(Received for publication, August 17, 1994; and in revised form, October 18, 1994)
From the
Hormonal regulation of the cystic fibrosis transmembrane
conductance regulator (CFTR) Cl channel is largely
mediated via cAMP-dependent protein kinase (PKA). CFTR contains 10
dibasic consensus sites for potential PKA phosphorylation
((R/K)(R/K)X(S*/T*)). Previous studies (Chang, X.-B.,
Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon, N.,
Hanrahan, J.W., and Riordan, J.R(1993) J. Biol. Chem. 268,
11304-11311) showed that approximately 25% of the CFTR wild-type
response to PKA activation remained upon inhibition of most detectable
phosphorylation by in vitro mutagenesis of all 10 dibasic
consensus sites (10SA CFTR). To identify potential additional sites
responsible for the residual activity, large amounts of this mutant
CFTR were phosphorylated with PKA using high specific activity
[
-
P]ATP. Cyanogen bromide cleavage
indicated that a large portion of the observed PKA phosphorylation
occurred within a 5.8-kDa fragment of the R domain between residues
722-773. Removal of serines at potential PKA sites in this
fragment showed that Ser-753 accounted for all of the
-
P labeling of the 5.8-kDa peptide. Replacement of
Ser-753 with alanine reduced the level of residual CFTR activity by a
further 40%, indicating that phosphorylation at this previously
unidentified site contributes to the activation of 10SA CFTR.
It is presently thought that the channel activity of the cystic
fibrosis transmembrane conductance regulator (CFTR) ()(1) is regulated by the concerted action (2, 3, 4, 5, 6) of ATP at
the nucleotide binding folds(7, 8, 9, 10, 11, 12, 13) and
phosphorylation and dephosphorylation of the central R
domain(14, 15, 16, 17, 18, 19, 20, 21) .
Although PKA is not the only kinase influencing CFTR, phosphorylation
by PKA is a potent stimulus for activation of the channel (15, 22) and is likely to mediate much of the
hormonally regulated Cl
secretion in CFTR-expressing
epithelia(5) . In CFTR there are 10 dibasic consensus sites for
recognition by PKA ((R/K)(R/K)X(S*/T*), asterisk indicates the
site of potential phosphorylation), nine of which are clustered within
the R domain. Four consensus sites were found to be predominantly
phosphorylated in vivo(14) . Chang et
al.(19) , however, demonstrated that a mutant that had all
10 dibasic sites eliminated (10SA CFTR) could still be activated by PKA
approximately 25% as effectively as the wild-type molecule, although
phosphorylation was no longer readily detectable. The aim of the
present work was to determine whether the residual sensitivity of the
10SA channel to PKA is mediated by phosphorylation of additional sites
within CFTR or might involve other mechanisms.
The strategy followed
was to first identify site(s) still phosphorylated by PKA in 10SA and
to then examine their contribution to the residual sensitivity to
activation. Upon expressing the 10SA variant at maximal levels in CHO
and BHK cells, in vitro phosphorylation could be detected
using PKA catalytic subunit and high specific activity
[-
P]ATP. Much of the phosphorylation was
localized to a 5.8-kDa phosphopeptide fragment corresponding to an R
domain segment between residues 722 and 773. Of the potential sites
within this fragment, Ser-753 was found to be the sole site of PKA
phosphorylation and to account for a substantial portion of the
residual 10SA CFTR activation. We were thus able to show that
PKAcatalyzed phosphorylation of a specific site at a level near the
limit of detection, is capable of significantly contributing to channel
activation.
Figure 1:
Detection of phosphorylation of 10SA
CFTR by PKA. CHO K1 cells were stably transfected with wild-type or
10SA CFTR. A, expression of 10SA CFTR protein was induced with
2 mM sodium butyrate and detected by Western
blotting(25) . Amounts (µg) of total cell protein applied
are indicated above each lane. Migration of M (
10
) markers is shown
on the right. This comparison indicates that sodium butyrate
induction elevates 10SA CFTR expression approximately 30-fold. B, CHO K1 untransfected control cells and 10SA CFTR CHO cells
were induced with 2 mM sodium butyrate; wild-type (WT) CFTR CHO cells were not induced. Immunoprecipitated CFTR
was incubated in phosphorylation buffer including 180 nM catalytic subunit of PKA and 10 µCi/100 µl of
[
-
P]ATP and visualized by autoradiography
following SDS-PAGE.
