Cell, Vol. 66, 1027-1036,

September

6. 1991, Copyright

0 1991 by Cell Press

Phosphorylation of the R Domain by CAMP-Dependent Protein Kinase Regulates the CFTR Chloride Channel Seng Ii. Cheng,” Devra P. Rich,t John Marshall,* Richard J. Gregory,* Michael J. Welsh,t and Alan E. Smith* ‘Genzyme Corporation One Mountain Road Framingham, Massachusetts 01701 tHoward Hughes Medical Institute and Department of Internal Medicine and Physiology and Biophysics University of Iowa College of Medicine Iowa City, Iowa 52242

Summary CFTR, the protein associated with cystic fibrosis, is phosphorylated on serine residues in response to CAMP agonists. Serines 660, 737, 795, and 613 were identified as in vivo targets for phosphorylation by protein kinase A. The SPQ fluorescence assay revealed that mutagenesis of any one of these sites did not affect Cl- channel activity. Indeed, concomitant mutagenesis of three of the four sites still resulted in CAMPresponsive Cl- channel activity. However, mutagenesis of all four sites abolished the response. One interpretation of these results is that the CFTR CIchannel is blocked by the R domain and that phosphorylation on serines by protein kinase A electrostatically repels the domain, allowing passage of Cl-. The four phosphorylation events appear to be degenerate: no one site is essential for channel activity, and, at least in the case of serine 660, phosphorylation at one site alone is suff icient for regulation of Cl- channel activity. Introduction One of the hallmarks of the genetic disease cystic fibrosis (CF) is the failure of CAMP to open Cl- channels in airway and other epithelia (for reviews see Quinton, 1990; Welsh, 1990). When the gene associated with CF was isolated, it was not surprising to find that the gene product, termed CFTR, was predicted to be a membrane-associated protein (Kerem et al., 1989; Riordan et al., 1989; Rommens et al., 1989). It seemed reasonable to suppose that defective CFTR protein was in some way responsible for the altered membrane properties of CF cells. Recently, we demonstrated that CFTR in recombinant and high producer nonrecombinant cells is present in two forms, a partially glycosylated band S form with an apparent molecular mass of 135 kd and a mature, fully glycosylated band C version with an apparent molecular mass of 150 kd (Gregory et al., 1990; Cheng et al., 1990). Analysis of the properties of CF-associated mutants indicated that several variant CFTRs including the most common, AF508, lacked the ability to generate the band C protein, suggesting that the defect associated with these mutants may be their inability

to traffic to the correct cellular location (Cheng et al., 1990; Gregory et al., 1991). Although it has been suggested that CFTR is a membrane pump by analogy with structurally related proteins such as the multidrug resistance or P-glycoprotein (Hyde et al., 1990) subsequent studies suggest that CFTR itself is a Cl- channel. Thus expression of recombinant CFTR in a variety of cells results in CAMP-regulated Cl- channel activity (Anderson et al., 1991a; Kartner et al., 1991) and mutagenesis of the transmembrane domains of CFTR changes halide ion selectivity of the channel (Anderson et al., 1991 b). The predicted amino acid sequence of CFTR includes a unique R domain. This region includes a number of potential phosphorylation sites for protein kinase C and the CAMP-dependent protein kinase A (PKA) (Riordan et al., 1989). Gregory et al. (1990) showed that CFTR is an acceptor of phosphate when PKA and [yJ2P]ATP are added to immunoprecipitates obtained using the monoclonal antibody (MAb) 13-l (directed against a fusion protein containing P-galactosidase and R domain sequences). Since isolated recombinant R domain could also be phosphorylated in a similar reaction in vitro (S. H. C., unpublished data), it seemed likely that at least some of the phosphate acceptor groups were present in R domain sequences. These observations are consistent with a model suggesting that the increased Cl- channel activity of CFTR observed in the presence of CAMP agonists (Rich et al., 1990; Drumm et al., 1990; Anderson et al., 1991a) is a direct consequence of PKA-mediated phosphorylation of the R domain. However, they do not preclude the possibility that CAMP itself may be involved, since direct channel regulation by cyclic nucleotides has been demonstrated for olfactory receptors (Nakamura and Gold, 1987) and the cardiac pacemaker channel (DiFrancesco and Tortora, 1991). Other experiments in which part of the R domain (corresponding to residues 708 to 835) was deleted from a CFTR cDNA indicated that expression of the variant protein in HeLa cells resulted in the appearance of Cl- channels but that such channels were active in the absence of added CAMP, that is, they were constitutively active and showed only a small additional response to CAMP (Rich et al., 1991). One reasonable explanation for all these results is that CFTR is a Cl- channel, that the R domain keeps the channel closed, and that phosphorylation of the R domain by CAMP-activated PKA reverses this inhibitory activity. We report here the results of experiments to test this hypothesis. We identify the sites on CFTR that are phosphorylated in vivo and show that phosphotylation at these sites regulates Cl- channel activity in recombinant cells expressing variant forms of CFTR. Results In Vivo Labeling of Wild-Type CFTR Earlier studies using COS cells transiently expressing ap-

Cell 1028

B.

