Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation

Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) channel is activated by PKA phosphorylation of a regulatory domain that interacts dynamically with multiple CFTR domains and with other proteins. The large number of consensus sequences for phosphorylation by PKA has naturally focused most attention on regulation by this kinase. We report here that human CFTR is also phosphorylated by the tyrosine kinases p60c-Src (proto-oncogene tyrosine-protein kinase) and the proline-rich tyrosine kinase 2 (Pyk2), and they can also cause robust activation of quiescent CFTR channels. In excised patch-clamp experiments, CFTR activity during exposure to Src or Pyk2 reached ∼80% of that stimulated by PKA. Exposure to PKA after Src or Pyk2 caused a further increase to the level induced by PKA alone, implying a common limiting step. Channels became spontaneously active when v-Src or the catalytic domain of Pyk2 was coexpressed with CFTR and were further stimulated by the tyrosine phosphatase inhibitor dephostatin. Exogenous Src also activated 15SA-CFTR, a variant that lacks 15 potential PKA sites and has little response to PKA. PKA-independent activation by tyrosine phosphorylation has implications for the mechanism of regulation by the R domain and for the physiologic functions of CFTR.—Billet, A., Jia, Y., Jensen, T., Riordan, J. R., Hanrahan, J. W. Regulation of the cystic fibrosis transmembrane conductance regulator anion channel by tyrosine phosphorylation.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a plasma membrane anion channel formed by 2 membrane-spanning domains, 2 nucleotide-binding domains (NBDs; 1 and NBD2), and a regulatory or R domain (1). CFTR activity depends on nucleotide binding by both NBDs and hydrolysis at NBD2 (2, 3) and on phosphorylation of an intrinsically disordered central region that interacts dynamically with NBD1 and the C terminus (4). The R domain possesses multiple consensus sequences for serine/threonine phosphorylation by PKA and PKC but is also phosphorylated by other kinases that modulate channel activity or trafficking (5, 6). Channel activation appears complex (7) and involves the ordered phosphorylation of both stimulatory and inhibitory sites (8–10). The large number of PKA consensus sites in the R domain and the robust activation by PKA suggest that it may mediate most CFTR activation by peptide hormones and transmitters (11), whereas PKC elicits a smaller (∼10%) response when added immediately after excision (12, 13) and enhances the response to PKA (14).

CFTR activity is also modulated by tyrosine kinases. p60c-Src (proto-oncogene tyrosine-protein kinase), a ubiquitously expressed tyrosine kinase hereafter referred to as Src, increases flickering and enhances the response to PKA in a manner reminiscent of PKC (15). Focal adhesion kinase (FAK) regulation of CFTR in response to osmotic stress has been reported in euryhaline fish (16). Another study showed that the drug spiperone can induce Cl⁻ currents in airway epithelial cells and CFTR-transfected Chinese hamster ovary cells (17). Spiperone was shown to act by stimulating the Ca²⁺-activated proline-rich tyrosine kinase 2 (Pyk2), which is related to FAK, and its effects on Cl⁻ current were abolished by the tyrosine kinase inhibitor tyrphostin A9, suggesting that Pyk2 mediates the response. More recently, we found that carbachol (Cch) binding to the muscarinic type 3 receptor can activate CFTR through the canonical PKA-dependent pathway and also through a second mechanism that is sensitive to both Src and Pyk2 inhibitors (18). Pyk2 is well known to form a complex with Src that promotes the autophosphorylation of Src during the activation of GPCRs by ligand binding (19); therefore, we proposed that Src mediates about half of CFTR response to Cch under these conditions.

To test this hypothesis more directly, the present work examines the effects of Src and Pyk2 on CFTR phosphorylation and channel activity in excised membrane patches. We report here that both Src and Pyk2 activate quiescent CFTR channels to a level approaching that induced by PKA when purified kinases are applied to the cytosolic face of the membrane. This gating response is associated with direct tyrosine phosphorylation of CFTR and is independent of PKA because both tyrosine kinases also activate a mutant that lacks 15 consensus PKA sites (15SA-CFTR) and is almost unresponsive to PKA. Taken together, the results suggest that direct tyrosine kinase phosphorylation causes robust activation of the CFTR channel.

