PKA holoenzyme is functionally coupled to CFTR by AKAPs

P. Huang1, K. Trotter2, R. C. Boucher1, S. L. Milgram2, and M. J. Stutts1

1 Departments of Medicine and CF/Pulmonary Research and Treatment Center and 2 Cell and Molecular Physiology, University of North Carolina, Chapel Hill, North Carolina 27599


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cystic fibrosis transmembrane regulator (CFTR) is reported to be preferentially regulated by membrane-bound protein kinase A (PKAII). We tested for close physical and functional association of PKA with CFTR in inside-out membrane patches excised from Calu-3 cells. In the presence of MgATP, 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) increased the product of CFTR channel number and open probability (from 0.36 ± 0.12 to 1.23 ± 0.57, n = 20, P < 0.0025), and this stimulation was abolished by PKI. Thus Calu-3 membrane isolated from cells retains PKA holoenzyme that is functionally coupled to CFTR. PKAII is anchored at specific subcellular sites by A kinase anchoring proteins (AKAPs). Exposure of excised patches to HT-31, a peptide that disrupts the association of PKAII and AKAPs, prevented CPT-cAMP stimulation of CFTR. Therefore, PKA holoenzyme in isolated membrane patches is bound to AKAPs. In whole cell voltage-clamp studies, intracellular dialysis of Calu-3 cells with HT-31 blocked the activation of CFTR by extracellular adenosine. These results suggest that AKAPs mediate PKA compartmentalization with CFTR and are required for activation of CFTR by physiological regulators.

cystic fibrosis transmembrane regulator; protein kinase A; adenosine 3',5'-cyclic monophosphate; adenosine; compartmentalization; A kinase anchoring proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

THE CYSTIC FIBROSIS TRANSMEMBRANE REGULATOR (CFTR) Cl- channel is regulated by phosphorylation/dephosphorylation of multiple conserved serines and requires ATP binding and hydrolysis at two intracellular nucleotide binding folds (17). Although multiple kinases and phosphatases contribute to this complex regulation of CFTR, cAMP-dependent protein kinase (PKA) is the dominant kinase activator of CFTR (24, 21, 34). The signal transduction elements utilized for cAMP-mediated regulation of endogenously expressed CFTR through PKA have not been fully characterized. Two recent studies found that CFTR mediated Cl- secretion in Calu-3 and T84 cells is preferentially activated by the membrane-associated isoform of PKA, PKAII (30, 31). This observation raised the possibility that physiologic regulation of Cl- conductance is mediated by PKA that is colocalized with CFTR. A principal mechanism for membrane localization of PKA is high-affinity binding between a region of the PKAII regulatory subunit (RII) and A kinase anchoring proteins (AKAPs) (10). AKAP-localized PKAII regulates several ion channels, including voltage-gated Na+ channels (27, 33), L-type Ca2+ channels (18), Ca2+-dependent K+ channels (13), and renal outer medulla K+ 1 channels (1).

We used CFTR's dependence on PKA activity and patch-clamp techniques to develop an assay that detects PKA holoenzyme in excised inside-out membrane patches. Using this assay, we tested the role of AKAPs in the functional association of PKA with isolated membranes. Moreover, we tested the role of anchored PKA in the activation of CFTR by the adenosine receptor coupled signaling pathway. Our results show that physiological regulation of CFTR by cAMP requires PKA that is functionally coupled to CFTR via an AKAP interaction.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cells. Human Calu-3 cells (ATCC HTT-55) were grown in MEM (GIBCO BRL) supplemented with 10% fetal bovine serum, 10% sodium pyruvate, 10% MEM non-essential amino acid solution (NEAA), and penicillin/streptomycin in an atmosphere of 95% air-5% CO2 at 37°C. Cells were passaged by 1:3 dilution every 5-7 days. For patch-clamp and whole cell voltage-clamp studies, 20,000 cells were seeded onto the center of plastic dishes (35 mm diameter, not coated) and used for experiments after 1-5 days.

Immunoblot analyses and RII overlays. Calu-3 cells were grown to confluence on Transwell filters before experiments were performed. Whole cell lysates prepared from the monolayer cultures were separated into soluble and particulate fractions by centrifugation at 436,000 g for 15 min at 4°C, and protein concentrations were determined using the bicinchoninic acid protein assay kit (Pierce; Rockford, IL). Equal amounts of soluble and particulate fractions were resolved on SDS-PAGE gels, transferred to Immobilon-P (Millipore), and analyzed by Western blot analysis as described (25). Rabbit anti-human PKA RIalpha , RIIalpha , and catalytic subunits were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). In control experiments, preincubation of antisera with blocking peptides specific for RI, RII, or the catalytic subunit abolished the signals observed on Western blots (not shown).