Figure 2:
Localization of residual 10SA CFTR
phosphorylation. Panel A, protein expression was induced with
2 mM sodium butyrate in untransfected K1 control cells and in
10SA CFTR cells (CHO). Immunoprecipitated CFTR protein was
phosphorylated in vitro with PKA, cleaved with CNBr, and
detected by autoradiography following SDS-PAGE (wild-type, WT). Panel B, upon in vitro phosphorylation
with PKA and CNBr cleavage of wild-type whole CFTR (C) and
isolated R domain (R; 24), bands 2 and 3 co-migrate, suggesting that these bands are derived from the R
domain. Band 1 has a lower mobility in the CFTR sample, which
may be attributed to the fact that in CFTR the final CNBr cleavage site
involving an R domain segment occurs at residue 837, which lies
C-terminal to the C terminus of the isolated R domain (residues
595-831), therefore generating a slightly larger fragment. The
additional 13 kDa band seen in the R domain preparation may correspond
to the 13-kDa natural cleavage product previously observed by Dulhanty
and Riordan (24) in the presence of SDS. PanelC, predicted CNBr cleavage map of the CFTR R domain. CNBr
cleaves C-terminal to methionine residues. Numbersabove the schematic indicate the position of each methionine, and numbersbelow the schematic show the predicted M of the resulting segments. The 5.8-kDa segment
(residues 722-773) is enlarged to show serines, which were
already replaced by alanines in 10SA CFTR (opencircles) and remaining serines (closedcircles). PanelD, immunoprecipitated
wild-type CFTR was labeled in vitro with PKA and cleaved with
CNBr. A control sample was immediately subjected to SDS-PAGE, whereas
the other two samples were reprecipitated with either P-Ab 6 (epitope:
724-746) or L11E8 (epitope: 341-702; 23) before separation
by SDS-PAGE and visualization by
autoradiography.
Figure 3: Identification of the site phosphorylated in the 5.8-kDa segment. Four different 11SA-CFTR mutants were created, each based on the 10SA CFTR construct (19) and each changing a different serine residue of the 5.8-kDa segment to an alanine (11SA-S728A, 11SA-S742A, 11SA-S753A, and 11SA-S756A). CFTR constructs were stably transfected into CHO K1 cells or BHK cells, and protein expression was induced by 2 mM sodium butyrate. Immunoprecipitated CFTR protein was labeled in vitro with PKA, cleaved with CNBr, and visualized by autoradiography following SDS-PAGE.
Figure 4:
Functional evaluation of 11SA-S753A CFTR. A, iodide efflux from BHK cells expressing mutant CFTRs.
+ indicates that from time 0 onward cells were stimulated with 10
µM forskolin dissolved in MeSO. The same
amount of Me
SO without forskolin was used in the control
samples(-). The profiles of control samples were similar, so that
only the efflux curve for 11SA-S753A CFTR(-) is presented in this
figure. The inset shows a Western blot (25) of various
CFTR mutants in BHK cells. Total protein applied per lane, 50 µg. B, activation of wild-type (WT), 10SA, and 11SA-S753A (11SA) CFTR channels in excised inside-out membrane patches
from BHK cells by the addition of 180 nM PKA and 1 mM ATP. V
= -30 mV. C, open probabilities (P
) of 10SA and
11SA-S753A CFTR channels upon exposure to 180 nM PKA and 1
mM ATP. 10SA CFTR channels expressed in BHK cells showed a P
of 0.346 ± 0.023, whereas 11SA-S753A
channels had a P
of 0.203 ± 0.012 (mean
± S.E., n = 5). The P
for
wild-type CFTR in BHK cells could not be accurately estimated because
gigaohm seals were lost during the vigorous response to PKA before
channel number could be determined
Phosphorylation and dephosphorylation are a common means of
modulating the function of ion
channels(14, 15, 16, 17, 18, 19, 20, 21, 27, 28, 29) .
However, in the cases of ligand and voltage-gated channels, this
modulation is secondary to the primary gating events, instead
influencing other channel properties such as inactivation
kinetics(30, 31, 32) . CFTR is thus far
unique among ion channels in that phosphorylation by PKA seems to exert
the primary control of activation of Cl conductance(15) . Previous studies showed that
mutagenesis of all 10 dibasic consensus sites for PKA interactions
(10SA CFTR) drastically diminished phosphorylation of CFTR by PKA and
decreased PKA-mediated activation to 25% of the wild-type
level(19) . Since phosphorylated sites in PKA-labeled proteins
do not always adhere to the dibasic
consensus(33, 34) , it seemed likely that additional
sites might also be present in CFTR, possibly accounting for the
residual sensitivity of 10SA CFTR. The experiments described in this
report demonstrate that this indeed is the case.
