©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
cAMP-dependent Protein Kinase-mediated Phosphorylation of Cystic Fibrosis Transmembrane Conductance Regulator Residue Ser-753 and Its Role in Channel Activation (*)

(Received for publication, August 17, 1994; and in revised form, October 18, 1994)

Fabian S. Seibert (1)(§) Joseph A. Tabcharani (2) Xiu-Bao Chang (3) Ann M. Dulhanty (1) Ceri Mathews (2) John W. Hanrahan (2) John R. Riordan (3)(¶)

From the  (1)Research Institute, The Hospital for Sick Children, and University of Toronto, Toronto, Ontario, M5G 1X8 Canada, the (2)Department of Physiology, McGill University, Montreal, Quebec, H3G 1Y6 Canada, and the (3)S. C. Johnson Medical Research Center, Mayo Clinic Scottsdale, Scottsdale, Arizona 85259

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

It is presently thought that the channel activity of the cystic fibrosis transmembrane conductance regulator (CFTR) (^1)(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.


MATERIALS AND METHODS

Phosphorylation of CFTR

Cells were grown at 37 °C in 5% CO(2) for 20 h in alpha-modified minimal essential medium (Life Technologies, Inc.) containing 8% dialyzed fetal bovine serum and (if indicated) 2 mM sodium butyrate. After cell lysis with RIPA buffer (1% Triton X-100, 1% deoxycholic acid, 0.1% SDS, 150 mM NaCl, 20 mM Tris-HCl, pH 8, 0.25 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotinin), CFTR was immunoprecipitated using 2 µg/ml monoclonal antibody (M3A7, generated against a fusion protein containing CFTR residues 1197-1480; (23) ) and protein G-Sepharose 4B beads (Sigma). CFTR was phosphorylated by incubating the beads in phosphorylation buffer (180 nM catalytic subunit of PKA (Promega), 20 µM ATP, 10 µCi of [-P]ATP (Amersham Corp.), 10 µg of bovine serum albumin, 140 mM NaCl, 4 mM KCl, 2 mM MgCl(2), 0.5 mM CaCl(2), 10 mM Tris, pH 7.4) for 15 min at room temperature. The labeled protein was solubilized in 2 times sample buffer (3% SDS, 5% 2-mercaptoethanol, 10% glycerol, 62.5 mM Tris-HCl, pH 6.8) and subjected to SDS-polyacrylamide gel electrophoresis (PAGE). Purified R domain was prepared as described (24) and labeled in solution under the same conditions as whole CFTR protein.

CNBr Cleavage of Phosphorylated CFTR

Following protein separation by SDS-PAGE, the labeled protein was transferred onto a polyvinylidene difluoride membrane using the buffer system described by Towbin et al.(25) . Membrane segments containing CFTR were incubated in the dark for 48 h in 15 mg/ml CNBr (Sigma) in 70% formic acid at room temperature. Cleaved protein was eluted from the membrane with two 100-min incubations in 2 µl/ml trifluoroacetic acid in 70% isopropyl alcohol. The cleavage and elution solutions were pooled, evaporated to dryness, and resolubilized in either 2 times sample buffer for SDS-PAGE or in RIPA buffer, pH 8, for a second immunoprecipitation.

Antibody Recognition of CNBr Cleavage Products

Radiolabeled and cleaved proteins were solubilized in RIPA buffer, pH 8, and subjected to a second immunoprecipitation (as above), the difference being that M3A7 was replaced with either P-Ab 6 (polyclonal antibody generated against a synthetic peptide corresponding to CFTR amino acids 724-746) or L11E8 (monoclonal antibody generated against a CFTR fusion protein containing CFTR residues 341-702; (23) ). The precipitated proteins were solubilized in 2 times sample buffer and subjected to SDS-PAGE.

In Vitro Mutagenesis of CFTR cDNA

To obtain various 11SA CFTR mutants, a fragment between the HindIII site at nucleotide 2189 and the HpaI site at nucleotide 2463 was replaced in the previously described 10SA pNUT-CFTR vector (19) by four different polymerase chain reaction fragments coding for four different serine to alanine changes(26) . The 11SA constructs created this way were 11SA-S728A, 11SA-S742A, 11SA-S753A, and 11SA-S756A. Sequences of the polymerase chain reaction fragments generated were verified after insertion into the vector using the CircumVent thermal cycle DNA sequencing kit (New England Biolabs). The CFTR pNUT expression vectors contained a mutant dihydrofolate reductase gene under the control of the SV40 early promoter. This facilitated stable transfection of the vectors into CHO-K1 cells and BHK cells using a methotrexate selection technique described previously(19) .