Plasmid pET11d.RIIalpha , encoding full-length mouse RIIalpha , was kindly provided by Dr. John Scott (Vollum Institute). Protein expression was induced in Escherichia coli BL21 (Novagen), and RIIalpha present in the soluble fraction was purified on cAMP agarose affinity columns as described (19). The purified protein was biotinylated using N-hydroxysuccinimide-LC-biotin (NHS-LC-biotin) according to the manufacturer's instructions (Pierce). Twenty micrograms of soluble and particulate fractions were electrophoresed on SDS-PAGE and transferred to nitrocellulose membranes. Nonspecific sites were blocked with 5% nonfat dry milk in Tris-buffered saline containing 0.05% Tween 20 (TBST) for 60 min at room temperature. Blocked membranes were washed three times for 15 min with TBST and incubated 14 h at 4°C with 15 nM RII-biotin with or without a 2 h preincubation with 4 µM HT-31 or 4 µM HT-31P peptides in TBST at room temperature. Membranes were then washed three times for 15 min with TBST and incubated with streptavidin-conjugated horseradish peroxidase (0.5 µg/ml) in TBST. Membranes were washed three times for 15 min with TBST and developed using enhanced chemiluminescence.

Single channel studies. Pipettes were pulled from glass capillaries (GC150TF-15, Clark Electromedical Instruments) and filled with 160 mM Tris · Cl and 30 mM sucrose (pH 7.0 adjusted with Tris). The ionic composition of the bath buffer was the same. Under these conditions, the resistance of an open pipette was about 4 MOmega . Slight negative pressure applied to the interior of a pipette touching the cell membrane resulted in high-resistance seals, typically in the range of 20-30 GOmega . CFTR Cl- channel activity was recorded at ± 40 and 60 mV pipette potential as current jumps with typical burstlike openings and flickery kinetics in the positive voltage range (15, 16, 20). Under these conditions, the slope conductance of CFTR in excised patches was ~7.0 pS.

To assay for PKA holoenzyme activity, we first identified CFTR Cl- channels in attached membrane patches. Patches were excised and, 5 min later, 100 µM 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) was added to the bath. Bath solution contained 1 mM MgATP and 1-2 nM human recombinant protein kinase C beta -1 (PKCbeta -1; Calbiochem), activated as follows. In 10 µl of bath buffer, PKC, phosphatidylserine (Avanti), 1,2-dioleoyl-sn-glycerol (Avanti), and CaCl2 were added to final concentration of 100 or 200 nM, 40 µg/ml, 12 µg/ml, and 0.5 mM, respectively. The mixture was incubated 5 min at 22°C and added to 1 ml of the patch bath solution. As indicated, PKA inhibitor peptide (10 µM PKI, BioMol P-204), AKAP disrupting peptide HT-31, or control peptide HT-31P (10 µM, Univ. of North Carolina Custom Peptide Synthesis Facility) were added to the bath solution before excision. Peptides were prepared as 10 mM stock solutions in DMSO.

Whole cell voltage-clamp studies. The pipette solution (32) for intracellular dialysis was 40 mM Tris · Cl; 100 mM potassium gluconate, 2 mM MgCl2; 5 mM TES; 1 mM EGTA; and 0.1 mM CaCl2, 1 mM MgATP, 0.1 mM Na2GTP (pH 7.4). Ca2+ activity was buffered to ~40 nM (1.0 mM EGTA and 0.1 mM CaCl2). The routine bath solution contained 150 mM Tris · Cl; 2 mM MgCl2; 1 mM CaCl2; 5 mM TES; 30 mM sucrose; and 10 mM D-glucose (pH 7.4 with Tris). Patch pipettes had a tip resistance of 2-3 MOmega with these solutions. CFTR-mediated whole cell conductance was studied as previously described (32). In the standard protocol, voltage was clamped to -40 mV and stepped to 80 mV every 10 s as current was recorded. Current voltage plots were obtained every 2 min from the current recorded in response to a series of voltage steps from -40 mV, covering the range from -100 to 100 mV in 20 mV increments. The whole cell conductance was calculated by the slope of a linear fit of the current-voltage relationship points from -100 mV and -40 mV. All membrane potential (Vm) shown are clamp voltages corrected for the measured junction potential of pipette and bath solution (-5 mV).

