marina r. picciotto, jonathan a. cohns, gloria bertuzzi ... · marina r. picciotto, jonathan a....

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 267, No. 18, Issue of June 25, pp. 12742-12752,1992 Printed in U.S.A.

(Received for publication, December 16, 1991)

Marina R. Picciotto, Jonathan A. CohnS, Gloria Bertuzzi, Paul Greengard, and Angus C. Nairn From the Laboratory of Molecular and Cellular Neuroscience, The Rockefeller University, New York, New York 10021 and the $Department of Medicine, Duke University and Veterans Administration Medical Center, Durham, North Carolina 22705

Regulation of epithelial chloride flux, which is defective in patients with cystic fibrosis, may be mediated by phosphorylation of the cystic fibrosis transmembrane conductance regulator (CFTR) by cyclic AMP- dependent protein kinase (PKA) or protein kinase C (PKC). Part of the R-domain of CFTR (termed CF-2) was expressed in and purified from Escherichia coli. CF-2 was phosphorylated on seryl residues by PKA, PKC, cyclic GMP-dependent protein kinase (PKG), and calcium/calmodulin-dependent protein kinase I (CaM kinase I). Direct amino acid sequencing and peptide mapping of CF-2 revealed that serines 660, 700, 737, and 813 as well as serine 768, serine 795, or both were phosphorylated by PKA and PKG, and serines 686 and 790 were phosphorylated by PKC. CFTR was phosphorylated in vitro by PKA, PKC, or PKG on the same sites that were phosphorylated in CF-2. Kinetic analysis of phosphorylation of CF-2 and of synthetic peptides confirmed that these sites were excellent substrates for PKA, PKC, or PKG. CFTR was immunoprecipitated from T84 cells labeled with 32Pi. Its phosphorylation was stimulated in response to agents that activated either PKA or PKC. Peptide mapping confirmed that CFTR was phosphorylated at several sites identified in vitro. Thus, regulation of CFTR is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by different second messenger pathways.

In patients with cystic fibrosis (CF),’ stimulation of chloride flux by cyclic AMP-dependent protein kinase (PKA) and by protein kinase C (PKC) is defective, leading to decreased chloride conductance in epithelial cells (Quinton, 1990; Welsh, 1990). Activation of PKA opens chloride channels in normal human epithelia but cannot stimulate chloride con-

* This work was supported by grants from the Cystic Fibrosis Foundation (to A. C. N. and J. A. C.) and the Veterans Administra- tion (to J. A. C.) and Grant DK 40701 (to J. A. C) from the 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 be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HEPES, N-2-hydrox- yethylpiperazine-N’-2-ethanesulfonic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PDBu, phorbol 20- oxo-20-deoxy-12,13-dibutyrate; PMA, 40-phorbol 12-myristate 13- acetate; PKA, CAMP-dependent protein kinase; PKC, protein kinase C; CaM kinase, Ca2+/calmodulin-dependent protein kinase; PKG, cGMP-dependent protein kinase; S-EC, S-ethyl cysteine; PMSF, phenylmethylsulfonyl fluoride; TPCK, tosylphenylalanyl chloromethyl ketone; HPLC, high performance liquid chromatography; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid.

ductance in CF-affected cells (Li et al., 1988, 1989; Hwang et al., 1989). PKC can also stimulate chloride flux in human epithelial cells in the presence of low concentrations of calcium, and this type of regulation is also defective in patients with CF (Li et al., 1988; Hwang et al., 1989). In contrast, at high concentrations of calcium, stimulation of PKC closes chloride channels. This activity is unaffected in patients with CF, however (Li et al., 1988), and may result from inhibition of a different chloride channel (Anderson and Welsh, 1991).

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) are the causative defect in patients with CF (Kartner et al., 1991; Riordan et al., 1989; Rich et al., 1990; Drumm et al., 1990). It is thought that CFTR is a chloride channel (Kartner et al., 1991; Frizzell and Cliff, 1991; Anderson et al., 1991c; Anderson et al., 1991b), and CFTR has been shown to be a substrate for PKA (Gregory et al., 1990). It is therefore likely that regulation of chloride flux by PKA is mediated by direct phosphorylation of CFTR (Cheng et al., 1991b; Tabcharani et al., 1991). While the effects of PKC on chloride channels are more complex, it is likely that by analogy with PKA, PKC regulates CFTR function via direct phosphorylation.

It is important to investigate the biochemical basis of the regulation of CFTR by phosphorylation. CFTR is a large, hydrophobic protein and has not been purified. Therefore, direct biochemical analysis of the protein has not yet been possible. There are many consensus sites for phosphorylation by either PKA or PKC present within a central domain of CFTR, and it has been proposed that phosphorylation of this so-called R-domain may be responsible for activation of CFTR (Riordan et al., 1989). To address this question, we have constructed a protein corresponding to a portion of the R-domain of CFTR (designated CF-2) which contains the most likely consensus sites for PKA and PKC phosphorylation. Given that there are many consensus sites for phosphorylation, it is also important to determine the kinetics of phosphorylation for these sites in order to determine which sites may be responsible for regulation of CFTR function.

Compared to other physiological substrates, CF-2 was found to be a very good substrate for phosphorylation by PKA and PKC and an excellent model for the study of phosphorylation of holo-CFTR. We have sequenced the sites of phosphorylation by PKA and PKC in CF-2 and find that within CF-2, and by implication within CFTR, not all consensus sites become phosphorylated. In addition, treatment of T84 cells with activators of PKA and PKC increase the phosphorylation of several of the sites in CFTR that have been identified in vitro.

EXPERIMENTAL PROCEDURES

Materials-Forskolin, ATP, Triton X-100, dithiothreitol, EDTA, Tris, Coomassie Brilliant Blue R-250, sodium dodecyl sulfate (SDS),

12742

Phosphorylation of CFTR 12743

histone type 111-S, Nonidet P-40, diacylglycerol, phosphatidylserine, and bovine serum albumin were from Sigma. HEPES and PMSF were from Calbiochem. Phorbol, 20-oxo-20-deoxy-12,13-dibutyrate (PDBu), and 4B-phorbol 12-myristate 13-acetate (PMA) were from LC Services. Glutamine was from Biofluids. [y-32P]ATP and ["PI orthophosphoric acid ("P,) were from Du Pont-New England Nu- clear. Tosylphenylalanyl chloromethyl ketone (TPCK)-treated trypsin was from Worthington. Waymouth's medium, fetal calf serum, Dulbecco's modified Eagle's medium, Ham's F-12 medium, penicillin, and streptomycin were from Gibco Laboratories. Phosphate-free modified Eagle's medium was from Flow Laboratories. Phosphocel- lulose P-81 paper was from Whatman. Trifluoroacetic acid was from Pierce. Acetonitrile was from Burdick-Jackson. Activated CH-Seph- arose beads were from Pharmacia LKB Biotechnology Inc. XAR-5 x- ray film and cellulose thin layer chromatogram sheets were from Eastman Kodak. An enhanced chemiluminescence kit was from Amersham International. Leupeptin, chymostatin, pepstatin, and antipain were from Chemicon.

The catalytic subunit of PKA was purified from bovine heart as described (Kaczmarek et al., 1980). Cyclic GMP-dependent protein kinase (PKG) was purified from bovine lung as described (Walter et al., 1980). Casein kinase I1 was purified from bovine brain as described (Girault et al., 1989). PKC was purified from rat brain essentially as described (Woodgett and Hunter, 1987). Calcium/calmodulin-dependent protein kinase I (CaM kinase I) was purified from bovine brain as described (Nairn and Greengard, 1987). CaM kinase I1 was purified from rat brain as described (McGuinness et aL, 1985). CaM kinase I11 was purified from rabbit reticulocytes as described (Nairn and Palfrey, 1987). Calmodulin was purified from rabbit brain as described (Grand et al., 1979).