MATERIALS AND METHODS

Plasmid and cell culture

Baby hamster kidney (BHK) cells stably expressing wild-type CFTR or the 15SA-CFTR mutant lacking 15 PKA consensus sequences (11) were plated on plastic coverslips at low density and cultured in minimum essential medium (Life Technologies, Burlington, ON, Canada) containing 5% fetal bovine serum and the selecting drug methotrexate (300–500 µM) for 2 d with 5% CO₂ at 37°C. An expression plasmid containing cDNA encoding v-Src or Pyk2 was cotransfected along with large T antigen plasmid into BHK cells using the GeneJuice Transfection Reagent (EMD Millipore, Billerica, MA, USA). v-Src plasmid was kindly provided by Dr. R. Huganir (Department of Neuroscience, Johns Hopkins University, Baltimore, MD, USA), and pKH3-Pyk2 was kindly provided by Dr. J. L. Guan (Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA).

Tyrosine phosphorylation

Cells stably expressing CFTR were transfected in 60 cm² dishes with 9 μg v-Src or Pyk2 plasmid DNA and 1 μg large T antigen expression plasmid using the GeneJuice Transfection Reagent. Two days later, the cells were incubated with the tyrosine phosphatase inhibitors orthovanadate (50 μM) and dephostatin (10 μM) in serum-free medium for 30 min, then rinsed, lysed in RIPA buffer, and immunoprecipitated with the anti-CFTR mAb M3A7 as described previously (20). Immunoblots were probed using antiphosphotyrosine antibody 4G10 (EMD Millipore), anti-CFTR antibody 23C5, or L12B4 [which recognizes an epitope between residues 386 and 412 (21)] as indicated in the figure legends. 23C5 was purified from hybridomas prepared using SJL (Swiss James Lambert) mice that had been immunized with 6His-R domain and recognizes an epitope between aa 751 and 765 of CFTR. It was generated in collaboration with the laboratories of D. Y. Thomas and G. Lukacs (McGill University) and Anne Marcil (Biotechnology Research Institute–National Research Council Canada, Montreal, QC, Canada), with support from the Breathe program (Cystic Fibrosis Canada).

To assess in vitro phosphorylation, CFTR was immunoprecipitated from RIPA lysates using M3A7 antibody and protein G-agarose beads (25 µl). The precipitates were washed and incubated at 30°C in 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-potassium hydroxide (pH 7.2), 8 mM MnCl₂, 2.0 mM MnCl₂, 1 mM DTT, 0.25 mM Na₃VO₄, 1 µM dephostatin, and 1 mM MgATP containing either recombinant Src (30 U for 30 min; EMD Millipore) or the catalytic domain of Pyk2 (30 U for 45 min; Cedarlane, Burlington, ON, Canada). After extensive washing, the eluates were electrophoresed on 8% polyacrylamide gels in sodium dodecyl sulfate buffer, transferred to PVDF membranes, immunoblotted using antiphosphotyrosine antibody (4G10; 1:500), exposed to secondary antibody conjugated to horseradish peroxidase (1:5000 for 45 min), and visualized by ECL (GE Healthcare, Pewaukee, WI, USA). The blots were then stripped and reprobed with anti-CFTR mAb 23C5.

Patch clamping

Microscopic CFTR currents were recorded from inside-out patches with the pipette potential held at +30 mV (i.e., membrane potential, −30 mV) and inverted for purposes of illustration. Initial bath and pipette solutions contained 150 mM NaCl, 2 mM MgCl₂, and 10 mM N-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid (pH 7.2). Currents were recorded at 500 Hz and filtered at 125 Hz using an amplifier (Axopatch 200B) and analog/digital interface (Digidata 1440A) and were analyzed with pCLAMP 10 software (all from Axon Instruments, Inc., Burlingame, CA, USA). Maximum currents were measured at the plateau using pCLAMP software. The number of functional CFTR channels was roughly estimated by adding adenylyl-imidodiphosphate (AMP-PNP) (1 mM; Sigma-Aldrich, St. Louis, MO, USA) at the end of the experiment to lock channels in the open state (22). Taking the ratio of the current activated by kinase in the absence and presence of AMP-PNP [which further increases open probability (P₀) to near 1] allowed CFTR responses to be normalized for variation in the number of channels per patch. PKA (final concentration, 75 nM to 30 U) was from Promega (Madison, WI, USA); MgATP (1 mM) was from Sigma-Aldrich.