Data acquisition and analysis. All hardware and software were from Axon Instruments. Single channel currents were filtered at 100 Hz, sampled at 1 kHz, and stored on the hard disk of a personal computer. Data were analyzed using Fetchan 6.0 and pStat 6.0. The product of CFTR channel number and open probability (NPo) was calculated for 90 s intervals, where N is the maximum number of channels observed, and Po is the single channel open probability (7). Whole cell voltage was clamped with Clampex 7.0. Whole cell currents were acquired at 500 Hz, filtered at 100 Hz, and analyzed with Clampfit 6.0. All the data were expressed as means ± SE. The Wilcoxon matched-pair sign-rank test and the Student's t-test were used for statistical analysis.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To directly test the hypothesis that membrane-associated PKA holoenzyme can regulate CFTR, we isolated CFTR-containing Calu-3 cell membranes by patch excision and applied maneuvers designed to selectively stimulate PKA activity. In the initial series of experiments, cell-attached patches containing active CFTR were excised into bath solution that contained MgATP, and were allowed to run down for 5 min. Application of the cAMP analog CPT-cAMP (100 µM) to the bath activated CFTR channels in 5 of 18 excised patches (~28%, not shown). From this observation, we tentatively concluded that PKA holoenzyme was associated with at least some excised membrane patches. To study this apparent association in more detail, we refined the conditions of the assay by including activated PKC in the bath, which was shown by Jia et al. to be a condition that permitted efficient activation of CFTR in excised patches by exogenous PKA catalytic subunit (21).

Accordingly, we next tested excised Calu-3 patches for PKA holoenzyme by excising CFTR containing patches into a bath that contained 1 mM MgATP and 2 nM activated PKC. In these experiments, we also compared the effects of CPT-cAMP on CFTR in the absence and presence of the specific PKA inhibitor peptide (PKI, 10 µM). After rundown for 5 min in patches incubated without PKI, 100 µM CPT-cAMP stimulated CFTR in 13 of 20 patches tested (~65%). The mean NPo for all patches in this series increased from 0.36 ± 0.12 before to 1.23 ± 0.57 (n = 20, P < 0.0025, sign-rank test) after the addition of 100 µM CPT-cAMP (Fig. 1, A and C). In contrast, when 10 µM PKI was continuously present in the bath, CPT-cAMP had no effect on NPo of CFTR (0.19 ± 0.15 before to 0.21 ± 0.15 after, n = 11, P > 0.1, sign-rank test; Fig. 1, B and C). This stimulation of CFTR by CPT-cAMP and its block by PKI shows that CFTR is regulated by PKA activity that remains associated with most membrane patches after excision.


View larger version (32K):
[in this window]
[in a new window]
 
Fig. 1.   Specific PKA inhibitor peptide (PKI) inhibition of 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) activation of cystic fibrosis transmembrane regulator (CFTR) channels in excised inside-out patches. Cell-attached patches with active CFTR were excised into bath solution containing either 1 mM MgATP and 2 nM activated PKC (A) or 1 mM MgATP and 2 nM activated PKC and 10 µM PKI (B). N, number of channels. Pipette voltage was held at -60 mV. A and B, right: all-points amplitude histograms of single channel traces before and after CPT-cAMP. C: product of CFTR channel number and open probability (NPo) was calculated from 90 s of data recorded up to addition of 100 µM CPT-cAMP and for 90 s of current recorded 3 min after addition of CPT-cAMP. * P < 0.0025, sign-rank test.

These observations are consistent with recent reports that CFTR is selectively regulated by PKAII, the isoform of PKA that is typically membrane associated (30, 31). Because PKAII associates with membrane surfaces of subcellular compartments by binding to AKAPs, we next studied the distribution of PKA regulatory subunit isoforms in Calu-3 cell fractions and tested for the presence of AKAPs by RII overlay. PKA RI was detected in the soluble fraction of Calu-3 cells, whereas RII was enriched in the particulate fraction (Fig. 2A). Biotinylated RII subunit bound to ~15 distinct bands present in the Calu-3 cell particulate fraction, as well as to several soluble Calu-3 cell proteins (Fig. 2B, left). The specificity of binding of RII to AKAPs in such overlay assays is typically demonstrated by competition with a synthetic peptide, HT-31, which is modeled on the RII binding domain of human thyroid AKAP (6). RII binding to Calu-3 cell proteins was completely blocked by 4 µM HT-31 peptide (Fig. 2B, right), but not by a control peptide, HT-31P (not shown) (6). Thus Calu-3 cell membranes contained both RII and multiple RII binding proteins, i.e., putative AKAPs.