Synthesis of Peptides-The following peptides corresponding to the indicated amino acids of human CFTR were synthesized by the Rockefeller University protein sequencing facility: FEKAKQNN- NNRKTSNGDDSLF (409-429); FSAERRNSILTETLHR (653- 668); FGEKRKNSILNPINSIRKFSIVQK (693-716); LERRLSLVP- DSEQGEAIL (732-749); LQARRRQSVL (761-770); IHRKTTAST- RKVSLA (783-797); IYSRRLSQETGL (807-818). Peptides were purified by preparative reversed-phase HPLC. All peptides were >95% pure as analyzed by HPLC and had the expected amino acid compositions (data not shown).

Preparation of Antibodies-Antibodies were produced in New Zea- land White rabbits against several synthetic peptides: FSAERRN- SILTETLHR (653-668) (antibody 396 (Marshall et al., 1991)); FGE- KRKNSILNPINSIRKFSIVQK (693-716) (antibodies CC23 and CC24); LERRLSLVPDSEQGEAIL (732-749) (antibodies CC25 and CC26); KEETEEEVQDTRL (1468-1480) (antibody a-1468 (Marino et al., 1991)). Purified peptides were coupled to bovine thyroglobulin using glutaraldehyde. Antisera CC23-CC26 were prepared by Cocalico Biologicals, Inc. The synthetic peptides were individually coupled to activated CH-Sepharose beads and used to affinity-purify the sera essentially as described (Yamagata et al., 1991). Briefly, serum was loaded on the respective affinity resin, and the column was washed. The anti-peptide antibody was eluted with 4.2 M magnesium chloride, dialyzed against HEPES-buffered saline, concentrated with a Centri- prep-30 concentrator (Amicon) to 0.2-1.0 mg/ml, and stored in aliquots at 4 "C.

Initial bleeds of antibodies CC23 and CC24 recognized only dephospho-CF-2 or dephospho-CFTR by immunoblotting (see Fig. 6) and were unable to immunoprecipitate phospho-CF-2 or phospho- CFTR. Following repeated boosting with coupled peptide, both of these antibodies were able to immunoprecipitate both phospho-CF-2 and phospho-CFTR (see Fig. 4).

Construction of CF-2, a CFTR R-domain Peptide-Two oligonucleotides, the first corresponding to bases 2057-2087 and the second corresponding to the complement of bases 2621-2651 of the CFTR message (Riordan et al., 1989), were synthesized by the Rockefeller University Protein Sequencing Facility. The first oligonucleotide was engineered with a restriction enzyme site for NcoI by including a C at position 2063 rather than a T. The second oligonucleotide was engineered with a restriction site for BglII by including a T at position 2637 rather than an A, and a G at position 2641 rather than a T. A cDNA encoding the relevant exons of CFTR, clone T16-4.5, was obtained from the American Type Culture Collection, and the two oligonucleotides described were used to amplify a 594-base pair product from 100 ng of this cDNA using the polymerase chain reaction method (Scharf et al., 1986; Saiki et aL, 1988). Amplification was performed using Taq polymerase (Promega), and the manufacturer's recommendations were followed. The conditions for polymerase chain

reaction were 20 cycles of the following: 94 "C for 1 min, 60 "C for 1 min, and 72 "C for 2 min with 5 s added per cycle to the elongation at 72 "C. The amplified product was purified by electrophoresis through low melting temperature agarose (FMC) and then by phenol extraction and ethanol precipitation. The DNA was cut with NcoI and BglII, and the fragment was subcloned into the NcoI and BarnHI sites of the PET-& expression vector (Studier et al., 1990). Several individual clones containing the insert were isolated and transformed into HBlOl containing the pLys-S plasmid encoding T7 polymerase (Studier et al., 1990). The bacteria were induced to express the protein encoded by the plasmid, corresponding to amino acids 645 to 835 of CFTR, by incubation for 3 h at 37 "C with 1 mM isopropyl p-D- thiogalactopyranoside (IPTG). Initiation of translation occurred at methionine 645 of CFTR and terminated at a stop codon directly following the insert in the expression vector.

Purification of CF-2-All clones tested produced a peptide of the predicted molecular weight. The peptide produced was recognized in every case by polyclonal antisera raised against peptides corresponding to epitopes within CF-2. Four liters of bacteria were induced to produce the protein. The cells were pelleted, resuspended in 25 ml of Buffer A (30 mM Tris, pH 7.5, 2 mM EDTA, 2 mM EGTA, 6 mM 0- mercaptoethanol, 100 pg/ml leupeptin, 100 p~ PMSF), and lysed by freezing at -20 "C. The suspension was diluted to 500 ml, then homogenized at 7,000 rpm with a Polytron homogenizer to shear the DNA in the sample. CF-2 was purified away from bacterial proteins by anion exchange chromatography using a DEAE-Sephacel (Phar- macia) column (2.5 X 25 cm) eluted with a 1-liter gradient of 0-0.3 M NaCl in Buffer A. The fractions containing CF-2 were identified by phosphorylation with PKC (details below). CF-2 was the major phos- phoprotein in the preparation using this assay. The peak fractions were pooled and subjected to hydroxylapatite chromatography using an HTP column (Bio-Rad, 2.5 X 15 cm) and eluted with a 1-liter gradient of 0-0.3 M potassium phosphate in Buffer A. Finally, the fractions containing CF-2 were pooled again, concentrated in an Amicon concentrator under nitrogen, and separated by gel filtration through an Ultragel AcA-44 column (LKB) using Buffer A with 0.3 M NaC1. The peak fractions were concentrated and stored at -70 "C.

Phosphorylation Assays-Phosphorylation assays were carried out for different amounts of time at 30 "C. The standard reaction mixture contained 50 mM HEPES, pH 7.5, 10 mM magnesium acetate, 1 mM EGTA, 0.5-5 r g of CF-2, and 50 p~ [y-32P]ATP. Reaction mixtures with PKC contained either 1.5 mM Ca2+ or no free calcium, 50 pg/ml L-a-phosphatidylserine, and 4 pg/ml 1,2-dioleoyl-sn-glycerol. Reac- tion mixtures with PKG contained 1 p~ cyclic GMP (cGMP), and reaction mixtures with CaM kinases I, 11, and I11 contained 20 pg/ml calmodulin and 1.5 mM Ca2+. The final concentrations of kinases in the reaction mixtures were as follows: PKA, 0.2 pg/ml; PKC, 0.125 Fg/ml; PKG, 3 pg/ml; CaM kinase I, 2.5 pg/ml; CaM kinase II,5 pg/ ml; CaM kinase 111, 0.5 pg/ml; casein kinase 11, 1 pg/ml. After 2 min of preincubation at 30 "C, reactions were initiated by the addition of [Y-~'P]ATP, specific activity 750 cpm/nmol. Reactions were terminated by the addition of 20 pl of SDS-containing stop buffer (final concentrations, 1% SDS, 60 mM Tris-HC1 (pH 6.8), 5% (v/v) glycerol, 0.2 M 0-mercaptoethanol, traces of pyronin Y). CF-2 containing samples were subjected to SDS-PAGE using 11.5% polyacrylamide according to the method of Laemmli (Laemmli, 1970). Gels were stained in Coomassie Brilliant Blue, destained, dried, and subjected to autoradiography.

The kinetics of phosphorylation of synthetic peptides by PKA and PKC were determined using the assay conditions described above with 200 p M ATP, specific activity 1.1 X 10' cpm/nmol. 20, 25, 33, 50, 100, and 200 p~ concentrations of peptides were tested using 20 ng of PKA or 12.5 ng of PKC per reaction. The incorporation of 32P was linear with these concentrations of peptides and kinases. Reac- tions were stopped after 5 min by the addition of 50 p l of 30% acetic acid, and aliquots were spotted on phosphocellulose filters (PSI, Whatman). Filters were washed in water for 10 min and then counted by Cerenkov counting. Lineweaver-Burk plots of l/cpm versus 1/ [peptide] were used to obtain values for K , and V,,,,,.