A. Forskolin:

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-

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69M

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1 Origin (-)

Figure 1. Metabolic

Labeling of CFTR in Transfected

COS-7 Cells with 32P,

(A) COS-7 cells were transfected with either wild-type CFTR (pMT-CFTR; lanes 1 and 2) or the mutants pMTCFTRS737A (lanes 3 and 4) and pMTCFTRS795A (lanes 5 and 8). Forty-eight hours posttransfection, the cells were labeled for 4 hr with Y. At the end of the labeling period, cells in lanes 2, 4, and 6 were treated with 20 pM forskolin and 100 nM IBMX for 15 min. CFTR was immunoprecipitated with MAb 13-1 (Cheng et al., 1990) and then analyzed on an SDS-polyacrylamide gel. The positions of bands B and C are indicated on the right margin. Quantitation of the labeled bands was achieved using a Betagen Betascope 603 b!ot analyzer. Autoradiography was for 24 hr at -70%. (B) Phosphoamino acid analysis. The band C protein obtained following immunoprecipitation of COS-7 cells transfected with pMT-CFTR, which had been unstimulated (lane 1) or stimulated with forskolin and IBMX (lane 2) using MAb 13-1 was hydrolyzed in vacua. The hydrolysates were applied to thin-layer chromatography plates and then separated by electrophoresis at pH 3.5. The origins are marked with an X. Spots corresponding to ninhydrin-stained amino acid standards are circled. Autoradiographywas for 5 days at -70°C.

propriate vectors have shown that CFTR can be detected either by immunoprecipitation of extracts of [%]methionine-labeled cells (Cheng et al., 1990) or by addition of PKA and [Y-~*P]ATP to immunoprecipitates of unlabeled cell extracts (Cheng et al., 1990; Gregory et al., 1990). We Table 1. Potential CFTR PKA Phosphorylation

sought to label CFTR with [32P]phosphate in whole cells followed by immunoprecipitation of the protein with MAb 13-l. Figure 1A shows that basal levels of phosphatelabeled CFTR were detected in control cells expressing the protein, but that the amount of labeling increased upon

Sites

Residue

Conserved Residue in Murine CFTR”

Conserved Consensus Sequence in Murine CFTR”

S422 S660 S686 s700 S712 s737 S768 T788 s795 SE1 3

Yes Yes Yes Yes Yes Yes Yes No Yes Yes

Yes Yes No Yes Yes Yes Yes No Yes Yes

Phosphorylation” In Vitro

In Vivo

-

-

++

+

+ ++ +++ +++ -

-

+++ +

++ +++

++ -

a The corresponding residues on murine CFTR were obtained from Tata et al. (1991). All residues except serine 422 are located within the R domain. ’ The consensus sequence for phosphorylation by PKA is K/R K/R X S/T where K = lysine, R = arginine, S = serine, T = threonine, and X is any amino acid. c A minus sign indicates no detectable phosphorylation; a plus sign indicates phosphorylation of the residee. Where indicated, +++ means the residues are more phosphorylated than residues with ++, which in turn are more phosphorylated than residues with +.

y;q&iation

of CFTR Cl- Channel Activity

treatment of the cells with forskolin and 3-isobutyl-lmethylxanthine (IBMX) 15 min prior to cell lysis. Quantitation of the material present in CFTR revealed an approximately 1.6-fold increase in label following stimulation. Phosphoamino acid analysis of the labeled bands (band C form) obtained in the absence or presence of added forskolin and IBMX showed that phosphate is attached exclusively to serine residues with no detectable counts associated with either threonine or tyrosine (Figure 1 B). This observed increase in phosphorylation on serines in response to forskolin is consistent with PKA-catalyzed phosphorylation rather than a direct interaction with CAMP being the mechanism of CFTR regulation. Mutagenesis of the Ten Putative PKA Sites We investigated the sites at which the protein was modified by mutating individually the ten potential PKA phosphorylation sites in CFTR (Table 1) using standard oligonucleotide-directed mutagenesis. The mutants were named to include the amino acid residue number preceded by the wild-type amino acid and followed by the amino acid to which the residue was changed using the single-letter amino acid code. Thus S737A means that serine residue 737 was changed to alanine. Mutated plasmids were introduced into a COS cell expression vector, and the ability of each mutant protein to respond to stimulation with forskolin and IBMX was measured by the whole-cell phosphate labeling method. Figure 1A shows that S737A and S795A both retained the ability to respond to forskolin and IBMX in vivo. A similar result was obtained with all ten mutants, with each mutant responding to CAMP agonists with an increase in phosphate content in the range of 1.5 to 2.0-fold (data not shown). These experiments also showed that each of the mutants is capable of producing the fully glycosylated band C version of CFTR. Thus the mutations did not introduce structural alterations that prevent normal protein trafficking as is commonly seen with mutations in other domains (Cheng et al., 1990; Gregory et al., 1991). We also tested the ability of the individual phosphorylation site mutants to induce CAMP-stimulated Cl- channels in HeLa cells. To do this, we used the vaccinia virus expression system described earlier (Elroy-Stein et al., 1969; Rich et al., 1990) and measured halide efflux by change in 6-methoxy-N-(3sulfopropyl)-quinolinium (SPQ) fluorescence. All ten mutants displayed no basal activity and an increase in SPQ fluorescence upon stimulation with forskolin and IBMX (Figure 2). The SPQ assay is not quantitative in that the absolute value of fluorescence and the absolute rate of change of fluorescence are affected among other things by the concentration of CFTR in each transfected, vaccinia-infected cell, by the size of the cell, and by the amount of SPQ loaded intoeach cell (discussed in Rich et al., 1990). This can be appreciated by comparing the results obtained with wild-type CFTR in Figures 2A, 28, and 2C. The important consideration for the data shown in Figure 2 is whether or not CAMP stimulated an increase in the rate of fluorescence change. We conclude from the data in Figure 2 that CAMP stimulated an increase in the rate of fluorescence change in