View larger version (50K):
[in this window]
[in a new window]
 
Fig. 2.   Distribution of RI, RII, and catalytic (CAT) subunit in Calu-3 cells. A: equal amounts of soluble (S) and particulate (P) fractions from Calu-3 cell lysates were electrophoresed on 10% SDS-PAGE and transferred to Immobilon-P membrane. Distributions of RI, RII, or CAT subunit were visualized by immunoblot analysis followed by horseradish peroxidase-conjugated secondary antisera and chemiluminescent detection. B: identification of A kinase anchoring proteins in Calu-3 cells. Equal amounts of soluble (S) and particulate (P) were electrophoresed on 10% SDS-PAGE and transferred to nitrocellulose membranes. Blots were incubated with 10 nM biotin-RIIalpha with or without 4 µM HT-31 peptide, and bound RIIalpha was detected after incubation with SA-HRP and chemiluminescent detection.

We reasoned that the disruption of the RII-AKAP interaction by HT-31 might provide a test of whether or not the PKA holoenzyme activity we detected functionally in excised membrane patches was bound to AKAPs. Accordingly, we tested the effect of 10 µM HT-31 on the activation of CFTR by CTP-cAMP. With 10 µM HT-31 in the bath solution, CPT-cAMP had no effect on NPo (from 0.39 ± 0.14 before to 0.39 ± 0.13 after, n = 29, P > 0.5, sign-rank test; Fig. 3, A and C). In contrast, in the presence of 10 µM HT-31P, a control peptide in which a proline substitution was introduced to decrease its affinity for endogenous RII, CPT-cAMP markedly increased CFTR NPo (from 0.27 ± 0.09 before to 1.48 ± 0.64 after, n = 34, P < 0.002, sign-rank test; Fig. 3, B and C). In previous studies utilizing these peptides, this pattern of results was interpreted as disruption of binding between PKA regulatory subunit and AKAPs (1, 37). Thus PKA holoenzyme functionally coupled to CFTR is specifically associated with isolated membrane patches of Calu-3 cells by binding to one or more AKAPs.


View larger version (28K):
[in this window]
[in a new window]
 
Fig. 3.   HT-31 inhibition of CPT-cAMP activation of CFTR channels in excised inside-out patches. Patches containing CFTR were excised into bath solution containing either 1 mM MgATP, 1 nM activated PKC, and 10 µM HT-31 (A) or 1 mM MgATP, 1 nM activated PKC, and 10 µM HT-31P (B, control). N, number of channels. Pipette voltage was held at -60 mV. C: summary data for effect of CPT-cAMP on CFTR NPo in presence of HT-31 and HT-31P (control). CPT-cAMP-induced change in NPo was calculated as described in Fig. 2. * P < 0.002, sign-rank test.