Protein Detection

Upon cell lysis with 2 times sample buffer, the lysate was cycled through a 30-gauge needle. Proteins were separated by SDS-PAGE and subjected to Western blotting(25) . Blots were probed with M3A7 as the primary monoclonal antibody. The secondary antibody was a goat anti-mouse antibody labeled with horseradish peroxidase, detected with the ECL kit (Amersham Corp.).

Iodide Efflux Studies

The same method was used as described previously(19) . Briefly, cells were incubated for 1 h at room temperature in loading buffer (136 mM NaI, 3 mM KNO(3), 2 mM Ca(NO(3))(2), 11 mM glucose, 20 mM HEPES, pH 7.4). After thoroughly washing the cells with efflux buffer (containing 136 mM NaNO(3) instead of NaI, 10 washes), four aliquots (equilibrated for 1 min in 1-ml aliquot) of efflux buffer were used to establish a stable baseline. Further aliquots contained an additional 10 µM forskolin in Me(2)SO or only Me(2)SO for control samples. The amount of iodide in each aliquot was determined with an iodide-specific electrode (hnu systems).

Patch-clamp Studies of CFTR-expressing CHO Cells

Cells were grown on glass coverslips at 37 °C in 5% CO(2) for 2-4 days before use. Single channel currents were measured in excised inside-out membrane patches as described previously(15) . The pipette and bath solutions contained 145 mM NaCl, 4 mM KCl, 2 mM MgCl(2), 10 mM TES, pH 7.4. Excised channels were activated by the addition of 1 mM MgATP and 180 nM of the catalytic subunit of PKA. Open probability was calculated for 10-s intervals during recordings that lasted at least 600 seconds. All experiments were carried out at room temperature. Data were presented as the mean ± S.E. Statistical significance was assessed at the 95% confidence level using paired Student's t tests.


RESULTS

10SA CFTR Is Still Phosphorylated by PKA

Previously we had not detected phosphorylation of 10SA CFTR in CHO cells(19) . In order to be able to visualize any remaining phosphorylation, the amount of CFTR protein accumulated in these cells was increased by sodium butyrate treatment, which induced CFTR synthesis approximately 30-fold (Fig. 1A). When 10SA CFTR from the induced cells was immunoprecipitated with a monoclonal antibody (M3A7; 23), -P phosphorylation by PKA was readily detectable but was much reduced compared with wild-type protein (Fig. 1B). To test whether the remaining activation of 10SA CFTR was mediated by this phosphorylation, it was necessary to identify labeled site(s) and to demonstrate their contribution to CFTR function.


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(r) (times10) 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.



Localization of PKA Phosphorylation in 10SA CFTR

In order to map the location of the remaining PKA phosphorylation site(s) to a specific area within the protein, -P-phosphorylated CFTR was cleaved by CNBr. Gel electrophoresis of the cleavage products produced three major labeled bands for both 10SA CFTR and wild-type CFTR (Fig. 2A). The segments generated from wild-type CFTR showed slightly less mobility, which may be due to an increase in molecular weight due to a larger number of phosphoryl groups attached. Such effects of phosphorylation on R domain mobility have been reported previously(17, 24) . All three observed bands most likely originated from within the R domain since wild-type R domain (produced as a recombinant protein in Escherichia coli and purified; 24) showed the same banding pattern as wild-type CFTR upon PKA phosphorylation and CNBr cleavage (Fig. 2B). Based on this information, it was possible to use antibodies to assign each band to a specific segment of the predicted CNBr cleavage map of the R domain (Fig. 2C). Fig. 2D illustrates that P-Ab 6, raised against a synthetic peptide containing residues 724-746, recognized band 3, suggesting that band 3 corresponds to the predicted 5.8-kDa segment (residues 722-773). The intermediate band 2 was recognized by the monoclonal antibody L11E8 (raised against a glutathione S-transferase fusion protein containing CFTR residues 341-702; 23), suggesting that band 2 contains the predicted 8.7-kDa segment (residues 646-721). Both antibodies interacted with band 1, indicating that it may represent a partial cleavage product containing the 5.8-kDa segment, the 8.7-kDa segment, and possibly other segments. Furthermore, there is evidence that within band 2 the predicted 7.3-kDa segment of the R domain co-migrates with the 8.7-kDa segment. (^2)The further search for a remaining phosphorylation site focused on the predicted 5.8-kDa segment (band 3). That this region may play an important role in CFTR regulation through PKA phosphorylation is consistent with the observation that most labeling of wild-type CFTR occurs within band 3 (Fig. 2A).