120 100 80

-c -

S737A S795A S813A

60 40 20 0

c.

180 160

g g

140 120

8 E

100 a0

sc 0 2

60 40 20 0

0 i

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io 12 14 16 18 TIME

(MIN)

Figure 2. Functional Assay of CFTR Phosphorylation SPQ Fluorescence

Site Mutants by

The change in fluorescence of SPQ is shown for HeLa cells expressing (A) CFTR (n = 9, where n = number of cells), S660A (n = 25) S737A (n = I l), S795A (n = 13) or S613A (n = 20); (6) CFTR (n = 6) S712A (n = 7) or S766A (n = 10); (C) CFTR (n = 19) T766A (n = 2), S641A (n = 11) S666A (n = IO), or S422A (n = 17). NOs was substituted for I- in the bathing medium at 0 min. Five minutes later (at the arrow), cells were stimulated with 20 uM forskolin and 100 uM ISMX to raise intracellular CAMP levels (CAMP). Data are mean + SEM of fluorescence at time t (Ft) minus the baseline fluorescence (Fo, average fluorescence measured in the presence of I- for 2 min prior to ion substitution). In some cases the standard error bars are smaller than the symbols.

cells expressing each of the single-site phosphorylation mutants. We interpret this to mean that no one potential PKA phosphorylation site is the predominant phosphate acceptor or is solely responsible for the increase in CIchannel activity following stimulation by CAMP. We assumed that this meant that multiple phosphorylation sites are involved in activating the CFTR Cl- channel and therefore sought to map such sites. Mapping the Tryptic Phosphopeptides of CFTR Labeled In Vitro To identify phosphopeptides associated with each poten-

Cdl 1030

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(795)7.

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(795) (700)

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0 6 (6601 06 1766) 9 (795)

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0 12(768)

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Figure 3. Two-Dimensional

Tryptic

Phosphopeptide

Maps of In Vitro PKA Phosphorylated

Wild-Type

and Mutant CFTFis

COS-7 cells were transfected with either wild-type or mutant CFTR cDNAs. lmmunoprecipitates (obtained using PAb 5x13) containing wild-type (A) or mutant CFTRs (S-H) that had been phosphorylated in vitro with PKA and [@*P]ATP were resolved on S$&polyacr$&mide gels. The immunopurified CFTRs (band C form) were gel purified, digested with trypsin, resolved by electrophoresis and’chiomatography in two dimensions,