Adenosine receptors coupled to adenylate cyclase were recently reported to activate CFTR as efficiently as forskolin, but generated only 20% as much increase in total cAMP (8). Because our excised patch data showed AKAPs to mediate both close physical association and functional coupling of PKA and CFTR, we hypothesized that AKAPs could be important in the efficient regulation of CFTR by adenosine receptors. To test this possibility, we compared the effects of HT-31 and HT-31P on CFTR mediated whole cell Cl- currents in Calu-3 cells (Fig. 4). We applied adenosine to the bath at 1 µM, the approximate half-maximal effective dose for adenosine activated Cl- secretion in Calu-3 cells in Ussing chamber studies (not shown). Both basal [105.3 ± 21.4 pA/pF/100 mV; reversal potential (Rp) = 29.6 ± 1.6 mV] and adenosine stimulated (221.5 ± 39.5 pA/pF/100 mV, Rp = 28.0 ± 1.4 mV, n = 15) currents had reversal potentials (ECl) close to -32 mV and were unaffected by the addition of 3 mM DIDS (not shown), consistent with CFTR mediated Cl- current (32). Cells dialyzed with HT-31 (10 µM) had virtually no increase in current in response to adenosine, whereas cells dialyzed with the control peptide, HT-31P (10 µM), had a similar increase in whole cell current in response to adenosine as the control cells. These results implicate anchored PKA holoenzyme in the physiologic regulation of CFTR by showing that disruption of the AKAP-PKA interaction breaks the coupling of adenosine receptors to activation of CFTR.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 4.   Adenosine activation of CFTR-mediated Cl- current in whole cell voltage-clamp studies. A: families of currents measured in response to voltage steps before and after 1 µM adenosine (ADO). Voltages were stepped from a holding potential of -40 mV to a range of voltage from -100 to +100 mV in 20-mV increments. Each clamp voltage was maintained for 850 ms, in which currents in last 300 ms were averaged and used for plotting current-voltage relations. B: plots of current-voltage data displayed in A. Reversal potentials were 27.2 mV before ADO and 27.0 mV after ADO. C: histogram of adenosine-stimulated CFTR currents in whole cells. Slope conductance of whole cell Cl- current was calculated as described in EXPERIMENTAL PROCEDURES. Slope conductance of Cl- current after addition of adenosine was normalized to slope conductance of Cl- current before addition of adenosine, and percentage increase is plotted. Control, n = 15; HT-31P, n = 12; HT-31, n = 13. P values were obtained by t-test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Our studies reveal that PKA holoenzyme is associated with CFTR-containing Calu-3 cell membranes, and that it is tightly coupled to the regulation of CFTR Cl- channel activity. We based our investigation on the premises that patch excision is a way to directly isolate CFTR-containing cell membranes and that CFTR NPo acts as a real time reporter of PKA activity in these excised patches. We speculated that this approach would reliably detect membrane-associated PKA because CFTR NPo is tightly regulated by PKA, and because highly specific and well established maneuvers are available to manipulate PKA activity. Because the principal hallmark of PKA holoenzyme is the activation of catalytic subunit caused by binding of cAMP at its tandem binding sites on each regulatory subunit (3), we first tested the effect on CFTR NPo of adding a cAMP analog to the cytoplasmic face of excised membrane patches. To ensure saturation of cAMP binding sites in excised membrane patches, we used a large excess of CPT-cAMP (100 µM), a cAMP analog that is both lipophilic and resistant to phosphodiesterases (26). The fundamental observation in our study is that exposure of excised membrane patches to CPT-cAMP, in the presence of MgATP, increased CFTR NPo in most excised patches. This observation is primary evidence that PKA holoenzyme remained associated with membrane patches displaced from cells by excision, and was physically close enough to CFTR to affect its gating. Importantly, the CPT-cAMP-induced change in CFTR gating was completely inhibited by PKI, a specific peptide inhibitor of PKA. Inhibition by PKI is a second hallmark of PKA activity (3). Because both CPT-cAMP and PKI are highly selective reagents that exert opposite effects on PKA activity, it is clear that PKA holoenzyme remains in membrane patches after excision, and furthermore, that this PKA is functionally coupled to CFTR.

A second important conclusion from our studies is that after physical isolation of Calu-3 membranes into an essentially infinite bath, the PKA activity in excised patches is associated with AKAPs. Our RII overlay experiments detected ~15 RII binding proteins in the particulate fraction of Calu-3 cells. This assay is based on RII binding to a 20-30 amino acid sequence present in AKAPs that forms an alpha -helical amphipathic domain (9, 10). This AKAP domain binds with high affinity to several isoleucines present in the amino terminus of the Type II isoform of RII (10). The amino terminal sequence of RII is not present in RI, although it has been recently reported that RI can bind AKAPs with lower affinity than RII (5). The high affinity interaction between RII and the alpha -helical domain of AKAPs is disrupted by HT-31, a 24 amino acid synthetic peptide based on the alpha -helical domain first identified in human thyroid AKAP (6). Our observation that HT-31, but not the control peptide HT-31P, prevented activation of CFTR by CPT-cAMP in excised membrane patches indicates that the PKA activity that regulates CFTR in excised membrane patches was bound by a highly specific molecular mechanism. By similar reasoning, this result also constitutes formal evidence that one or more of the AKAPs present in Calu-3 membranes are retained in excised patches and are required for PKA to regulate CFTR.