Statistics

Results are the means ± se of the number (n) of observations. Data were compared using the Student’s t test. Differences were considered statistically significant at P < 0.05. All statistical tests were performed using GraphPad Prism, version 6.0 (GraphPad Software, La Jolla, CA, USA).

RESULTS

Phosphorylation of CFTR by Src in situ and in vitro

To test if CFTR can be phosphorylated on tyrosines in situ, Src activity was first elevated by transfection with constitutively active Src (p60^V-Src or v-Src). Figure 1A, upper panel, shows Western blots of the total cell lysates and CFTR immunoprecipitates probed with mouse mAb against phosphotyrosine. As expected, many proteins were phosphorylated on tyrosine in cells expressing v-Src (Fig. 1A, lane 1). A strongly phosphorylated band at ∼170 kDa was detected when CFTR was immunoprecipitated from v-Src-transfected cells and the blots were probed with mouse monoclonal antiphosphotyrosine antibody (Fig. 1A, lane 2). This band was not detected when v-Src or CFTR was not expressed (Fig. 1A, lanes 3 and 4, respectively). These data suggest that constitutively active Src phosphorylates tyrosine residues on CFTR in situ.

Figure 1.

Phosphorylation of CFTR by Src in intact cells and in vitro. A) Representative immunoblot (IB) probed with antiphosphotyrosine antibody (upper panel) or CFTR antibody (lower panel). Lane 1 shows whole-cell lysate. In lane 2, CFTR immunoprecipitated using mAb M3A7 is recognized by the same antiphosphotyrosine antibody when v-Src and CFTR were coexpressed. No phosphotyrosine protein is detected when either v-Src (lane 3) or CFTR (lane 4) is not expressed. B) In vitro phosphorylation of CFTR by Src. The immunoblot was probed with antiphosphotyrosine antibody (upper panel), then stripped and reblotted using anti-CFTR antibody 23C5 (lower panel). Lanes marked “L” are total cell lysate controls; lanes marked “Ctl IP” are nonimmune controls for nonspecific binding of CFTR to the beads. The phosphotyrosine (phosphoTyr; indicated by arrows) and CFTR (lower blots) bands are superimposable when CFTR-expressing cells are treated with 30 U active Src for 30 min. A representative of 3 experiments for each condition is shown.

To assess tyrosine phosphorylation in vitro, CFTR was immunoprecipitated with M3A7 antibody on protein G-agarose beads, incubated with active Src and 1 mM MgATP for 30 min at 30°C, washed, and immunoblotted. To detect tyrosine-phosphorylated protein, blots were probed using the antiphosphotyrosine mAb 4G10. Then, to identify CFTR and control for its level of expression, the same blots were stripped and reprobed using a different anti-CFTR mAb from the one used for immunoprecipitation (IP), 23C5 (Fig. 1B). The left panels in Fig. 1B show a strong phosphotyrosine band at the mass expected for CFTR only after incubating M3A7 immunoprecipitates with active Src (30 U; compare lanes 3 and 4), suggesting that the phosphotyrosine was on CFTR. No CFTR or phosphotyrosine signals were detected if the IP was performed using cells that had not been transfected with CFTR (Fig. 1B, lane 2). To further confirm that phosphotyrosine was on CFTR, cells transfected or not with CFTR were immunoprecipitated using M3A7 and immunoblotted for phosphotyrosine and CFTR. Cells devoid of CFTR yielded only a faint nonspecific band at the position of CFTR when blots were probed with antiphosphotyrosine antibody relative to precipitates from CFTR-expressing cells (Fig. 1B, right panel), further indicating that the phosphoprotein was indeed CFTR. Together, these results confirm that CFTR is indeed phosphorylated by Src in vitro.