Cell Culture"T84 cells were grown as monolayers in 60- and 100- mm culture dishes in a 1:l mixture of Dulbecco's modified Eagle's medium (high glucose) and Ham's F-12 medium, supplemented with (final concentration) 15 mM HEPES buffer, pH 7.5,lO mM NaHC03, 100,000 units of penicillin, 100 mg of streptomycin per liter, and 5% fetal calf serum. Confluent monolayers were subcultured by trypsin- ization with 0.1% trypsin, virus- and mycoplasma-free, and 1 mM EDTA in Ca2+- and Mg2"free phosphate-buffered saline.

Radiolabeling of T84 Cell.-The T84 cells used were slightly sub-

12744 Phosphorylation of CFTR confluent. Cells were washed twice with phosphate-free Dulbecco's modified Eagle's medium and then incubated with phosphate-free Dulbecco's modified Eagle's medium containing 32Pi (0.5-1 mCi/ml). After an 8-12-h incubation at 37 "C under 5% Con, cells were washed twice with phosphate-free medium and then subjected to the appro- priate treatment. Forskolin, PDBu, or PMA was diluted in phosphate- free Dulbecco's modified Eagle's medium, and cells were incubated with these agents for various times. At the end of the incubation period, the medium was aspirated, washed with Dulbecco's modified Eagle's medium, and 1 ml (60-mm plates) or 2 ml (100-mm plates) of 1% SDS containing 100 pg/ml leupeptin and 100 p~ PMSF was added, and the cells were allowed to solubilize for 5 min. The solubilized cells were scraped from the plates and transferred to 15-ml polypropylene tubes and sonicated for 30 s using a Microson Ultra- sonic Cell Disrupter (Heat Systems Ultrasonics) (setting 14).

Immunoprecipitation of CFTR-CFTR was immunoprecipitated from SDS-solubilized T84 cell cultures as follows. The SDS-solubilized extracts containing equal amounts of protein were diluted 2-fold with a concentrated stock solution to give the following final concentrations: 25 mM Tris-HC1, pH 7.5, 200 mM NaCl, 5 mM EDTA, 100 mM NaF, 20 mM sodium pyrophosphate, 2.5% Nonidet P-40, and 0.5% SDS. The diluted samples were incubated with 20-100 pl of Sepharose-protein A beads (10% w/v) for 15 min, centrifuged for 1 min at 10,000 X g, and the supernatants were incubated with 10-50 pl of purified anti-CFTR antisera (0.2-0.5 mg/ml) as indicated for 2- 3 h. The samples were then incubated with 20-50 pl of Sepharose- protein A beads for 30-60 min with frequent vortexing. The samples were centrifuged for 1 min at 10,000 X g and washed by resuspension with 1 ml each of Buffer B: 10 mM Tris-HC1, pH 8.0, 500 mM NaCl, 0.5% Nonidet P-40, 0.05% SDS; Buffer C: 10 mM Tris-HCI, pH 8.0, 150 mM NaC1, 0.5% Nonidet P-40, 0.05% SDS, 0.5% deoxycholate; Buffer D: 10 mM Tris-HC1, pH 8.0, 0.05% SDS. The washed beads were resuspended in 100 p1 of SDS stop solution and vortexed several times over a period of 30-60 min at room temperature. The samples were centrifuged and the supernatants were separated by SDS-PAGE (5.5% gels), the gels were stained, destained, and dried, and autoradiography was performed. The incorporation of 32P into CFTR was quantified by excision of the protein bands from the dried gels using the autoradiogram as a guide followed by liquid scintillation spec- trometry.

Immunoprecipitation of CFTR and Back-phmphorylation-T84 cells were washed twice with phosphate-buffered saline, and cells were scraped from the plates into phosphate-buffered saline and centrifuged for 5 min at 2000 X g. The cells were resuspended in 20 mM Tris-HC1, pH 7.5, 1 mM EDTA, 1 mM EGTA plus 100 pg/ml leupeptin, and 100 p~ PMSF and sonicated as described above. The sonicated cells were centrifuged at 300,000 X g for 15-30 min using a Beckman TL-100 centrifuge. Pellets were resuspended in 1% SDS plus 100 pg/ml leupeptin and 100 p~ PMSF and sonicated.

CFTR was immunoprecipitated with antibody a-1468 as described (Cohn et al., 1992), and the washed beads were further washed twice with 1 ml of 20 mM Tris-HC1, pH 7.5, 1 mM EDTA, 1 mM EGTA. CFTR attached to the beads was phosphorylated as described above except that PKA (2.5 pg), PKC (5 pg), and [32P]ATP (5 pCi) with no cold ATP were used. Samples were incubated for 30-60 min with frequent vortexing. Samples were washed using buffers B, C, and D as described above for immunoprecipitation of CFTR. The beads were resuspended in SDS stop solution, and SDS-PAGE was performed as described above.

Two-dimensional Phosphopeptide Mapping-Gel pieces containing "'P-labeled CF-2 or CFTR were excised from dried SDS-polyacrylamide gels, washed with two changes of 10% acetic acid/30% methanol, three changes of 50% methanol, and lyophilized. Phosphorylated peptides were separated from unincorporated [32P]ATP by chromatography over a 1-ml column of Dowex AG 1-X8 (acetate form, 100- 200 mesh) in 50 mM acetic acid and then lyophilized. 1 ml of 50 mM NH,HC03, pH 8.0, containing TPCK-trypsin (50 pg/ml) was added to the dried gel pieces or peptides, and the mixture was incubated at 37 "C for 20 h. The gel pieces were washed with 0.5 ml of 50 mM NH4HCOa at 37 "C for 4 h, and the collected supernatants were lyophilized. Dried samples were resuspended in electrophoresis buffer (10% acetic acid, 1% pyridine, pH 3.5) and spotted 10 cm from the right and 4 cm from the bottom on thin layer cellulose sheets (20 X 20 cm, Eastman Kodak). Phosphopeptides were separated by electrophoresis at 400 V for 90 min in the first dimension, followed by chromatography in the second dimension in a buffer containing pyridine:l-butano1:water:acetic acid (10:15:12:3, v/v). Dried sheets were subjected to autoradiography.

HPLC and Sequencing-After phosphorylation by either PKA or PKC for 20 min, CF-2 (100 pg) was electrophoresed using an 11% polyacrylamide gel. The gel was dried without fixing, and autoradiography was performed. Gel pieces containing radioactive CF-2 were excised, rehydrated in 1 ml of water, and then frozen in liquid nitrogen. Water was added to a volume of 10 ml, and the thawed gel pieces were homogenized using a glass Teflon homogenizer. The resulting slurry was spun at 2500 X g for 10 min to pellet the acrylamide, and the supernatant was harvested. The pellet was re- extracted with 10 ml of water and re-spun, and the supernatants were then spun at 10,000 X g for 20 min. The supernatants were freeze- dried and then resuspended in 85% acetone, 5% triethylamine, 5% glacial acetic acid, 5% water. After 1 h at -20 'C, the protein was pelleted by centrifugation for 20 min at 10,000 X g. The pellets were dissolved in 5 pl of 8 M urea, 400 mM ammonium acetate, 10 p1 of 8 M guanidine HC1, 7.5 pl of acetonitrile. Water and NH4HC03, pH 8.0, were added to a final volume of 150 pl and 25 mM NH4HC03. CF-2 was then digested with 10 pg/ml of sequencing grade V8 protease (Boehringer) for 48 h at 37 "C. The resulting peptides were separated by reversed phase HPLC on a C-18 column using a gradient of 0- 60% acetonitrile (over 60 min) in 0.1% TFA. Peptides containing greater than 5% of total radioactivity were selected for further char- acterization. The selected peptides were further purified by HPLC using a C-18 column. Since the amount of acetonitrile needed to elute each peptide was known for the first elution (n%), each peptide was eluted from n - 4% to n + 4% over 40 min. The resulting pure peptides were analyzed by two-dimensional peptide mapping. The phosphoserine residues were derivatized to S-ethyl-L-cysteine (S-EC) with ethanethiol as described (Holmes, 1987). Derivitized peptides were sequenced by Edman degradation, and S-EC was detected by comparison to a standard. The amino acid sequence of 32P-labeled tryptic phosphopeptides was determined as described (Hunkapillar et al., 1983) using an Applied Biosystems (Foster City, CA) AB-470 gas- phase sequencer. Identification of phenylthiohydantoin-amino acid derivatives was accomplished by on-line CIa HPLC analysis and was semiquantitative .