~e&ulation

of CFTR Cl Channel Activity

tial phosphorylation site in CFTR, each of the mutant proteins was expressed in COS cells and labeled using PKA and [yJ2P]ATP. The labeled CFTR (mature band C form) was excised from polyacrylamide gels and digested with trypsin. The consensus site for phosphorylation by PKA is preceded by two basic residues (Kemp, 1979), and since trypsin cleaves at arginine or lysine residues, we expect each phosphopeptide to contain only one phosphate residue. Thedigested protein was fingerprinted byelectrophoresis at pH 8.9 followed by ascending chromatography (Hunter and Sefton, 1980; Cheng et al., 1988). Figure 3A shows the two-dimensional tryptic phosphopeptide map of wild-type in vitro phosphorylated CFTR. About 13 phosphopeptides are visible. To assign each peptide to a particular serine residue, the individual mutant proteins were labeled, digested, and fingerprinted in the same way. By comparison of the mutant and wild-type fingerprints (Figures 3A-3H) we were able to assign all labeled spots to seven of the potential phosphorylation sites. For example, the most acidic phosphopeptide in the wild-type fingerprint (spot number 13) is present in all mutant versions of CFTR except S813A (Figure 3H). We therefore identified this phosphopeptide as including residue 813. The data are summarized in Table 1. Some sites generated multiple phosphotryptic peptides (for example, serine 795; Figure 3G). We believe that this means that some peptides represent partial digestion products perhaps resulting from incomplete cleavage adjacent to some amino acid or phosphoserine residues. Alternatively, such peptides may reflect the presence of other protease activities in the trypsin. We are not certain why we were unable to identify three of the peptides predicted to be PKA phosphorylation targets by the primary amino acid sequence. One possibility is that the peptides are insoluble and do not fractionate under our digestion and fingerpinting conditions. Another possibility is that the sites are fully phosphorylated as isolated from cells and therefore are unable to accept additional phosphate in vitro. Inspection of Table 1, however, reveals another, possibly more likely explanation: these peptides may not be detected because they are not substrates for PKA. Serine 422 lies outside the R domain, possibly in a region not accessible to enzyme. Serine 686 is present in a consensus sequence that is not conserved in mouse CFTR (Tata et al., 1991) and amino acid 788 contains a threonine residue rather than serine and again lies within a sequence not conserved in mouse CFTR. Furthermore, that threonine 788 is unlikely to be phosphorylated by PKA is corroborated by our inability to detect phosphothreonine from either in vitro PKA-phosphorylated CFTR (S. H. C., unpublished data) or from CFTR in recombinant cells (Figure 1 B).

-___

A

“s;

B

CFTR

CFTR

t Forskolin

s’

Figure 4. Tryptic Phosphopeptide Maps of In Vivo Labeled CFTR in the Presence and Absence of Forskolin COS-7 cells were transfected with pMT-CFTR. Forty-eight hours posttransfection, the cells were labeled for 4 hr with =P, and then either untreated (A) or treated (6) with forskolin and IBMX for 15 min. CFTRS from these lysates were immtmopurified by precipitation using MAb 13-l. The labeled CFTR (band C form) was digested with trypsin, and the resultant phosphopeptides were resolved in two-dimensions by as outlined in Experimental Proelectrophoresis and chromatography cedures The origins are marked with an X. The letter S in the autoradiograms signifies peptides phosphorylated in the basal state. The correlation of spots and peptides was based on the results obtained in Figure 3. The identity of spots corresponding to peptides containing residues 613 and 660 was further confirmed by fingerprinting the respective in viva labeled mutant proteins (data not shown). Autoradiography was for 2 weeks.

Mapping In Vivo Phosphorylation Sites in CFTR Since we could map seven potential PKA phosphorylation sites in CFTR labeled in vitro, we sought to identify those sites phosphorylated in vivo. COS cells were transfected with wild-type CFTR and labeled with [32P]phosphate in the presence or absence of forskolin and IBMX. The labeled proteins (mature band C form) were extracted, immunoprecipitated, and fingerprinted under the conditions used above for in vitro labeled material. Figure 4A shows that five minor tryptic phosphopeptides were detected in the unstimulated cells. None of these corresponded to the PKA phosphopeptides identified in Figure 3. Upon stimulation with forskolin and IBMX, however, peptides comigrating with those containing residues 660,737,795, and 813 were apparent (Figure 4B). The data in Figure 46 also show that some of the background phosphopeptides also increase in intensity upon CAMP treatment. Since these do not correspond to peptides containing prototype PKA recognition sequences characterized in Figure 3, we assume they represent other phosphorylation events, perhaps ones that respond indirectly to enhanced intracellular levels of CAMP. We interpret these data to mean that the R domain of CFTR is phosphorylated by PKA in response to increased CAMP

-~

and then analyzed by autoradiography. Electrophoresis at pli 6.9 was first performed in the horizontal dimension with the anode followed by ascending chromatography. The origins are marked with an X. Spot numbering is as follows: peptide containing Ser-700 containing Ser-766 (2, 3, 6, and 12); peptide containing Ser.712 (4); peptide containing Ser.660 (6); peptides containing Ser-795 (5, peptide containing Ser-737 (10); and peptide containing Ser-613 (13). Exposure time was for 3 days, A schematic representation phosphopeptides is shown In (I).

on the right, (1); peptides 7, 9, and 11); of the tryptic

Cell 1032

A.