Effect of Src on CFTR activity

To study the effect of tyrosine phosphorylation on CFTR channel activity, silent patches were excised from BHK cells stably expressing CFTR and immediately exposed to PKA (75 nM) or phospho-Src (30 U/ml) in the presence of 1 mM MgATP. PKA activated large currents in all patches tested, whereas Src activated channels in 23 of 26 patches. Figure 2A, B shows representative traces of CFTR activation by PKA and Src, respectively. These currents were only observed in BHK cells when the cells were transfected with CFTR (data not shown). To further confirm that Src-activated currents were carried by CFTR, we examined the unitary conductance. Figure 2C shows the mean unitary current-voltage (i/V) relationship in symmetric high-Cl⁻ solutions. The linear i/V relationship of Src-activated currents (blue line and symbols in Fig. 2C) and the mean slope conductance of 6.3 pS are consistent with CFTR (23) and are similar to the i/V relationship obtained during stimulation by PKA (black symbols and dotted line).

Figure 2.

Effect of Src on CFTR channel activity. A and B) Recordings obtained immediately after membrane patches were excised from an unstimulated cell into bath solution containing (A) 1 mM MgATP and 75 nM PKA or (B) 30 U/ml Src. Pipette voltage was +30 mV. Upward deflections represent channel openings. C) Single-channel i/V relationship determined in symmetric high-Cl⁻ solutions. Symbols represent means ± se of 3 experiments. D) Control experiment under same conditions as in (A) and (B) except with MgATP plus a 5 nM concentration of the PP2A inhibitor Calyculin A. E) Membrane patch excised into bath solution containing 1 mM MgATP and Src (30 U/ml). AMP-PNP (1 mM) was added subsequently as indicated.

Src inhibits the serine/threonine phosphatase protein phosphatase 2 (PP2A) (24), which has been shown to deactivate CFTR channels (25–27); therefore, we tested if the action of Src could be mediated, at least in part, through its effects on PP2A. Inside-out patches were excised from BHK cells stably expressing CFTR, and channel activity was recorded in the presence of Calyculin A, a potent inhibitor of PP1 and PP2A. As shown in Fig. 2D, 5-min exposure to 5 nM Calyculin A in the presence of 1 mM MgATP did not increase the Cl⁻ current. More importantly, subsequent exposure to Src still increased the current, which demonstrates that Src activation is unaffected by inhibition of serine/threonine phosphatase while confirming that functional channels were present in the membrane patch.

Another diagnostic property of CFTR is the ability of AMP-PNP, a nonhydrolyzable ATP analog, to cause PKA-activated channels to become locked in an open state [i.e., P₀ ≈ 1 (22, 28, 29)]. When 1 mM AMP-PNP was added to patches in the presence of 1 mM ATP after exposure to Src rather than PKA, the macroscopic current increased by >2-fold (Fig. 2E). Currents were not observed when the enzymatic activity of Src had been destroyed prior to the experiment by heating to 100°C for 10 min or when patches from control cells lacking CFTR were studied (data not shown). Taken together, the linear i/V relationship, low unitary conductance, and effect of AMP-PNP on gating indicate that CFTR channels carry the Src-activated current.

Effect of Pyk2 on CFTR phosphorylation and activity

We found previously that Cch stimulation of CFTR is partially inhibited by tyrphostin A9, a relatively specific Pyk2 inhibitor (18). Because Pyk2 is known to regulate Src (19), we wondered if the inhibition by tyrphostin A9 was caused by inhibition of Pyk2 itself or was an indirect consequence of inhibiting Src. To investigate this, we examined CFTR phosphorylation and activity induced by Pyk2.

Phosphorylation of CFTR by Pyk2 was tested in vitro as described above for Src. After exposure to Pyk2 catalytic domain in the presence of MgATP, a strong band was detected by the antiphosphotyrosine antibody 4G10 at ∼170 kDa only when the kinase and CFTR were both present (Fig. 3A, lanes 3 and 6), indicating that phosphorylation was due to Pyk2 activity, and the phosphoprotein substrate was CFTR.

Figure 3.

