE. The gel was dried and exposed
to x-ray film. CFTR migrates as a broad band of 180 kDa
(``band C''; see (14) ). The 86- and 74-kDa
bands represent residual amounts of autophosphorylated cGKII (lanes
5 and 6, intact 86-kDa form + 74-kDa proteolytic
fragment; cf. (17) ) and cGKI (lanes 3 and 4, intact 74-kDa form), respectively, remaining following the
washing steps. Panel B, Lineweaver-Burk plots of CF-2
phosphorylation by equal concentrations (25 nM) of cAK, cGKI,
and cGKII. The experimental conditions needed to ensure linear rates of
P incorporation are specified under ``Experimental
Procedures.'' The inset shows the kinetic constants (K
, V
) calculated
from the Lineweaver-Burk plots. A.U., arbitrary units. Data
represent the mean of three experiments.
Figure 4:
Two-dimensional tryptic phosphopeptide
maps of CF-2 and CFTR. Upper and lower panels,
phosphopeptide maps of CF-2 and CFTR, respectively, showing
phosphorylation by cAK (A and D), cGKI (B and E), and cGKII (C and F),
respectively. Purified CF-2 and immunoprecipitated CFTR were
phosphorylated for 40 min at 30 °C in the presence of cAK (2
milliunits/ml), cGKI (7.5 milliunits/ml), or cGKII (9.4 milliunits/ml)
as described under ``Experimental Procedures.'' The P-labeled proteins were separated by 6% (CFTR) or 12%
(CF-2) SDS-PAGE and visualized by autoradiography. Radioactive bands of
CF-2 (pooled middle and upper bands running at 30 and 32 kDa,
respectively; cf. (26) ) and CFTR (see Fig. 3A) were excised from gels, washed, and digested
for 20 h with 50 µg/ml L-1-tosylamido-2-phenylethyl
chloromethyl ketone-treated trypsin. Tryptic digests were separated by
electrophoresis in the first dimension at 400 V in 10% acetic acid, 1%
pyridine, pH 3.5, and by chromatography in the second dimension using
pyridine:1-butanol:water:acetic acid (10:15:12:3%). O, origin. Left, +; right, -. Phosphopeptides are
numbered from 1 to 9, and correspond to those in (26) . The
results shown are representative of three independent experiments. In
two other sets of CF-2 maps, an additional phosphopeptide migrating
closely to peptide 9 could be distinguished, indicating that the
peptide 9 spot may contain more than one peptide or more than one
phosphoacceptor site in one peptide, occasionally giving rise to
alternative tryptic digestion.
In this study a recently cloned cGK isoform,
cGKII(20, 21) , expressed at high levels in the
luminal membrane of intestinal epithelial cells(16, 17, 18, 19, 21, 41) and to
a lower extent in kidney and brain(21) , is identified as a
novel potential regulator of the CFTR-Cl channel.
Using a reconstitution assay consisting of a detergent-free preparation
of solubilized and purified cGKII, inside-out membrane patches from
CFTR-transfected intestinal cells (IEC-CF7), or NIH-3T3 fibroblasts,
Mg-ATP and cGMP, the enzyme could almost fully mimic the effect of cAK
on CFTR-Cl
channel opening, albeit with a slower time
course. It is possible, however, that the rate-limiting step in the in vitro assay is the anchoring of the cGKII to the membrane,
and that this delayed opening does not necessarily imply a similar
kinetic disadvantage for cGKII in vivo, considering its
colocalization with CFTR in the luminal
membrane(19, 41) . The amount of Cl
channel current reached (78 ± 24% of that measured after
the subsequent addition of cAK) was considerably greater than the
current level reported after addition of another activating kinase,
PKC, using similar assay conditions (15 ± 8% of cAK; (38) ). In accordance with these functional data, the
phosphopeptide maps made from CF-2 and CFTR phosphorylated in vitro by cGKII and cAK were virtually identical and clearly different
from the pattern generated by PKC(26) . The plateau level of
phosphate incorporation into CFTR reached with cAK and cGKII was also
similar (Fig. 3A), although the rate of CF-2
phosphorylation by cGKII was slower (Fig. 3B). However,
such a close correlation between in vitro phosphorylation and
functional data should be interpreted with caution: cGKI
, another
mammalian cGK isoform expressed in many non-intestinal cell types (30, 31) failed to activate CFTR-Cl
channels in this and an earlier study (38) or to
potentiate cGKII activation of the channel, in spite of its ability to
phosphorylate in vitro three of the four major cAK/cGKII sites
in CFTR (Fig. 4).