Miscellaneous Procedures-Proteins were transferred to nitrocellulose filters as described (Towbin et al., 1979). Immune complexes were detected by 1251-protein A or by enhanced chemiluminescence. Phosphoamino acid analysis was performed as described (Nairn and Greengard, 1987). Briefly, tryptic digests were hydrolyzed with 6 M HCl, and phosphoamino acids were separated by electrophoresis at pH 1.9 in 8.7% acetic acid, 2.5% formic acid followed by electrophoresis in the same direction at pH 3.5 in 10% acetic acid, 1% pyridine.

RESULTS

Phosphorylation of CF-2-A fragment of the R-domain of CFTR corresponding to amino acids 645 to 835 was expressed in and purified from E. coli. Its predicted molecular weight was 21,000, but its apparent molecular weight on SDS-PAGE was close to 28,000. CF-2 was a very good substrate for PKA (Fig. 1A), and phosphorylation of CF-2 by PKA caused a decrease in its mobility on SDS-PAGE. At 1 min of phosphorylation, a doublet was already apparent, consisting of a lower band that ran at approximately 30,000, and a middle band that ran at about 32,000. The amount of radioactivity in the lower band decreased through the time course while the intensity of the middle band increased. In parallel, there was a shift of the Coomassie Blue staining material, indicating that there was stoichiometric phosphorylation of the site(s) that caused the shift (data not shown). A third, upper band of approximately 34 kDa, which is slightly visible in Fig. L4 at 15 min of PKA phosphorylation, appeared later. This band was phosphorylated to a stoichiometry of -5 mol/mol (data not shown). There were two or three sites phosphorylated by PKA that caused a shift in the mobility of CF-2 (see below), and the middle and upper bands represent molecules that were phosphorylated stoichiometrically at one or more of these sites.

CF-2 was also a very good substrate for PKC (Fig. L4 ) and was phosphorylated to a stoichiometry of -2 mol/mol (data not shown). A mobility shift on SDS-PAGE was not observed when CF-2 was phosphorylated by PKC to 2 mol/mol al-


A. PKA PKC MW ( k W

- 45

"-30

- 21

1' 5' 1 5 1' 5 15

B. PKC

ca*+ P K G y i l - T C K I I

CamK

- +

- 30 - 30

- 21

FIG. 1. Phosphorylation of CF-2 with PKA, PKC, PKG, and CaM kinase I. Purified CF-2 was phosphorylated as described under "Experimental Procedures." Reactions were stopped with 10 pl of stop buffer, and the entire sample was electrophoresed using an 11.5% SDS-polyacrylamide gel. Gels were dried and exposed to x-ray film. A , CF-2 was incubated with PKA in the standard reaction mixture for 1, 5, or 15 min. CF-2 was incubated with PKC in the standard reaction mixture plus 1.5 mM Ca'+, SO pg/ml phosphatidylserine, and 4 pg/ml dioleoyl glycerol for 1, 5, or 15 min. R, CF-2 was incubated for 5 min in the standard reaction mixture containing phosphatidvl- serine and dioleoyl glycerol and either no calcium (-) or 1.5 mM Ca" (+). C, CF-2 was incubated with PKG, CaM kinases I, 11, or 111, or casein kinase I1 for 5 min. The PKG reaction mixture contained 1 PM cGMP, and the CaM kinase reaction mixtures contained 20 pg/ ml calmodulin and 1.5 mM Ca".

though a t later time points with large amounts of kinase an upper band appeared (data not shown). The calcium depend- ence of phosphorylation of CF-2 by PKC was anomalous (Fig. 1B). While relatively high levels of phosphate were incorporated into CF-2 under standard assay conditions (-0.5 mM free calcium), slightly higher levels of phosphorylation were observed when no calcium was present (1 mM EGTA). Phos- phopeptide maps of CF-2 phosphorylated under the two conditions were very similar (data not shown).

Calcium has been shown to regulate chloride conductance in epithelial cells independent of PKC activation (Boucher et al., 1989; Schoumacher et al., 1990). To determine whether this effect might involve phosphorylation of CFTR, we examined whether any of the CaM kinases could phosphorylate CF-2. The protein was phosphorylated by CaM kinase I, but not by CaM kinase I1 or CaM kinase I11 (Fig. IC). Casein kinase I1 and PKG were also tested for their ability to phosphorylate CF-2, since consensus sites (Kennelly and Krebs, 1991) for these kinases are present within CF-2. CF-2 was not phosphorylated by casein kinase 11, but was highly phosphorylated by PKG. Like PKA, PKG caused two distinct mobility shifts in CF-2 to 30 kDa (data not shown) and 32 kDa (Fig. IC). The extent of the mobility shift with PKG was a result of the high concentration of enzyme used.

Analysis of the kinetics of phosphorylation by PKA, PKC, and PKG indicated that CF-2 was a very good substrate by comparison to DARPP-32, a known substrate for these kinases. In addition, CF-2 was a better substrate for phosphorylation by CaM kinase I than synapsin I, the best known substrate for this kinase. In each case, the K , values were between 1 and 10 PM (data not shown).

Two-dimensional Phosphopeptide Mapping of CF-2 Phos- phorylated by PKA, PKC, PKG, and CaM Kinase I-Tryptic (Fig. 2, Panel I ) , chymotryptic, and thermolytic (data not shown) two-dimensional peptide maps of CF-2 phosphoryl-

ated in uitro by PKA contained several phosphopeptides. Tryptic maps of the lower band of CF-2 contained peptides 1-6. These sites were already prominent in a map of CF-2 phosphorylated for 1 min. In addition, maps of the middle band contained peptides 7 and 8, and maps of the upper band contained peptides 7, 8, and 9. Tryptic maps of CF-2 phosphorylated by PKC (Fig. 2, Panel ZZ) contained peptides 1-4 which were identical with peptides 1-4 in maps of CF-2 phosphorylated by PKA. In addition, peptides 11, 12, and 16 were prominent and were phosphorylated rapidly. These peptides were not found in the maps of CF-2 phosphorylated with PKA and represent distinct sites phosphorylated by PKC. CaM kinase I phosphorylated only one major peptide (Peptide 17 in Fig. 2, Panel ZZZ), and phosphorylation of this site did not cause a shift in mobility. Peptide 17 co-localized with peptide 6 in mixing experiments (data not shown), but we have not determined unequivocally whether these two peptides are identical. The map of CF-2 phosphorylated by PKG (Fig. 2, Panel ZZZ) was almost identical with that of CF-2 phosphorylated by PKA (Fig. 2, Panel I ) . It is likely that under the conditions used, PKA and PKG phosphorylated the same sites in CF-2. Phosphoamino acid analysis indicated that CF-2 was labeled only on seryl residues by all the kinases discussed above (data not shown).