Effect of Pyk2 on CFTR channel activity. A) In vitro phosphorylation of CFTR by Pyk2. An immunoblot (IB) probed with antiphosphotyrosine antibody (upper panel), then stripped and reblotted using anti-CFTR antibody 23C5 (lower panel) is shown. The phosphotyrosine (phosphoTyr; indicated by arrows) and CFTR (lower blot) bands are superimposable when CFTR-expressing cells are incubated for 45 min with 30 U active Pyk2. A representative of 3 experiments is shown. B) Recording obtained immediately after membrane patch was excised from an unstimulated cell into bath solution containing 1 mM MgATP and 30 U/ml of the catalytic domain of Pyk2. Pipette voltage was +30 mV. Upward deflections represent channel openings. C) Single-channel i/V relationship determined in symmetric high-Cl⁻ solutions. Symbols represent means ± se of 3 experiments. D) Membrane patches excised into bath solution containing 1 mM MgATP and Pyk2 (30 U/ml). AMP-PNP (1 mM final concentration) was added as indicated.

The effect of Pyk2 on CFTR activity then was tested using quiescent, inside-out patches excised from BHK cells stably expressing wild-type CFTR. Figure 3B shows a typical recording obtained following application of the active form of Pyk2 (30 U/ml) in the presence of 1 mM MgATP. A similar current was recorded for 20 of the 24 patches tested. The enlarged segment shows CFTR opening/closing events having a characteristic low unitary current of ∼0.4 pA at −30 mV. The linear i/V relationship of Pyk2-activated single-channel currents (Fig. 3C, red line and symbols) and mean slope conductance of 6.4 pS are consistent with the known properties of CFTR. Application of AMP-PNP after activation by Pyk2 caused a further increase in the overall current without changing the amplitude of unitary currents (Fig. 3D). These biochemical and functional results indicate that Pyk2 can phosphorylate and activate CFTR channels.

Comparison of CFTR activation by tyrosine kinases and PKA

To compare the magnitude of CFTR currents stimulated by tyrosine kinases with those produced by serine/threonine phosphorylation, we normalized for patch-to-patch variation in the number of channels by determining the ratio of currents before and after adding 1 mM AMP-PNP. Figure 4A shows the results for patches exposed to PKA and Src. The mean ratio for current stimulated by tyrosine kinase alone-to-tyrosine kinase plus AMP-PNP was 0.40 ± 0.05 and 0.42 ± 0.04 for Src and Pyk2, respectively, as compared with 0.54 ± 0.08 with patches stimulated using PKA (Fig. 4B). Src and Pyk2 (both 30 U/ml) responses had similar time courses (10–12 min to reach the maximal current), which were somewhat slower than the response to PKA (5–7 min; 75 nM). These results suggest that both tyrosine kinases elevate P₀ to ∼80% of the level attained by PKA, albeit more slowly.

Figure 4.

Comparison of CFTR activity stimulated by Src, Pyk2, and PKA. A) Membrane patches excised into bath solution containing 1 mM MgATP and 75 nM PKA (left trace) or 30 U/ml Src (right trace). Src (left) or PKA (right) was then added where indicated. Recording configuration is the same as in Figs. 2 and 3. B) Left histogram shows a comparison of the mean ratio of kinase-stimulated currents before and after addition of 1 mM AMP-PNP. Hatched bars represent PKA:AMP-PNP current ratios after Src or Pyk2 exposure (30 U/ml for each): Src or Pyk2 was present initially, and PKA (75 nM) was added after the maximal tyrosine kinase-induced current had been reached. Error bars show se for the number of cells indicated. ns, no significant difference. *P < 0.05 with Student’s t test. Right histogram shows a comparison of the mean ratio of the PKA or Pyk2-stimulated currents measured before and after adding 1 mM AMP-PNP; cells were bathed for 30 min in 10 µM SrcInh 1 prior to the addition of kinases.

Exposing CFTR channels to Src after they had already been strongly activated by PKA caused no further increase (Fig. 4A, left trace), although further stimulation by Src was observed when channels were only partially activated using a low concentration of PKA (data not shown). On the other hand, exposure to PKA after maximal activation with Src or Pyk2 did cause a further increase in CFTR current (Fig. 4A, right trace), although the mean ratio of currents before and after addition of AMP-PNP did not exceed that induced by PKA alone (Fig. 4B, left histogram).

Because PKA and Pyk2 are known to regulate Src activation by direct phosphorylation (30, 31), we tested if a fraction of the PKA or Pyk2-dependent CFTR current was not due to the activation of a residual or associated Src kinase under the patch pipette. Src inhibitor (SrcInh) was added before PKA or Pyk2 addition, and there was no difference in the mean current ratio in the presence of SrcInh compared with normal conditions (Fig. 4B, right histogram).