0

2

4

6

8

10 12 14 16 18

TIME (MIN) Figure 5. Functional

Analysis

of CFTR Variants

0

2

4

6

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10 12 14 16 18

TIME (MIN) Using the SPQ Fluorescence

0

2

4

6

8

10 12 14 16 18

TIME (MIN) Assay

The change in SPQ fluorescence is shown for HeLa cells expressing (A) CFTR (n = 27) (6) CFTRAR (n = IO), and (C) S660AKFTRAR (n = 7) after substitution of NOs- for I- at 0 min. Cells were stimulated with forskolin (20 PM) and IBMX (100 uM) at the arrow (CAMP). Data are mean f SEM fluorescence at time t (Ft) minus the baseline fluorescence (Fo, average fluorescence measured in the presence of I- for 2 min prior to ion substitution).

and that four serine residues are so modified (serines 660, 737, 795, and 813). Mutagenesis of All the In Vivo PKA Phosphorylation Sites Since studies in which we had mutated singly each of the in vivo phosphorylation sites had shown that abolition of phosphorylation of anyone site did not measurably reduce the ability of CFTR to respond to CAMP in the SPQ fluorescence assay, we created mutants with multiple mutations within the R domain. We made a triple (8737/795/813A) and a quadruple (S660/737/795/813A) phosphorylation site mutant. Furthermore, since we had earlier created a mutant in which we had deleted part of the R domain (including residues 708-835), we used this mutant, CFTRAR (Rich et al., 1991) to generate a combined S660AICFTRAR mutant: To establish that the mutants bearing multiple mutations were still able to mature, we used the in vitro PKA method to label immunoprecipitates of extracts of COS cells transiently expressing S737/795/813A and S660/737/795/ 813A. These experiments showed that both mutants retain the ability to make band C CFTR (data not shown). We next tested the ability of all four mutants to induce halide efflux using the SPQ assay. We particularly wished to ascertain whether the CFTR variants induced Cl- channel activity in the absence of added CAMP (that is, whether they had constitutive activity) and whether there was an increase in the rate of fluorescence change in response to added CAMP. We have previously shown that CFTRAR displayed active Cl- channels even without addition of CAMP agonist and that channel activity increased further on addition of forskolin (Rich et al., 1991; Figure 58). We interpreted this result to mean that the R domain plays a major role in regulating channel activity but that an additional site responsive to CAMP mapped outside the deletion. S660AICFTRAR (Figure 5C) like CFTRAR (Figure 58) is constitutively active, but in this case there is no detectable additional change in the rate of change of fluorescence in response to CAMP, at least as measured by the SPQ assay. We interpret these data to mean that phos-

phorylation at serine 660 is responsible for the additional responsiveness of CFTRAR to CAMP and its absence causes the lack of response to CAMP in S660AICFTRAR. The data shown in Figures 6A and 6B lead to the same conclusion. 3737/795/813A, which retains serine 660, has no basal activity but responds to forskolin, whereas S66Ol 737/795/813A, which lacks all in vivo phosphorylation sites, has no basal activity and does not respond to forskolin. In other words, S660/737/795/813A fails to induce CAMP-responsive Cl- channels. The data showing that S660AICFTRAR and 873717951813A are able to form CIchannels imply that no gross structural alterations in the molecules have occurred that prevent activity. The difference in activity between the two AR mutants and between the triple and quadruple mutants clearly indicates that S660 alone is able to render CFTR responsive to CAMP in transfected cells. It also implies that in the absence of phosphorylation of at least one site, the CFTR Cl- channel fails to open. We believe that the S66Ol737l 795l813A mutant is inactive because its channel is constitutively blocked and cannot be opened by phosphorylation rather than because it is denatured. This hypothesis would be consistent with the finding that the mutant traffics normally to form mature band C and with results obtained with S660A/CFTRAR, which far from being denatured, is constitutively active. Discussion

*

CFTR Is Phosphorylated In Vivo by PKA The data presented here show that CFTR is phosphorylated as expressed in recombinant cells in response to CAMP agonists. The phosphate detected is attached exclusively to serine, and at least four sites that act as phosphate acceptors have been identified. These sites are serines 660, 737, 795, and 813. Since each of these sites is part of a consensus PKA recognition sequence, which is preceded at residues -2 and -3 with basic amino acids, this result also strongly suggest&hat CAMP acts to regulate Cl- channels via PKA rafher than by direct binding of

y;q;lation

of CFTR Cl- Channel Activity

Figure 6. Functional Analysis of Triple and Quadruple Point Mutants using the SPQ Fluorescence Assay

A. 100 -o-

CFrR TRIPLE

60

t

CFrR QUAD

40

1CAMP 0

2

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10 12 14

TIME (MIN)

16 16

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The change in SPQ fluorescence is shown for HeLa cells expressing (A) CFTR (n = 15) or S737/795/613A (TRIPLE; n = 15); (B) CFTR (n = 12) or 8660/737/795/613A (QUAD; n = 13) after substitution of NO,- for I- at 0 min. Cells were stimulated with forskolin (20 PM) and IBMX (100 KM) at the arrow (CAMP). Data are mean f SEM of fluorescence at time t (Ft) minus the baseline fluorescence (Fo. averaae fluorescence measured in the presence of-lfor 2 min prior to ion substitution).