I t is possible that the shift in the mobility of CF-2 was a result of an accumulation of charge due to phosphorylation to a threshold stoichiometry. However, it is more likely that peptides 7, 8, or 9 contain the specific phosphorylated residue(s) that caused the shift in mobility on SDS-PAGE. These peptides were only observed when the mobility of CF-2 was shifted following phosphorylation by PKA or PKG. Further- more, under initial rate conditions, peptides 7, 8, and 9 were not phosphorylated by PKC or CaM kinase I, and no mobility shift was observed.

Sequencing of Phosphorylation Sites in CF-2-To determine the amino acid sequence of the phosphorylation sites, CF-2 was incubated with ["'PIATP and PKA or PKC for 20 min, purified by SDS-PAGE, and cleaved with V8 protease. The resulting radioactive peptides were separated by reversed phase HPLC, and peptides containing 5% or more of the total incorporated radioactivity were sequenced. Phosphorylated serines were derivatized with ethanethiol to prevent ambiguity in determining the phosphorylated residues, and derivatized peptides were sequenced by Edman degradation. All sequences obtained were consistent with the predicted amino acid sequence of CFTR, and the first residue of each peptide coin- cided with recognition sites for V8 protease. Results of amino acid sequencing are summarized in Table I.

Four peptides phosphorylated by PKA contained greater than 75% of total radioactivity. The peptide spanning residues 693-725 contained 20%, the peptide spanning residues 727- 743 contained 20%, the peptide spanning residues 747-804 contained 25%, and the peptide spanning residues 805-815 contained 8% of the total counts. The rest of the radioactivity was spread across the entire run or contained in small peaks. S-EC was detected in three peptides a t residues 700, 737, and 813. I t was not possible to sequence enough residues of the peptide spanning amino acids 747-804 to determine whether S-EC was incorporated only at residue 768, only at residue 795, or at both residues. This was the most radioactive peptide, and it is possible that both residues were phosphorylated.

When CF-2 was phosphorylated by PKC, two peptides contained greater than 60% of total radioactivity. The peptide spanning residues 682-691 contained 28% of total radioactivity, and the peptide spanning residues 747-804 contained 35% of the radioactivity. S-EC was detected at residue 686. It was

12746 Phosphorylation of CFTR

FIG. 2.--continued

FIG. 2. Two-dimensional tryptic phosphopeptide maps of CF-2 and CFTR. Radioactive bands of CF-2 from the experiment in Fig. 1, A and C, were excised, washed, and digested overnight with 50 Fg/ml trypsin. CFTR was immunoprecipitated from T84 cells, phosphorylated with PKA or PKC, and run on 5.5% gels as described (Cohn et al., 1992). Radiolabeled CFTR was excised from gels, washed, and digested as described above. Tryptic digests were separated by electrophoresis in the first dimension a t 400 V in 10% acetic acid, 1% pyridine, pH 3.5, and by chromatography in the second dimension using pyridine:l-butano1:water:acetic acid (10:15:12:3, v/v). 0, origin. Left, positive; right, negative. Phosphopeptides are numbered from 1-17, and corresponding phosphopeptides have the same number in each panel. Panel I , peptide maps of CF-2 and CFTR phosphorylated by PKA. a, CF-2 lower band. b, CF-2 middle band. c, CF-2 upper band. d, CFTR. Panels b and d have been used in a modified form to verify the specificity of CFTR antibody a-1468 (Cohn et al., 1992). Panel I I , peptide maps of CF-2 and CFTR phosphorylated by PKC. a, CF-2. b, CFTR. Panel I I I , peptide maps of CF-2 phosphorylated with CaM kinase I or PKG. a, CaM kinase I. b, PKG.

not possible to sequence enough residues from the peptide spanning amino acids 747-804 to determine the site at which S-EC was incorporated; however, we were able to identify this phosphorylation site as serine 790 by peptide mapping (see below).

Phosphorylation of Synthetic Peptides-To identify the residues phosphorylated in tryptic phosphopeptides in the two- dimensional phosphopeptide maps of CF-2, peptides were synthesized containing phosphorylation consensus sites. Pep- tides that contained consensus sites flanked by basic residues were phosphorylated under the same conditions used to phosphorylate CF-2. Peptides were then digested with trypsin, and phosphopeptide mapping was performed. Mixing experiments were performed with the peptide and CF-2 digested with trypsin. Using this method, we determined, for example, that peptides 2 and 4 contained serine 700 (Fig. 3) and that peptide 3 contained serine 660 (data not shown). This latter peptide was not highly phosphorylated in peptide maps of CF-2 phosphorylated with PKA (Fig. 2, Panel I ) and must represent less than 5% of the phosphate incorporated into CF-2 in the sequencing experiment. There were six prominent peptides in the tryptic maps of CF-2 phosphorylated by PKA, and two were unequivocally identified (2 and 4). Peptides 1, 7, 8, and 9 each contained greater than 5% of total radioactivity, and the relative intensities of all four peptides appeared to be independent of one another (Fig. 2, Panel Z, a, 6, and c ) . Therefore, this implies that these peptides contained the other sequenced serines (737, 768, 795, and 813). We were unable to identify peptides 5 and 6 as they were not highly phosphorylated. In similar mixing experiments of CF-2 phosphorylated with PKC (data not shown), peptides 11 and 12 were found to contain serine 790. This implies that peptide 16, the remaining prominent peptide, contained serine 686.

To assess the phosphorylation of each of the multiple sites in CF-2 independently, the kinetics of phosphorylation of synthetic peptides by PKA, PKC, or PKG were compared


TABLE I Sequences of phosphoylated peptides purified from a V8 digest of CF-2 (645-836)

CF-2 was labeled with PKA or PKC for 20 min as described under “Experimental Procedures.” The labeled protein was digested with V8 protease, and the peptides were separated by reversed phase HPLC. Phosphorylated serines were derivatized with ethanethiol. All peaks containing 5% or more of the total radioactivity were sequenced by Edman degradation. Column 1, sites of V8 cleavage. Numbers refer to residue positions in CFTR. Column 2, sequence of peptides. Phosphorylation consensus sites are bold and underlined. Column 3, residues for which sequence was obtained. Column 4, position of residue that was derivatized as S-ethylcysteine (S-EC).

Residues Sequence Residues S-EC detected sequenced residue

PKA phosphorylation 693-725 FGEKRKNSILNPIN 693-716 700

SIEIVQKTPLQhlNGIE 727-743 DSDEPLERRRRLVPDSE 727-743 737 747-804 AILPRISVISTGPTLQARRRQSVLNLMTH 747-761 ND“

SVNQGQNIHRKTTASm>LAPQANLTE 805-815 LDIYSEsQE 805-815 813

PKC phosphorylation 682-691 TKKQSFKQTE 682-691 686 747-804 AILPRISVISTGPTLQARRRQSVLNLMTH 747-777 ND *

SVNQGQNIHRKTTASTRKVSLAPQANLTE Ser-768 or Ser-795 or both are phosphorylated. Ser-790 was not sequenced but was determined unequivocally to be phosphorylated from peptide mapping.

(Table 11). The K , and V,,, values were determined for the peptides with each kinase. The kcat was calculated from V,,,, and kc.,/Km was used as an index of the effectiveness of each peptide as a substrate. The values we have obtained for kat and K, compare favorably, for example, with values obtained using synthetic peptides based on the sequence for DARPP- 32, a very good substrate for PKA, PKC, and PKG ((Hem- mings et al., 1990) and data not shown). The peptide containing serine 768 (residues 761-770) was the best substrate for PKA, and all peptides corresponding to sites phosphorylated in CF-2 in vitro were very good substrates for the kinase. A peptide containing serine 422 of CFTR (residues 409-429) was also tested with PKA. This peptide had a consensus site for phosphorylation by PKA, but was a poor substrate for the kinase (data not shown). Therefore, not every consensus site was a good substrate for PKA. Using PKC, the peptide containing serine 790 (residues 783-797) was the best substrate tested, and the peptides containing serines 700 and 712 (residues 693-716), as well as the peptide containing serine 768 (residues 761-770), were moderately good substrates. Unlike PKA, however, PKC phosphorylated some peptides poorly. Using PKG, the peptide containing serine 768 (residues 761-770) was the best substrate. The other peptides were phosphorylated with similar kinetics.