The third major finding in our study is that the AKAP-PKA interaction must be intact for activation of CFTR by adenosine receptors. It was recently reported that A2 receptors stimulated CFTR-mediated Cl- conductance more efficiently than forskolin (8). A2 receptors generate cAMP by coupling to adenylate cyclase through stimulatory G protein (Gs), but few other specifics of the signal transduction elements linking A2 receptors to activation of CFTR are known (8, 23). We found that the activation of CFTR mediated Cl- current by 1 µM adenosine, a concentration we selected to mimic a physiologic stimulus, was specifically abolished by dialyzing cells with HT-31. This finding suggests minimally that functional coupling of PKA to CFTR by AKAPs is not a phenomenon detected only in excised membrane patches, but is a requisite step in the signal transduction path between adenosine receptors and CFTR in whole cells. Importantly, these whole cell data reveal that functional coupling of PKA to CFTR is important at physiologic levels of cAMP.

The role of membrane-associated PKA for physiologic regulation of CFTR by cAMP suggests that AKAPs may be important in compartmentalization of signal transduction mechanisms in epithelia. There is growing awareness that such compartmentalization, which has been studied for several years in neural specializations, may play a prominent role in how the functions of polarized epithelial cells are regulated (11). We, and others, have reported that CFTR binds, via its conserved COOH terminus, the subapical PDZ scaffold protein, EBP50 (29, 35). Whereas no specific function has been identified for the CFTR-EBP50 interaction, it is attractive to speculate that it could mediate some aspect of CFTR regulation. In fact, EBP50 binds to ezrin, an actin binding protein, which is prominently involved in cytoskeletal reorganization (12). Moreover, ezrin was reported to function as an AKAP (12), in vitro. These separate observations prompt additional speculation that the binding of CFTR to EBP50 represents secondary binding of CFTR to an AKAP, which could be relevant to our present results. It is important to point out, however, that there is so far no evidence that ezrin functions as an AKAP in cells, or if it does, whether it is an AKAP that is important for regulation of CFTR.

Through the identification and characterization of individual apical AKAPs that mediate regulation of CFTR by compartmentalized PKA, it may be possible to learn the molecular identity of additional signaling elements important for the regulation of CFTR. For example, CFTR is coactivated by an unknown isoform of PKC, and is dephosphorylated by one or more protein phosphatases that have not been definitively identified (14, 24). Several AKAPs that have already been characterized have been found to anchor not only PKAII, but other signal transduction elements including PKC and phosphatases (22). Moreover, CFTR appears to affect the function of numerous other ion channels or transporters that can be present in the apical membrane of epithelia (28). Full characterization of an AKAP that anchors signal transduction elements that regulate CFTR could help define molecular or functional linkages between CFTR and other channels or transporters that appear to be coordinately regulated with CFTR.


    ACKNOWLEDGEMENTS

This study was supported by Cystic Fibrosis Foundation Grant CFF9710, and National Heart, Lung, and Blood Institute Grants HL-42384 and HL-533094.


    FOOTNOTES

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: P. Huang, 6021 Thurston Bowles Bldg., Campus Box 7248, Univ. of North Carolina, Chapel Hill, NC 27599-7248 (E-mail: Pingbo_Huang{at}med.unc.edu).

Received 10 August 1999; accepted in final form 27 September 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

1.   Ali, S., X. Chen, M. Lu, J. Z. Xu, K. M. Lerea, S. C. Hebert, and W. H. Wang. The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels. Proc. Natl. Acad. Sci. USA 95: 10274-10278, 1998[Abstract/Free Full Text].

2.   Becq, F., T. J. Jensen, X. B. Chang, A. Savoia, J. M. Rommens, L. C. Tsui, M. Buchwald, J. R. Riordan, and J. W. Hanrahan. Phosphatase inhibitors activate normal and defective CFTR chloride channels. Proc. Natl. Acad. Sci. USA 91: 9160-9164, 1994[Abstract/Free Full Text].

3.   Beebe, S. J. The cAMP-dependent protein kinases and cAMP signal transduction. Semin. Cancer Biol. 5: 285-294, 1994[ISI][Medline].

4.   Berger, H. A., S. M. Travis, and M. J. Welsh. Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by specific protein kinases and protein phosphatases. J. Biol. Chem. 268: 2037-2047, 1993[Abstract/Free Full Text].

5.   Burton, K. A., B. D. Johnson, Z. E. Hausken, R. E. Westenbroek, R. L. Idzerda, T. Scheuer, J. D. Scott, W. A. Catterall, and G. S. McKnight. Type II regulatory subunits are not required for the anchoring-dependent modulation of Ca2+ channel activity by cAMP-dependent protein kinase. Proc. Natl. Acad. Sci. USA 94: 11067-11072, 1997[Abstract/Free Full Text].