TIME (MIN)

CAMP to CFTR. Similar conclusions have been reached by Berger et al. (1991) based on addition of the catalytic subunit of PKA to inside-out patches from cells expressing recombinant CFTR followed by measurement of Cl- channel activity. Whether CFTR contains phosphate at other sites in the basal state and whether these sites respond to CAMP are less certain, since the serine residues phosphorylated under these conditions (Figure 4) have not been mapped to CFTR. One possibility is that the unmapped peptides are from other proteins that comigrate with CFTR. At this stage, all we can conclude is that the basal state peptides do not represent PKA sites in CFTR since of the ten putative sites, seven have been identified and the remaining three are either not phosphorylated even in vitro or the phosphopeptides they generate cannot be detected by our methods. Similarly, we have not yet examined the ability of protein kinase C to phosphorylate CFTR either in vitro or in vivo. The effect of protein kinase C on the CFTR CIchannel activity is somewhat controversial and less easy to predict (Boucher et al., 1989; Hwang et al., 1989; Li et al., 1989). Nevertheless, it remains possible that additional sites, responding to different agents and resulting in other means to regulate CFTR activity, remain to be discovered. Regulation of CFTR Cl- Channel by Phosphorylation Several lines of evidence now strongly suggest that CFTR is a Cl- channel regulated by the action of the R domain. First, expression of CFTR in a wide variety of cells results in the presence of CAMP-responsive Cl- channels (Anderson et al., 1991 a; Kartner et al., 1991). Second, mutagenesis of charged residues within the transmembrane domains of CFTR in some cases changes the ion selectivity of the channel (Anderson et al., 1991 b). Third, deletion of part of the R domain of CFTR results in a constitutively activecl-channel, whichcan befurtheractivated by CAMP (Rich et al., 1991). The present finding that CFTR is multiply phosphorylated on the R domain by PKA provides experimental evidence to support our earlier suggestion that the observed change in Cl- channel activity in response to CAMP is directly mediated by phosphorylation of the R domain (Rich et al., 1991). Although we have no supporting experimental data, the phosphorylation event could open the channel by providing an electrostatic force to repel the

R domain away from the membrane and prevent it from otherwise plugging the pore. Alternatively, it could cause a conformational change in the R domain and/or surrounding regions of CFTR that allows passage of Cl- ions through the channel. This model, invoking the R domain as constituting a plug that occludes the ion channel pore, has also been proposed by Kartner et al. (1991) and is analogous to that proposed for the Drosophila Shaker K’ channel (Hoshi et al., 1990; Zagottaet al., 1990). In the case of the Shaker K+ channel, it is proposed that the aminoterminal portion of the molecule interacts with the open channel to cause closure or inactivation. It is notable, however, that the domain involved in the Shaker K+ channel is considerably smaller than the R domain defined here. CFTR Phosphorylation Is Degenerate One interesting finding of this study is that at least four phosphorylation sites are present on the R domain that respond to changes in CAMP. No one of these is essential for activation since mutagenesis of each individually to alanine results in a CFTR molecule still able to respond to CAMP, at least as measured in transfected cells by the SPQ method. Indeed, mutagenesis of three of the sites (737, 795, and 813) still produced a Cl- channel that responded to CAMP. This result is somewhat analogous to that obtained with the CFTRAR deletion mutant in that the deletion removed the same three phosphorylation sites but left the serine 860 site intact. The CFTRAR channel was constitutively active but nevertheless still responded to CAMP agonists. We believe that these results mean that the phosphorylation-dependent regulation of the CFTR Cl- channel is degenerate. In other words, more than one site is normally involved, but no one site is essential. Furthermore, one site alone can be sufficient. Evidence that one site is sufficient is illustrated by CFTR variants in which only serine 660 of the normal in vivo phosphorylation sites remains (8737/795/813A and CFTRAR): an increase in CAMP still stimulates Cl- channel activity in these mutants. That this same sufficient site is not necessary is illustrated by the variant that lacks serine 660 but retains sites 737, 795, and 813; this mutant also responds to CAMP. Only mutagenesis of all four sites results in a molecule that lacks CIchannel activity. Other examples of regulation of protein activity by degenerate phosphorylation at multiple sites

Cell 1034

include the insulin receptor (Zhang et al., 1991) and the membrane-associated proto-oncogene product pp60c-src (Shenoy et al., 1989; Espino et al., 1990).