Phosphorylation of CFTR in Vitro-CF-2 was a very good substrate for PKA and PKC; however, it was necessary to confirm that CFTR was phosphorylated on the same sites as CF-2. Previous studies had shown that CFTR immunoprecipitated from T84 cells could be phosphorylated by exogenous PKA (Gregory et al., 1990; Cheng et al., 1991b; Cohn et al., 1992). Using antibodies raised against synthetic peptides, CFTR was immunoprecipitated from T84 cells and phosphorylated with PKA as described (Cohn et al., 1992) (data not shown). Two-dimensional peptide mapping showed that the number and position of peptides in the map of the middle band of CF-2 and the map of CFTR were very similar (Fig. 2, Panel I ) . Mixing experiments indicated that all the peptides except 7 co-localized. This demonstrated that all the major sites of phosphorylation by PKA in CFTR were contained within CF-2. Peptide mapping of CFTR phosphorylated in vitro by PKG indicated that the sites phosphorylated in CF- 2 by this kinase were also phosphorylated in CFTR (data not shown). PKC was also used to phosphorylate immunoprecipitated CFTR. A tryptic map of CFTR contained the major

peptides present in tryptic maps of CF-2 phosphorylated by PKC but also contained peptides 10, 14 and 15 (Fig. 2, Panel 11). Phosphoamino acid analysis indicated that CFTR was labeled only on seryl residues by PKA, PKG, and PKC (data not shown).

Phosphorylation of CFTR in T84 Cells-To determine whether CFTR could be phosphorylated in intact cells and that the sites phosphorylated in vitro were also phosphorylated in vivo, CFTR was immunoprecipitated from T84 cells prelabeled with 32Pi. Incubation of T84 cells with forskolin (an activator of PKA) or PDBu (an activator of PKC) increased the amount of 32P incorporated into CFTR compared to control (Fig. 4A). Forskolin caused a 3.7-fold increase, and PDBu caused a 2.3-fold increase in the phosphorylation of CFTR (Fig. 4B). Phosphoamino acid analysis indicated that CFTR was phosphorylated only on serines under all conditions analyzed (data not shown).

Peptide mapping of CFTR phosphorylated in T84 cells indicated that a subset of the sites labeled in vitro by PKA and PKC were phosphorylated in vivo (Fig. 5). When T84 cells were stimulated with forskolin, peptide 1, peptides 2 and 4 (serine 700), peptide 3 (serine 660), peptide 8, and peptide 9 were phosphorylated in CFTR. Forskolin also stimulated the phosphorylation of one peptide that we have not identified in vitro. PDBu stimulated phosphorylation of peptide 1, peptide 2 (serine 700), and peptide 16 (serine 686) in CFTR. One peptide, c, that was not observed in the in vitro experiments was present in phosphopeptide maps of CFTR from unstim- ulated T84 cells and was not affected by the various treatments. Two different sets of sites are phosphorylated in CFTR in response to activation of PKA and PKC, respectively, This confirms that the increases in phosphorylation indicated in Fig. 4 were specifically due to phosphorylation of CFTR by PKA or PKC.

We have also examined the phosphorylation state of CFTR in vivo using an antiserum which recognized the dephospho- form of CFTR. Analysis of antiserum CC23, which was raised against a synthetic peptide containing serine 700, indicated that early bleeds were dephospho-specific. The specificity of CC23 was demonstrated by immunoblotting of CF-2 phosphorylated by PKA to varying stoichiometries (Fig. 6A). The antiserum preferentially recognized the lower band of CF-2, which contained nonphosphorylated CF-2, and its affinity for the protein decreased with longer times of incubation. Thus,

12748 Phosphorylation of CFTR

Mix

FIG. 3. Identification of the phosphorylated residue in phosphopeptides 2 and 4. CF-2 and a synthetic peptide containing residues 693-716 of CFTR were phosphorylated with PKA under standard assay conditions (see "Experimental Procedures"). CF-2 was run on an 11.5% polyacrylamide gel, excised, and washed as described. The peptide was separated from unincorporated ["'PIATP 1)y chromatography over a minicolumn of Dowex AGl-X8 (acetate form) and lyophilized to dryness. Both samples were digested overnight with 50 pg/ml TPCK trypsin. Tryptic digests were separated as described in Fig 2. 0, origin. Top panel, tryptic digest of CF-2. Middle panel, tryptic digest of the phosphorylated peptide containing residues 693-716 of CFTR alone. Bottom panel, tryptic digests of CF- 2 and the peptide combined and run together.

CC23 responded to the phosphorylation state of several sites and not just serine 700. When CC23 was used to blot CFTR in extracts from T84 cells incubated with forskolin or PMA, there was a decrease in antibody binding to CFTR compared to control conditions (Fig. 6B). To ensure that there were no differences in the total amount of CFTR present in each lane, duplicate blots were incubated with a later bleed of CC23 that did not distinguish between the phosphorylated and dephos- phorylated forms of the protein. No difference was seen after the various treatments using the control antibody (data not

shown). In contrast to the '"P-labeling experiments which indicate relative changes in the phosphorylation of CFTR, the increased phosphorylation of CFTR measured using the dephospho-specific antibodies indicates that a substantial proportion of the protein is being phosphorylated in response to activation of PKA and PKC.

DISCUSSION

We have examined the phosphorylation of CFTR by several protein kinases i n vitro and in vivo. CF-2, a portion of the R- domain produced in Escherich.ia coli was an excellent model for the phosphorylation of intact CFTR, and, based on the data obtained using CF-2, the sites phosphorylated in CFTR in response to activation of PKA and PKC i n vivo were identified. These data imply that regulation of chloride flux in the apical membrane of epithelial cells is likely to occur through direct phosphorylation of the R-domain of CFTR by protein kinases stimulated by several different second messenger pathways.

Amino acid sequencing and peptide mapping showed that CF-2 was phosphorylated by PKA i n vitro on serines 660, 700, 737, 813 and most likely on both serines 768 and 795. Kinetic analysis of these sites individually using synthetic peptides indicated that each of the serines phosphorylated i n vitro in CF-2 were very good substrates for phosphorylation by PKA. The phosphorylation of CFTR by PKA i n vitro is very similar to that of CF-2, based on the similarity of peptide maps of CF-2 and CFTR, indicating that CF-2 can be used as a model for CFTR phosphorylation.

PKC phosphorylated serines 686 and 790 in CF-2. The phosphopeptide map of CFTR phosphorylated in vitro with PKC contained more phosphorylated peptides than that of CF-2. This could be due to sites that lie outside the domain included in CF-2. For example, there are consensus sites for phosphorylation by PKC in the carboxyl-terminal region of CFTR (Riordan et al., 1989; Marshall et al., 1991). These sites do not seem to be phosphorylated i n vivo, however (see below). Analysis of phosphorylation of synthetic peptides indicated that a peptide containing serine 790 was the only very good substrate for PKC; however, a peptide containing serine 686 was not tested. Other synthetic peptides that were good substrates for PKA, for example the peptide containing serine 700, were also phosphorylated by PKC, although a t lower rates. Consistent with these data, CFTR was phosphorylated to a low level by PKC i n vitro and i n vivo on sites, including serine 700, that were also phosphorylated by PKA.