Effect of v-Src or Pyk2 overexpression on CFTR activity in excised patches

To test if CFTR channel could be phosphorylated by overexpression of Pyk2, we performed biochemical experiments similar to those above with v-Src (see Fig. 1A). As shown in Fig. 5A, tyrosine phosphorylation of CFTR could be detected when Pyk2 was overexpressed in cells stably transfected with CFTR.

Figure 5.

Spontaneous CFTR activity in excised patches from v-Src or knockdown-Pyk2-transfected cells. A) Representative immunoblot (IB) probed with antiphosphotyrosine (phosphoTyr; upper panel) or CFTR (lower panel) antibody. Lane 1 shows whole-cell lysate. In lane 2, CFTR immunoprecipitated using mAb M3A7 is recognized by the same antiphosphotyrosine antibody when pKH3-Pyk2 and CFTR were coexpressed. No phosphotyrosine protein was detected when either Pyk2 (lane 3) or CFTR (lane 4) was not expressed. B) Patch recording from CFTR-expressing cells transiently transfected with large T antigen and control (Ctl) plasmid lacking v-Src. C and D) CFTR activity in patches excised from cells expressing v-Src (C) or pKH3-Pyk2 (D). MgATP (1 mM) was present throughout the experiment. Dephostatin (10 µM) was added at the arrows.

Furthermore, when BHK cells stably expressing CFTR were transfected with v-Src or pKH3-Pyk2, a low level of spontaneous channel activity was observed in excised patches bathed with solution containing 1 mM MgATP without addition of exogenous kinases (Fig. 5C, D). By contrast, spontaneous channel activity was not observed when patches were taken from control cells expressing CFTR and bathed in the same solution without v-Src or Pyk2 (Fig. 5B). These results suggest that heterologous v-Src or Pyk2 can become associated with the membrane and phosphorylate CFTR in the excised patch.

CFTR activity increased when patches from v-Src or Pyk2-transfected cells were exposed to the specific protein tyrosine phosphatase (PTPase) inhibitor dephostatin (10 µM; n = 4; Fig. 5C, D). When added to control patches lacking heterologous tyrosine kinase expression, neither MgATP (1 mM, present throughout the recording) nor dephostatin (10 µM, added acutely) stimulated CFTR activity (Fig. 5B). These results suggest that stimulation by heterologously expressed, constitutively active tyrosine kinases is opposed by an endogenous, membrane-associated phosphatase activity. This tonic PTPase activity is apparently sufficient to suppress CFTR stimulation by endogenous c-Src and Pyk2 under control conditions.

Src and Pyk2 activate mutant 15SA-CFTR channels, which do not respond to PKA

PKA is the only kinase reported to cause robust activation of quiescent CFTR channels when added to patches. To exclude the possibility that Src stimulation occurs indirectly through a mechanism that involves serine/threonine phosphorylation, we examined the effects of Src and Pyk2 on a CFTR mutant lacking 15 potential monobasic and dibasic PKA consensus sites [15SA; (10, 32)]. Activation of this mutant by PKA was attenuated ∼90% compared with wild-type channels, when currents were normalized by the AMP-PNP response (current ratio, 0.074 ± 0.021 vs. 0.54 ± 0.08 for wild-type; P < 0.01; see Fig. 6A). By contrast, exogenous application of active Src (Fig. 6B) or Pyk2 (Fig. 6C) in the presence of MgATP induced a robust activation of the mutated channels. The currents displayed a linear unitary i/V relationship and conductance as expected for CFTR channels.

Figure 6.

PKA independence of the phosphotyrosine stimulation. Recordings start immediately after excision. MgATP (1 mM) is present throughout the experiments. A) Effect of PKA on channel activity in a patch excised from a cell that is stably expressing the 15SA mutant. Left panel shows a representative recording of membrane patches excised into bath solution containing 1 mM MgATP and 75 nM PKA. Right panel shows a comparison of the PKA:AMP-PNP current ratio for wild-type (wt)- and 15SA-CFTR channels. B and C) Effect of exogenous Src (30 U/ml) (B) and Pyk2 (30 U/ml) (C) on activity of the 15SA mutant. Each recording is representative of 4 experiments.