The molecular basis for the differential
activation of the CFTR-Cl channel by cGK isotypes is
presently unclear. One possibility is that cGKI
may not recognize
one or more phosphoacceptor sites in CFTR that are crucial for its
activation. However, the results from the two-dimensional peptide
mapping studies indicate that the pattern of phosphorylation of CF-2 is
similar for cGKI and cGKII and essentially the same as that of cAK.
Furthermore, the pattern of phosphorylation of CFTR by the three
kinases was also similar with the exception that one peptide (peptide
9) was phosphorylated to only a very low level by cGKI (Fig. 4).
Although initial mutagenesis studies suggested that the multisite
phosphorylation of the R domain is degenerate and that no single
phosphorylation site is critical for CFTR-Cl
channel
function(14) , more recent studies provide evidence for a
distinct role of subsets of phosphorylation sites in controlling the
function of the two nucleotide binding domains in CFTR(39) ,
and for the existence of stimulatory and inhibitory phosphorylation
sites in the R domain(42) . However, the possible presence of a
predominant inhibitory site phosphorylated by cGKI
but not by
cGKII or cAK seems unlikely since CFTR-Cl
channel
activation by cGKII or cAK was not inhibited by cGKI (Fig. 1C). A second possibility is that the CFTR
protein in its natural membrane environment is differentially
accessible to the cGK isoenzymes and that this difference is lost
following immunoprecipitation or in CF-2 peptide studies. Conceivably,
differences in size or quaternary structure between the monomeric cGKII
protein (17, 18, 19) and the cGKI dimer do
not play a major role because limited trypsinization of cGKI
generating a monomeric C-terminal fragment failed to improve
CFTR-Cl
channel activation. On the basis of secondary
structure analysis, it has been argued that the structural determinants
accounting for the tight association of the type II cGK with membranes
may reside in the N-terminal region(21) . Earlier topological
studies in intestinal brush borders have also pointed to a role of a
15-kDa N-terminal fragment in anchoring cGKII to the microvillar
cytoskeleton(17, 18) . In particular the presence of a
consensus sequence for N-terminal myristoylation in cGKII (21) may have functional significance. This sequence is absent
in cGKI
(43) , which may explain in part its inability to
activate CFTR. As a third possibility, cGK activation of the
CFTR-Cl
channel may depend on the additional
phosphorylation of a CFTR-associated regulatory protein ubiquitously
expressed in cells of epithelial origin (IEC-CF7) and non-epithelial
cells (3T3 fibroblasts). Small phosphorylatable proteins have been
recently implicated in the regulation of a different class of
Cl
channels (44, 45) . However, the
opening of CFTR-Cl
channels in lipid bilayers by cAK
does not require an auxiliary protein (46) . Similar studies
performed with the cGK isotypes are clearly needed to discriminate
between a direct and indirect model of CFTR regulation.
The
demonstration of cGKII regulation of CFTR-Cl channels
in membrane patches together with the high expression level of cGKII
and virtual absence of cGKI in intestinal epithelium (17, 18, 19, 41) strongly support a
model depicting cGKII as the major effector of the action of cGMP and
cGMP-linked secretagogues (ST, guanylin) in this tissue. Recent
immunological and ion transport experiments (41) also show a
tight correlation between cGKII expression and 8-Br-cGMP-provoked, but
not 8-Br-cAMP-provoked Cl
secretion in rat intestinal
segments and human colonic cell lines (T84, CaCo-2). With one possible
exception (47) , the latter cell lines do not express
detectable levels of cGKII (41) but are still able to activate
CFTR in response to ST, apparently as a consequence of excessive cGMP
accumulation followed by cross-activation of
cAK(11, 12, 13) . However, in a previous
study no low affinity binding of cGMP to cAMP receptors in the brush
border membrane could be detected following luminal exposure of rat
small intestine to ST in vivo(48) . Moreover, in
contrast to 8-Br-cAMP, ST did not further enhance Cl
secretion in rat small intestinal mucosa and proximal colon in vitro beyond the level reached in the presence of exogenous
cGMP analogues(41) . The apparent need for coexpression of
cGKII and CFTR may also explain why cGMP activation of Cl
secretion is not universally observed in other CFTR-expressing
cell types, including human airway epithelium(38) .