Recognition of a phosphorylation site by PKC is influenced by basic residues on the amino-terminal side or the carboxyl- terminal side of the phosphorylated residue, and better substrates have basic residues on both sides of the phosphorylated residue (Kennelly and Krebs, 1991). The consensus site sur- rounding residue 686, KKQSFK, conforms to the pattern found in better substrates for PKC. In addition, the sequence SFK is found in three phosphorylation sites in the MARCKS protein (Seykora et al., 1991). Interestingly, the sites in the MARCKS protein are the best substrates for PKC yet studied (Graff et al., 1991), suggesting that the SFK motif may be significant in influencing the substrate specificity of PKC.

One interesting observation was that -0.5 mM free calcium inhibited the phosphorylation of CF-2 by PKC. Other substrates for PKC, including DARPP-32' and protamine (Bazzi and Nelsestuen, 1987), have been found to be phosphorylated in the absence of calcium. CFTR is unusual, however, in that its phosphorylation by PKC is inhibited by calcium. This

' J.-A. Girault, A. C. Nairn, and P. Greengard, unpuhlished results.


TABLE I1 Kinetics of phosphoplation of CFII'R peptides b?) PKA and PKC

Synthetic peptides were made which contained consensus sites for phosphorylation. The kinetics of phosphor.vlation of these peptides was determined for PKA, PKC, and PKG. K,, and V,,,,, were determined from Lineweaver-Rurk plots, and h,,, was calculated from V,,,,,. Column 1, position of peptide within CFTR. Column 2, sequence of peptide. Consensus sites for phosphorylation are bold and underlined. Column 3, kinetics of PKA phosphorylation. Column 4, kinetics of PKC phosphorylation. Column 5, kinetics of PKG phosphorylation.

Residues I'KA

Peptide sequence PKC PKG

K,,, k,,., k d K n f K,,, kt .,,, k,,,,/K,, K,, k,,,, k , J K n S p M s-' p M 5" p M s"

653-668 FSAEBILTETLHR 19 21.0 1.11 50 1.0 0.020 100 2.5 0.025 693-716 FGEKRKNSILNPINSIRKFSIVQK 30 17.0 0.57 40 1.5 0.038 125 6.0 0.048 782-749 LERRLSI.VPDSEQGI<AII. 48 12.0 0.25 45 0.3 0.007 50 2.7 0.0.54 761-770 LQARRRQsVL 8 14.0 1.75 40 2.3 0.058 14 2.9 0.21 783-797 IHRKTTASTRKVSLA 5 0 I .2 0.15 15 2.3 0.153 55 6.0 0.11 807-818 IYSRRLSQETGL 40 15.0 0.38 0 0 0.0 83 3.5 0.043

".

A. T04 - IP B.

C F Ph ," " ~ ~ ~~

F Ph - 1

M W 400 , i

FIG. 4. In vivo phosphorylation of CFTR. A, T84 cells were incuhated with inorganic phosphate overnight to label endogenous ATP pools. Cells were then incuhated with forskolin or PDRu for 10 min. Cells were lysed and CFTR was precipitated with late hleeds of either CC23 or CC24 that were not dephospho-specific, separated by electrophoresis on a 5 5 s SDS-polyacrylamide gel, dried, and exposed t o x-ray film. CFTR is observed as a broad band a t 170 kDa. H , quantitation of several separate experiments examining the effects of 50 p~ forskolin (5-15 min of incuhation) and 1 p~ PDRu (5-10 min of incuhation). Data are expressed as percentages of control. The mean control value was 52 f 4 cpm (f S.E., n = 5). The percentage increases were: forskolin, 376 ? 56 ( n = 8) ; PDRu, 230 f 26 ( n = 6). (', control; F, forskolin; Ph, PDRu.

observation may explain why studies using apical membrane patches from epithelial cells indicated that PKC could open chloride channels in the absence of calcium but not in the presence of high calcium (Li et al., 1989). This may also explain the small effects obtained by different groups examining phosphorylation of CFTR by PKC in cells transfected with CFTR (Tabcharani et al., 1991).

PKG and CaM kinase I also phosphorylated CF-2. This

raises the possibility that CFTR is regulated by second messenger pathways that use cyclic GMP or calcium to transduce extracellular signals. Phosphopeptide maps of CF-2 phosphorylated by PKG are almost identical with those of CF-2 phosphorylated by PKA. It is likely, therefore, that PKG is phosphorylating the same sites as PKA in CF-2 and could have similar effects on regulation of the channel under the correct conditions. A low conductance chloride channel that may be CFTR, based on its conductance properties, is stimulated by cGMP in T84 cells (Lin et al., 1992), and this regulation may be mediated by phosphorylation of CFTR by PKG. Calcium has been reported to affect chloride flux in epithelial cells (Boucher et al., 1989; Schoumacher et al., 1990), and it has been proposed that this regulation might be mediated by direct phosphorylation of CFTR by CaM kinase I1 (Wagner et al., 1991). We have found that the R-domain of CFTR is not phosphorylated by CaM kinase I1 in vitro, although CFTR may be phosphorylated by this enzyme on some other site. In addition, it is likely that CaM kinase I1 activates a chloride channel other than CFTR; for example, the calcium-dependent outwardly-rectifying chloride channel (Worrell and Frizzell, 1991). The R-domain of CFTR is phosphorylated efficiently by CaM kinase I, however. Therefore, it is feasible that CaM kinase I may play a role in the regulation of CFTR function by calcium.

CFTR was phosphorylated in T84 cells in response to treatment with forskolin, which activates PKA, or in response to treatment with PDRu which activates PKC. We have determined unequivocally that serines 660 and 700 are phosphorylated in CFTR in response to forskolin treatment. In addition, forskolin stimulated phosphorylation of peptide 1 and peptides 8 and 9. We can deduce from the CF-2 sequenc-

FIG. 5 . Phosphopeptide maps of CFTR phosphorylated in vivo. Radioactive hands containing CFTR from the gel shown in Fig. 4.4 were excised, washed, and digested overnight with 50 pg/ml trypsin. Tr-yptic digests were separated as described in the legend to Fig 2 . Some radioactivity remained at the origin (indicated by an arrow in the lower left of each panel). Peptides are numbered from 1-16, and corresponding spots have the same numbers as used in Fig. 2. c, phosphopeptide ohserved only in CFTR labeled in V ~ O O . Con. control CFTH immunoprecipitated from untreated cells. Forsh, CFTR immunoprecipitated from cells treated for 10 min with forskolin. Phorb, CFTR immunoprecipitated from cells treated for 10 min with PDRu.

12750 Phosphorylation of CFTR A. CF-2 0. T84

I 1' 2 5 1u C F P h

Dephosphwpeciflc~

FIG. 6. Dephospho-specific antibodies. CF-2 was used to test the dephospho-specificity of an early bleed of antibody CC23, and that antiserum was used to detect CFTR in extracts of T84 cells. A, duplicate samples of CF-2 were incubated with PKA under standard conditions with [y3'P]ATP (1) or with cold ATP only (2) for 1, 2, 5, and 10 min. The reactions were stopped, and the samples were electrophoresed using an 11.5% SDS-polyacrylamide gel. 1, the part of the gel containing the radioactive samples was dried and autora- diographed. 2, the part of the gel containing the nonradioactive proteins was transferred to nitrocellulose and blotted with antibody CC23 (1:lOO). Immunoreactive bands were visualized by incubation with ''%protein A and autoradiography. Only the parts of the auto- radiograms containing CF-2 are shown. B, T84 cells were treated for 10 min with 50 p~ forskolin or 10 p~ PMA. Samples were electrophoresed using a 5.5% SDS-polyacrylamide gel and then transferred to nitrocellulose. The filter was blotted with antibody CC23 (l : lOO), and immunoreactive bands were visualized with enhanced chemiluminescence. Only the part of the immunoblot containing CFTR (170 kDa) is shown. Densitometry was used to quantitate the amount of immunoreactive CFTR. In six separate experiments, the average decrease in response to forskolin was 35% and in response to PDBu was 25%. Due to the nonlinearity of the enhanced chemiluminescence method, these results are semiquantitative.

ing data that these three peptides can only contain serines 737,768,795, or 813, implying that one of these serines is not phosphorylated in vivo in response to forskolin treatment. Stimulation with forskolin also resulted in phosphorylation of one peptide that was not observed when CFTR was phosphorylated by PKA in vitro. There are several possible expla- nations for this observation. CFTR could be phosphorylated by PKA in vivo on a seryl residue that is not phosphorylated in vitro, another kinase could be activated in vivo, either directly or indirectly, in response to forskolin, or the new phosphopeptide might be a result of alternative cleavage of an identified site. We cannot distinguish between these possibilities at this time.