6.   Carr, D. W., Z. E. Hausken, I. D. Fraser, R. E. Stofko-Hahn, and J. D. Scott. Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain. J. Biol. Chem. 267: 13376-13382, 1992[Abstract/Free Full Text].

7.   Chalfant, M. L., J. S. Denton, B. K. Berdiev, I. I. Ismailov, D. J. Benos, and B. A. Stanton. Intracellular H+ regulates the alpha -subunit of ENaC, the epithelial Na+ channel. Am. J. Physiol. Cell Physiol. 276: C477-C486, 1999[Abstract/Free Full Text].

8.   Clancy, J. P., F. E. Ruiz, and E. J. Sorscher. Adenosine and its nucleotides activate wild-type and R117H CFTR through an A2B receptor-coupled pathway. Am. J. Physiol. Cell Physiol. 276: C361-C369, 1999[Abstract/Free Full Text].

9.   Colledge, M., and J. D. Scott. AKAPs: from structure to function. Trends Cell Biol. 9: 216-221, 1999[ISI][Medline].

10.   Dell'Acqua, M. L., and J. D. Scott. Protein kinase A anchoring. J. Biol. Chem. 272: 12881-12884, 1997[Free Full Text].

11.   Dransfield, D. T., A. J. Bradford, and J. R. Goldenring. Distribution of A-kinase anchoring proteins in parietal cells. Biochim. Biophys. Acta 1269: 215-220, 1995[ISI][Medline].

12.   Dransfield, D. T., A. J. Bradford, J. Smith, M. Martin, C. Roy, P. H. Mangeat, and J. R. Goldenring. Ezrin is a cyclic AMP-dependent protein kinase anchoring protein. EMBO J. 16: 35-43, 1997[Abstract/Free Full Text].

13.   Esguerra, M., J. Wang, C. D. Foster, J. P. Adelman, R. A. North, and I. B. Levitan. Cloned Ca2+-dependent K+ channel modulated by a functionally associated protein kinase. Nature 369: 563-565, 1994[ISI][Medline].

14.   Fischer, H., B. Illek, and T. E. Machen. Regulation of CFTR by protein phosphatase 2B and protein kinase C. Pflugers Arch. 436: 175-181, 1998[ISI][Medline].

15.   Fischer, H., and T. E. Machen. CFTR displays voltage dependence and two gating modes during stimulation. J. Gen. Physiol. 104: 541-566, 1994[Abstract].

16.   Fischer, H., and T. E. Machen. The tyrosine kinase p60c-src regulates the fast gate of the cystic fibrosis transmembrane conductance regulator chloride channel. Biophys. J. 71: 3073-3082, 1996[Abstract].

17.   Gadsby, D. C., and A. C. Nairn. Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis. Physiol. Rev. 79: S77-S107, 1999[Medline].

18.   Gao, T., A. Yatani, M. L. Dell'Acqua, H. Sako, S. A. Green, N. Dascal, J. D. Scott, and M. M. Hosey. cAMP-dependent regulation of cardiac L-type Ca2+ channels requires membrane targeting of PKA and phosphorylation of channel subunits. Neuron 19: 185-196, 1997[ISI][Medline].

19.   Gray, P. C., V. C. Tibbs, W. A. Catterall, and B. J. Murphy. Identification of a 15-kDa cAMP-dependent protein kinase-anchoring protein associated with skeletal muscle L-type calcium channels. J. Biol. Chem. 272: 6297-6302, 1997[Abstract/Free Full Text].

20.   Haws, C., W. E. Finkbeiner, J. H. Widdicombe, and J. J. Wine. CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance. Am. J. Physiol. Lung Cell. Mol. Physiol. 266: L502-L512, 1994[Abstract/Free Full Text].

21.   Jia, Y., C. J. Mathews, and J. W. Hanrahan. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272: 4978-4984, 1997[Abstract/Free Full Text].

22.   Klauck, T. M., M. C. Faux, K. Labudda, L. K. Langeberg, S. Jaken, and J. D. Scott. Coordination of three signaling enzymes by AKAP79, a mammalian scaffold protein. Science 271: 1589-1592, 1996[Abstract].

23.   Lazarowski, E. R., S. J. Mason, L. Clarke, T. K. Harden, and R. C. Boucher. Adenosine receptors on human airway epithelia and their relationship to chloride secretion. Br. J. Pharmacol. 106: 774-782, 1992[Abstract].