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(r) 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.



Mutagenesis of Possible Phosphorylation Sites within the 5.8-kDa Segment

PKA-catalyzed phosphate incorporation in CFTR has only been detected on serine residues(14, 17) . The 5.8-kDa segment of the R domain contains 6 serines at positions 728, 737, 742, 753, 756, and 768, two of which (Ser-737 and Ser-768) have already been changed to alanines in 10SA CFTR (Fig. 2C). To investigate whether any one of the remaining 4 serines was phosphorylated by PKA, each one was individually converted to an alanine in a 10SA CFTR construct (19) by in vitro mutagenesis, thus creating four different 11SA CFTR mutants (11SA-S728A, 11SA-S742A, 11SA-S753A, and 11SA-S756A). The mutants were stably expressed in CHO cells. When CFTR from each 11SA variant was phosphorylated with PKA and cleaved with CNBr, it was observed that in 11SA-S728A, 11SA-S742A, and 11SA-S756A CFTR the 5.8-kDa segment (band 3) was still labeled (Fig. 3). The 5.8-kDa segment in 11SA-S753A CFTR, however, was not phosphorylated, indicating that the only remaining PKA phosphorylation site within this fragment was Ser-753. In 11SA-S753A CFTR, the labeling of the partially cleaved band 1 was greatly diminished, suggesting that Ser-753 made a major contribution to its phosphorylation. A 16SA CFTR variant was also created in which all remaining serines (Ser-728, Ser-742, Ser-753, and Ser-756) and threonines (Thr-757 and Thr-760) within the 5.8-kDa segment were simultaneously mutated to alanines. This mutant produced results coinciding with those presented for 11SA-S753A CFTR, i.e. the 5.8-kDa segment (band 3) of 16SA CFTR could not be phosphorylated by PKA (data not shown). In both 11SA-S753A and 16SA CFTR, band 2 still gets labeled, illustrating that there is further residual phosphorylation outside the 5.8-kDa segment of the R domain.


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.



Evaluation of the Functional Effect of Serine 753 Mutagenesis

To address the functional role of Ser-753, 10SA CFTR, 11SA-S753A CFTR and wild-type CFTR were stably transfected into BHK cells, which consistently express similar amounts of CFTR protein for different mutants (Fig. 4A, inset), thereby facilitating a rigorous quantitative functional comparison of cell lines expressing different CFTR variants. Fig. 3confirms that in BHK cells, Ser-753 also accounts for all the labeling within the 5.8-kDa segment of the R domain (band 3), indicating that this phosphorylation is not cell type-specific. Iodide efflux assays demonstrated that forskolin-stimulated channel activity was reduced in the cell line expressing 11SA-S753A CFTR relative to the cell line expressing 10SA CFTR (Fig. 4A). Furthermore, upon stimulation with forskolin, 16SA BHK cells showed no further decrease in iodide efflux activity compared with 11SA-S753A BHK cells (data not shown), indicating that Ser-753 was the single functionally-active site within this fragment. In addition, the chloride channel activity of 11SA-S753A CFTR was evaluated in comparison to 10SA CFTR using single-channel patch-clamping (Fig. 4B). In inside-out membrane patches excised from BHK cells expressing CFTR variants, both 10SA and 11SA-S753A channels demonstrated typical CFTR characteristics of low conductance and nonrectification (data not shown). Significantly, the open probability (P(o)) in the presence of 1 mM ATP and 180 nM PKA was 40% lower for 11SA-S753A CFTR channels when compared with 10SA CFTR channels (Fig. 4C). This result was consistent with the iodide efflux data and showed that Ser-753 does significantly contribute to the residual activity of 10SA CFTR.


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 Me(2)SO. The same amount of Me(2)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(o)) 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(o) of 0.346 ± 0.023, whereas 11SA-S753A channels had a P(o) of 0.203 ± 0.012 (mean ± S.E., n = 5). The P(o) 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




DISCUSSION

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. (^3)

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.


FOOTNOTES

*
This work was supported by the Cystic Fibrosis Foundations of the United States and Canada and the NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a National Science and Engineering Research Council studentship.

To whom correspondence should be addressed. Tel.: 602-301-6206; Fax: 602-301-7017.

(^1)
The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PKA, cAMP-dependent protein kinase; BHK, baby hamster kidney; CHO, Chinese hamster ovary; PAGE, polyacrylamide gel electrophoresis; CNBr, cyanogen bromide; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.

(^2)
F. S. Seibert, J. M. Rommens, and J. R. Riordan, unpublished data.

(^3)
CF Genetic Analysis Consortium, unpublished data.


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