When T84 cells were treated with PDBu, an activator of PKC, peptide 1 and serine 686 were phosphorylated in CFTR. Stimulation of PKC in vivo caused phosphorylation of some sites, for example peptide 1, that were also phosphorylated by PKA in vitro. All the sites phosphorylated in CFTR by stimulation of PKC in vivo appeared in phosphopeptide maps of CF-2 phosphorylated in vitro; therefore, sites outside the R-domain do not appear to be phosphorylated by PKC in vivo. Treatment of T84 cells with forskolin or phorbol ester induced a 35% or a 25% decrease, respectively, in the binding of a dephospho-specific antibody to immunoprecipitated CFTR. These data imply that a significant proportion of the CFTR present in T84 cells is phosphorylated in response to activation of PKA or PKC.

The results we have obtained concerning the phosphorylation of CFTR by PKA in vitro agree well with the data presented in the recent paper by Cheng and co-workers (Cheng et al., 1991b), in which site-directed mutagenesis was used to change seryl residues in the R-domain of CFTR to alanyl residues. Their study showed that when mutant proteins were phosphorylated with PKA, specific phosphopeptides were absent in two-dimensional phosphopeptide maps. However, their study did not include direct amino acid se-

quencing, an analysis of the relative rates of phosphorylation of the various sites, or examination of the phosphorylation of CFTR by kinases other than PKA. Cheng and co-workers also determined using phosphopeptide mapping that serines 660, 737, 795, and 813 were phosphorylated in response to forskolin in experiments using cells transfected with CFTR and that changing all these serines to alanine resulted in unregulated chloride flux (Cheng et al., 1991b). They did not identify serine 700 in these studies; however, several peptides whose phosphorylation was stimulated by forskolin were un- assigned in their phosphopeptide map. Since we have used different conditions for our two-dimensional peptide mapping, it is impossible to compare our data directly with that of Cheng and co-workers. It is possible that phosphorylation of serine 700 might have functional significance in vivo.

CFTR appears to be an apical membrane chloride channel which is regulated by phosphorylation (Tabcharani et al., 1991; Anderson et al., 1991c, 1991b). We have shown that CFTR is phosphorylated directly by PKA and PKC in vivo. In CF-affected cells, neither PKA nor PKC can activate chloride conductance. One explanation that has been proposed for this lack of regulation is that mutations lead to a defect in processing and transport of CFTR (Cheng et al., 1991a). This might lead to inaccessibility of CFTR to kinases or might prevent CFTR from reaching the apical membrane. Another possibility is that mutations cause a gross conformational change in CFTR (Thomas et al., 1991). A change in conformation might result in an unphosphorylatable protein. This does not seem to be the case as the A508 mutation of CFTR, the most common defect in patients with cystic fibrosis, as well as several other mutations of CFTR, can be phosphorylated by PKA (Riordan et al., 1989; Cheng et al., 1991a; Gregory et al., 1991). However, peptide mapping and kinetic analysis of the phosphorylation of the mutant proteins have not yet been performed. Another possibility is that, although CFTR is phosphorylated in CF-affected cells, mutations in the protein disrupt the ability of phosphorylation to open the chloride channel. In support of this hypothesis, it has been reported that phosphorylation by PKA is necessary to allow ATP hydrolysis by CFTR and that ATP hydrolysis is necessary for channel opening (Anderson et al., 1991a).

Our data and that of Cheng and co-workers (Cheng et al., 1991b) show that all the serines phosphorylated in CFTR lie within a regulatory region called the R-domain (Riordan et al., 1989). The R-domain is an inhibitory domain of CFTR, and phosphorylation of the R-domain removes this inhibition, allowing activation of chloride flux. Mutations in CFTR that remove most of the R-domain result in a constitutively active chloride channel (Rich et al., 1991). We have shown that phosphorylation by PKA can cause a shift in the mobility of the R-domain peptide CF-2 on SDS-PAGE. The serines that cause the mobility shift in CF-2, phosphopeptides 8 and 9, are phosphorylated in CFTR in vivo in response to forskolin treatment. In addition, the binding to CFTR of dephospho- specific antibodies raised against one consensus site changed in response to the phosphorylation of CFTR in vivo on several different sites. These observations imply that phosphorylation results in a conformational change in the R-domain, consistent with a role for the R-domain in the regulation of CFTR function.

We have shown that there are up to six different sites phosphorylated in the R-domain in response to activation of PKA or PKC in vivo. All the seryl residues phosphorylated in CFTR in vivo are phosphorylated rapidly in CF-2. In addition, kinetic data using synthetic peptides have shown that all the seryl residues phosphorylated by PKA are very good sub-


strates for the enzyme. Furthermore, it is necessary to mutate several seryl residues to alanyl residues in CFTR to eliminate the effect of PKA on chloride flux (Cheng et al., 1991b). This indicates that there is a great deal of redundancy in the regulation of CFTR by phosphorylation. It is unusual to have so many phosphorylatable residues clustered in one domain. It is possible that inactivation of chloride flux may involve differential dephosphorylation of these residues by one or more protein phosphatases.

The model for regulation of CFTR by PKA described above could also be extended to regulation of CFTR by PKC. PKC has been shown to increase chloride flux in apical membrane patches from epithelial cells, and this regulation is absent in CF-affected cells (Li et al., 1989). In cells transformed with CFTR, small increases in chloride flux have been observed in isolated membrane patches in the presence of PKC (Tab- charani et al., 1991). Finally, PKC phosphorylated CFTR in uiuo on one unique residue as well as some of the same residues as PKA. The effects of PKC on chloride conductance are complex, however. Phosphorylation of CF-2 by PKC is inhibited by calcium, and this may explain why it has been difficult t o show consistent effects of PKC on activation of CFTR. It has also been reported that PKC can potentiate the effect of PKA on CFTR (Tabcharani et al., 1991). Phosphorylation of serines 686 or 790 by PKC may facilitate the phosphorylation of the other PKA sites. PKC may also be able to potentiate the phosphorylation of CFTR by other kinases such as PKG or CaM kinase I. CF-2 can be used as a substrate in further experiments to examine these possibilities.

In conclusion, we have shown that regulation of CFTR, a putative chloride channel, is likely to occur through direct phosphorylation of the R-domain by protein kinases stimulated by several different second messenger pathways. Phos- phorylation events regulate the opening and closing of several other ion channels including the nicotinic acetylcholine recep- tor (Hopfield et al., 1988), the dihydropyridine-sensitive calcium channel (Chang et al., 1991), the voltage-dependent sodium channel (Numann et al., 1991), and the Shaker potassium channel (Moran et al., 1991). All the channels mentioned above are large, hydrophobic, integral membrane proteins. These proteins are extremely difficult to study using tradi- tional biochemical techniques and the approach used here, synthesis of a soluble regulatory domain, is one method that could be used to study their regulation by second messengers.

Acknowledgments-We wish to thank Dr. Andrew Czernik for help with HPLC and Dr. Kiertisen Dharmsathophorn and Dr. Michael Welsh for T84 cells. Peptide synthesis and peptide sequencing were performed by the Rockefeller University protein sequencing facility.

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