Multidomain Regulation Channel Activity

of CFTR CI-

We believe the most likely ex’planation of our results is that the R domain of CFTR acts to plug the channel formed by the transmembrane domains and that phosphorylation of all four serine residues (660, 737, 795, and 813) by PKA provides an electrostatic force to repel the R domain and of Cl- ions. Althus open the channel and allow passage though other experiments combining missense and the CFTRAR mutations have shown an interaction between the R domain and at least nucleotide-binding domain 2 (Rich et al., 1991), this study leaves unresolved the role of the nucleotide-binding domains in the regulation of the CFTR Cl- channel. Possibly, they have some role in physically moving the R domain; alternatively, perhaps CFTR has a second functional activity in which these domains participate. Furthermore, it is highly likely that a phosphatase will dephosphorylate the R domain and thereby return the CFTR Cl- channel to its quiescent state. The nature of such a phosphatase and its mechanism of action remains to be established. Experimental Procedures In Vitro Mutagenesis of CFTR-Encoding cDNA and Plasmid Constructions Oligonucleotide-directed mutagenesis of the CFTR cDNA was performed according to standard procedures (Kunkei. 1985; Sambrook et al., 1989) and essentially as described previously (Cheng et al., 1990; Gregory et al., 1991). All mutations were introduced into the high copy number CFTR plasmid pTM-CFTR-4 (Cheng et al., 1990). Mutants containing multiple changes, except for the quadruple mutant (S660/ 737/795/813A), were generated by simultaneous inclusion of all the appropriate mutagenic oligonucleotides in the same reaction. Mutants were first identified by colony hybridization using appropriate probes, and the mutations were then verified by DNA sequencing, restriction enzyme analysis, and in vitro transcription and translation assays (Gregoryet al., 1990). Thequadruple mutantwasassembled byreplacing the approximately 1.5 kb EcoRl fragment of pTM-CFTR-S680A with that of the triple mutant pTM-CFTR-S737/795/813A. Mutants constructed in the pTM-CFTR-4 backbone were tested directly for functional activity using the SPQ assay described below. For expression in COS-7 cells, the CFTR cDNA mutants were transferred into the eukaryotic expression vector pMT-CFTR (Cheng et al., 1990; Gregory et al., 1991). This was accomplished by cloning the 3.5 kb Xbal and BstXl restriction fragment from pTM-CFTR-4 between the unique Xbal and BstXl sites within the CFTR cDNA portion of pMT-CFTR. In pMTCFTR, expression of CFTR is controlled by the flanking mouse metallothionein-1 promoter and simian virus 40 early polyadenylation signal. Transient Expression of CFTR in COS-7 Cells Transient expression in COS-7 cells was performed as described by Sambrook et al. (1989) with the following modifications. Exponentially growing COS-7 cells were rinsed twice with sterile PBS. Approximately 5-10 pg of the mutant DNAs in 0.5 mglml DEAE-dextran was added to the cells and incubated at 37% for 15 min. The DNA and DEAEdextran mixture was removed, the cells were washed twice with PBS,

and complete medium containing 100 m M chloroquine diphosphate was added. After 3 hr at 37OC, the medium was replaced with complete medium containing 10% fetal bovine serum, and the cells were incu-

bated for a further 48 hr prior to analysis. Metabolic labeling was performed using either 250 FCilml [35S]methionine or 300 bCi/ml =P, for 4 hr. In viva labeling with 32P,was performed in the presence of 50 PM