Using large amounts
of protein, we were able to show that 10SA CFTR is still phosphorylated
by PKA (Fig. 1). We traced a considerable amount of this
phosphorylation to residue Ser-753 within a 5.8-kDa segment of the R
domain (Fig. 3) and demonstrated a substantial decrease in the
PKA activation of 10SA CFTR upon removal of Ser-753 (Fig. 4). It
is striking that phosphorylation of a site at a level near the limit of
detection can contribute significantly to function. This indicates that
there need not be a correlation between the extent of phosphorylation
at a site and the magnitude of the functional consequence. Among the
four remaining serines in the 5.8-kDa segment of 10SA CFTR, Ser-753 was
the most likely candidate for PKA-catalyzed phosphorylation since it is
the only serine having a basic residue in close proximity
(PRIS). Rich et al.(20) , as a result
of experiments conducted on an R domain deletion mutant, had already
speculated that Ser-753 may be a PKA substrate. Our results support
this speculation, although it has to be noted that Ser-753 was found to
contribute to function in a 10SA CFTR mutant, devoid of all other major
phosphorylation sites. This does not prove that Ser-753 phosphorylation
contributes to activation of the wild-type protein. It has, however,
been difficult to evaluate the functional importance of individual
phosphate acceptors in vivo since CFTR phosphorylation sites
appear to act in a redundant manner so that removal of a single site
has little or no effect on function. This redundancy may explain the
intriguing observation that to date none of the mutations reported to
cause cystic fibrosis have mapped to a CFTR phosphorylation site
including Ser-753. (
)
Cyanogen bromide cleavage of CFTR demonstrated that the 5.8-kDa segment of the R domain is the most strongly PKA phosphorylated portion of wild-type CFTR (Fig. 2A, band3). This segment contains 2 serines (Ser-737 and Ser-768), which were removed in the 10SA CFTR mutant and must therefore account for the large difference in the amount of phosphorylation between the 5.8-kDa segments of wild-type and 10SA CFTR. This is in agreement with the results of Picciotto et al.(17) , who demonstrated that Ser-737 is phosphorylated in vitro by PKA. Furthermore, these authors showed that either Ser-768 or Ser-795 was the most strongly phosphorylated site in the R domain but could not distinguish between the 2 residues. Our studies suggest that Ser-768 is this strongly phosphorylated residue since it lies within the heavily labeled 5.8-kDa segment of the R domain, whereas Ser-795 does not. This is consistent with the report that, relative to other PKA consensus sites, the phosphorylation kinetics are the fastest for a synthetic peptide containing the PKA consensus site around Ser-768(17) . We also note that Ser-737 is one of the four serines initially reported to account for a large fraction of the overall PKA phosphorylation in vivo(14) .
Since the structural consequences of
phosphorylationinduced changes in the R domain for the entire molecule
are not understood thus far, there is as yet little insight as to why a
multiple-site mechanism is employed rather than a single-site switch.
We have speculated that a multiple-site mechanism may provide for a
graded response to hormonal stimulation of Cl secretion(3, 19) . Indications that greater
activation can be achieved by higher levels of phosphorylation were
reported by Drumm et al.(16) , who showed that in Xenopus oocytes elevations of intracellular cAMP resulted in
an increase in the overall Cl
conductance of
wild-type and mutant CFTR variants, presumably due to the stimulation
of PKA-mediated hyperphosphorylation. An observed increase in total
cell Cl
conductance should correspond to an increase
in the average number of channels open at any given instant, since none
of the phosphorylation site mutants studied thus far have altered
single channel conductances(19, 20) , suggesting that
once a channel is opened, its conductance is not influenced by the
degree of phosphorylation. Sites such as Ser-753 at which it is
difficult to detect labeling may generally undergo less phosphorylation
either because of a reduced rate of phosphorylation or a more rapid
rate of dephosphorylation. Conditions of elevated cellular cAMP
levels(16) , which increase the potential of phosphorylation
events to occur, could overcome unfavorable phosphorylation kinetics at
a given site. The utilization of sites such as Ser-753 to contribute to
channel activation might in this way increase the potential for a
metered response of total cell Cl
conductance.
This study illustrates the substantial functional contribution of phosphorylation at Ser-753, which represents a relatively small proportion of total CFTR labeling relative to some of the previously described phosphorylation sites. By removing Ser-753, we have made an additional step toward producing a CFTR mutant that cannot be phosphorylated or activated by PKA. The small functional response remaining in 11SA-S753A channels may be attributable to the residual phosphorylation observed in band 2 of the cyanogen bromide cleavage (Fig. 3). Several observations suggest that the predicted 7.3- and 8.7-kDa segments of the R domain co-migrate within band 2. Hence, any one or several of the 9 serines and 11 threonines within the two fragments could potentially account for the remaining PKA phosphorylation. Similar strategies to those presented here are being used to identify which of these sites are responsible. It seems reasonable to anticipate that the results may implicate one or more sites with basic amino acids in close proximity such as Ser-670, Thr-690, Thr-787, or Ser-790. However, this still remains to be determined.