24.   Luo, J., M. D. Pato, J. R. Riordan, and J. W. Hanrahan. Differential regulation of single CFTR channels by PP2C, PP2A, and other phosphatases. Am. J. Physiol. Cell Physiol. 274: C1397-C1410, 1998[Abstract/Free Full Text].

25.   Milgram, S. L., R. C. Johnson, and R. E. Mains. Expression of individual forms of peptidylglycine alpha -amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules. J. Cell Biol. 117: 717-728, 1992[Abstract].

26.   Ogreid, D., R. Ekanger, R. H. Suva, J. P. Miller, P. Sturm, J. D. Corbin, and S. O. Doskeland. Activation of protein kinase isozymes by cyclic nucleotide analogs used singly or in combination. Principles for optimizing the isozyme specificity of analog combinations. Eur. J. Biochem. 150: 219-227, 1985[Abstract].

27.   Rosenmund, C., D. W. Carr, S. E. Bergeson, G. Nilaver, J. D. Scott, and G. L. Westbrook. Anchoring of protein kinase A is required for modulation of AMPA/kainate receptors on hippocampal neurons. Nature 368: 853-856, 1994[ISI][Medline].

28.   Schwiebert, E. M., D. J. Benos, M. E. Egan, M. J. Stutts, and W. B. Guggino. CFTR is a conductance regulator as well as a chloride channel. Physiol. Rev. 79: S145-S166, 1999[Medline].

29.   Short, D. B., K. W. Trotter, D. Reczek, S. M. Kreda, A. Bretscher, R. C. Boucher, M. J. Stutts, and S. L. Milgram. An apical PDZ protein anchors the cystic fibrosis transmembrane conductance regulator to the cytoskeleton. J. Biol. Chem. 273: 19797-19801, 1998[Abstract/Free Full Text].

30.   Singh, A. K., K. Tasken, W. Walker, R. A. Frizzell, S. C. Watkins, R. J. Bridges, and N. A. Bradbury. Characterization of PKA isoforms and kinase-dependent activation of chloride secretion in T84 cells. Am. J. Physiol. Cell Physiol. 275: C562-C570, 1998[Abstract/Free Full Text].

31.   Steagall, W. K., T. J. Kelley, R. J. Marsick, and M. L. Drumm. Type II protein kinase A regulates CFTR in airway, pancreatic, and intestinal cells. Am. J. Physiol. Cell Physiol. 274: C819-C826, 1998[Abstract/Free Full Text].

32.   Stutts, M. J., S. E. Gabriel, J. C. Olsen, J. T. Gatzy, T. L. O'Connell, E. M. Price, and R. C. Boucher. Functional consequences of heterologous expression of the cystic fibrosis transmembrane conductance regulator in fibroblasts. J. Biol. Chem. 268: 20653-20658, 1993[Abstract/Free Full Text].

33.   Tibbs, V. C., P. C. Gray, W. A. Catterall, and B. J. Murphy. AKAP15 anchors cAMP-dependent protein kinase to brain sodium channels. J. Biol. Chem. 273: 25783-25788, 1998[Abstract/Free Full Text].

34.   Vaandrager, A. B., A. Smolenski, B. C. Tilly, A. B. Houtsmuller, E. M. Ehlert, A. G. Bot, M. Edixhoven, W. E. Boomaars, S. M. Lohmann, and H. R. de Jonge. Membrane targeting of cGMP-dependent protein kinase is required for cystic fibrosis transmembrane conductance regulator Cl- channel activation. Proc. Natl. Acad. Sci. USA 95: 1466-1471, 1998[Abstract/Free Full Text].

35.   Wang, S., R. W. Raab, P. J. Schatz, W. B. Guggino, and M. Li. Peptide binding consensus of the NHE-RF-PDZ1 domain matches the COOH terminal sequence of cystic fibrosis transmembrane conductance regulator (CFTR). FEBS Lett. 427: 103-108, 1998[ISI][Medline].

36.   Wang, Z. W., and M. I. Kotlikoff. Activation of KCa channels in airway smooth muscle cells by endogenous protein kinase A. Am. J. Physiol. Lung Cell. Mol. Physiol. 271: L100-L105, 1996[Abstract/Free Full Text].


Am J Physiol Cell Physiol 278(2):C417-C422
0363-6143/00 $5.00 Copyright © 2000 the American Physiological Society