sodium orthovanadate, a phosphatase inhibitor. Where stated, cells were treated with 20 PM forskolin and 100 PM IBMX for 15 min immediately prior to cell lysis. lmmunoprecipitation and Biochemical Analysis of CFTR Our procedures for preparation of cell lysates, immunoprecipitation of proteins using the anti-CFTR antibodies PAb Ex13 and MAb 13-1 (Gregory et al., 1990; Cheng et al., 1990), and SDS-polyacrylamide gel electrophoresis have all been described elsewhere (Cheng et al., 1988, 1990; Gregory et al., 1990, 1991). For in vitro phosphorylation of CFTR, immunoprecipitates were incubated with 20 ng of PKA (Sigma) and 10 @Zi of [y-32P]ATP in 50 ~1 of kinase buffer (50 m M TrisI-ICI [pH 7.5],10 m M MgCI,, 100 Kg/ml bovine serum albumin) at 30°C for 60 min. The reaction was stopped by the addition of 0.5 ml of RIPA buffer (50 m M Tris-I-ICI [pH 7.51, 150 m M NaCI, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS). To remove nonspecifically bound proteins, immunoprecipitates were additionally washed twice with RIPA containing 1 M NaCI. Phosphoamino Acid Analysis and Two-Dimensional Tryptic Phosphopeptide Mapping Extraction and recovery of radioactive proteins from gels were performed as described previously (Cheng et al., 1988, 1990). Briefly, proteins were eluted from the gel by shaking at 37OC in a buffer containing 50 m M ammonium bicarbonate, 0.1% SDS, and 0.2% 2-mercaptoethanol. Eluates were filtered and recovered by trichloroacetic acid precipitation. Following centrifugation. the protein pellets were washed with cold acidic acetone and then dried under vacuum. Phosphoamino acid analysis of the recovered proteins was performed following hydrolysis under vacuum at 110°C for 3 hr in 4 M HCI (Hunter and Sefton, 1980). The acid hydrolysates were dissolved in a marker mixture containing 2.5 m M each of phosphoserine, phosphothreonine, and phosphotyrosine. spotted onto cellulose thin-layer chromatography plates. and then analyzed by electrophoresis at pH 3.5 for 90 min at 750 V in glacial abetic acid-pyridine-H20, 50:5:945 (v/v). The markers were visualized with ninhydrin. Two-dimensional tryptic phosphopeptide mapping was conducted essentially as described by Cheng et al. (1988) following the conditions for electrophoresis and chromatography described by Hunter and Sefton (1980). Recovered proteins were resuspended in 100 ~1 of freshly prepared 1% (w/v) ammonium bicarbonate containing a 1~20 (w/w) ratio of TPCK-treated trypsin to protein and incubated for 12 hr at 37°C. The digested products were suspended and lyophilized several times by using 0.5 ml of HzO. The final samples were resuspended in water containing marker dyes, spotted ontocellulose thin-layerchromatography plates, and separated in two dimensions by electrophoresis at pH 8.9, followed by ascending chromatography. Electrophoresis was performed for 27 min at 1 kV in freshly prepared 1% ammonium carbonate, and the chromatography solvent used was n-butanol-pyridine-glacial acetic acid-H,O. 75:50:15:60 (v/v). SPQ Assay of Functional Activity CAMP-dependent Cl- channel activity was assessed using the halidesensitive fluorophore (SPQ), as previously described (Rich et al., 1990, 1991). We used the vaccinia virus-T7 hybrid expression system developed by Moss and colleagues (Elroy-Stein et al., 1989) to express pTM-CFTR-4 and mutants thereof in HeLa cells. HeLa cells were plated at approximately 5 x t04~m2 on collagen-coated glass coverslips 24 hr prior to infection. Recombinant vaccinia virus vTF7-3 was added to the cells for 1 hr in serum-free medium at a high multiplicity of infection (10 lo 20 moi). Cells were then transfected with recombinant plasmids (5 pg of plasmid per IO6 cells)39Nith lipofectin (Bethesda Research Laboratories) (20 pg of lipid @er IOn cells) and incubated at 37’YZ (Rich et al., 1990; Anderson ei al., 1991a). Cells were studied 9-18 hr after transfection. Cells were loadedwith SPQ by including 10 m M SPQ in the medium for 9 10 12 hr. SPQ fluorescence was initially quenched by incubating cells for 25 to & min in a buffer containing 135 m M Nal. 2.4 m M K&fPOI, 0.6 m M KHzPO,, 1 m M MGSO,, 1 m M CaSOI, IO m M HEPES (pH 7.4), and 10 m M ddxtrose. After meayring fluqfescence for at least 2 min, the 135 m M Nal solption w~,leplacedby one containing 135 m M NaN03 (time 0), and flubres&nCe was measured for another 17.5 min; SPQ fluorescenc& quenched by I- but not by N03-. To

Regulation 1035

of CFTR Cl- Channel Activity

increase intracellular CAMP levels, forskolin (20 KM) and IBMX (100 FM) were added 5 min after the anion substitution. In this assay, an increase in halide permeability results in a more rapid increase in SPQ fluorescence. It is the rate of change in fluorescence that is important in assessing anion permeability, not the absolute change; differences between groups in final absolute values of fluorescence may reflect quantitative differences between groups in SPQ loading, size of cells, or number of cells studied (Illsley and Verkman, 1987). Fluorescence of SPQ in single cells was measured with a Nikon inverted microscope, a SPEX digital imaging system, and a DAGE SIT 88 camera. Excitation was at 350 nm and emission was at >410 nm. Studies were conducted at room temperature. Cells were chosen for quantitation of fluorescence without knowledge of the rate of change; the area of measurement was chosen from the last fluorescence image. Depending on theareaandcell density,Eto 100cellswerestudied per microscopic field. Three microscopic fields were examined for each experimental condition on a given day, and studies under each condition were repeated on at least three different days. Data shown are representative of responses obtained in each condition. Because the expression system produces a heterogeneous response (i.e., not all cells express CFTR) (Rich et al., 1990; Anderson et al., 199la), the data shown are for the 25% of cells in each field with the greatest response. Data are presented as mean + SEM of fluorescence at time t (Ft) minus the baseline fluorescence (Fo, average fluorescence measured in the presence of I- for 2 min prior to ion substitution). Acknowledgments We thank Gary White for synthetic oligonucleotides and Aurita Puga, Elizabeth Burton, and Rob Seilei for technical assistance. We also thank John McPherson and Kathy Klinger for commenting on the manuscript. This work was supported in part by grants from the Cystic Fibrosis Foundation (A. E. S. and M. J. W.) and the National Heart Lung and Blood Institute (A. E. S. and M. J. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement’ in accordance with 18 USC Section 1734 solely to indicate this fact. Received June 28, 1991; revised July 28, 1991. References Anderson, M. P., Rich, D. P., Gregory, R. J., Smith, A. E., and Welsh, M. J. (1991a). Generation of CAMP activated chloride currents by expression of CFTR. Science 257, 879-882. Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991 b). Demonstration the CFTR is a chloride channel by alteration of its anion selectivity. Science 253, 202-205

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