DISCUSSION

The ATP-dependent gating cycle of CFTR is regulated by the phosphorylation of multiple serine residues in the R domain between Ser660 and Ser813; serines at positions 660, 670, 686, 700, 712, 753, 790, 795, and 813 increase channel activity, whereas phosphorylation at 737 and 768 is inhibitory (10, 20, 33). Phosphorylation modulates dynamic association of the R domain with other domains within CFTR, notably NBD1 (34–36), and also with the large CFTR interactome, which includes adaptor and scaffold proteins such as 14-3-3 (37) and Na⁺/H⁺ exchanger regulatory factor-1 (38). It is widely accepted that CFTR channel activity is determined mainly by PKA phosphorylation, with modulation exerted by PKC, AMPK, and deactivation due to several protein phosphatases (PP2A and PP2C) [see for review Bozoky et al. (39)]. Early circular dichroism experiments with the isolated R domain showed that its low α-helical content was further reduced upon phosphorylation by PKA (40), and more recently, elegant NMR studies have revealed a reduction in helical propensity near several PKA sites, including those between aa 805 and 820, which are proposed to shift the equilibrium of domain interactions toward channel opening. The finding that direct phosphorylation on tyrosine strongly activates CFTR is unexpected because there is only one tyrosine residue (Tyr808) in the domain between Ser660 and Ser813 where most PKA and PKC sites are situated. Phosphorylation of Tyr808 could potentially have similar effects on local secondary structures as PKA phosphorylation of Ser795 and Ser813, although it is not in a known consensus sequence for phosphorylation by tyrosine kinases. Other candidate tyrosines near the R domain include Tyr625 and Tyr627; however, they are also not in the classic consensus for Src: E(2-3)-I/V-Y-G/E-X-F (41). Phosphorylation of Tyr512 by the src family kinases (SFKs) Syk and Lyn modulates the regulation of CFTR by casein kinase 2 (42, 43), and phosphorylation of Tyr515 has also been reported (44). CFTR was immunoprecipitated by a phosphotyrosine antibody but was also shown to be in a tyrosine-phosphorylated protein complex; thus, it was not clear if the precipitation was due to phosphotyrosine on CFTR itself (17). It has been shown that isolated NBD1 from the CFTR can be phosphorylated by tyrosine kinases from the SFKs (Fyn, Lyn, and Fgr) in vitro (43, 45). In the present work, phosphotyrosine was clearly demonstrated on full-length CFTR after in vitro exposure to Src and Pyk2. Studies are underway to localize the phosphotyrosines responsible.

Coexpressing v-Src led to phosphorylation of tyrosine residues on CFTR that were not detectable in control BHK cells expressing only endogenous Src and Pyk2, in agreement with the absence of phosphotyrosine on CFTR noted previously in COS cells (46). It is possible that expression of these kinases may be too low in these cell lines to give a detectable CFTR signal. Alternatively, endogenous Src and Pyk2 may need to be activated by the muscarinic type 3 receptor (47) or by some other upstream stimulus. Oxidant stress stimulates Src, and bacterial infection causes a massive stimulation of tyrosine kinases and would be consistent with a role of CFTR in airway innate immunity. The present results indicate that Src and Pyk2 are surprisingly potent regulators of CFTR channel activity, each capable of producing CFTR currents that are ∼80% as large as those stimulated by PKA. They had no effect after being heated to 100°C to destroy their kinase activity. The nonadditivity of maximal responses to tyrosine kinases and PKA suggests that they share a common step in channel activation (48). Robust activation of quiescent CFTR channels by Src may not have been observed previously due to the use of lower Src activity (1–20 vs. 30 U/ml) or perhaps different cell lines (NIH 3T3 fibroblasts vs. BHK), which may differ in their endogenous kinase and phosphatase activities (15).

Possible role of tyrosine phosphorylation

Although CFTR channels are activated by the serine/threonine kinases PKA and PKC, many “noncanonical” regulatory mechanisms have been described that are intriguing but remain poorly understood, including G proteins (49) and GPCRs that signal through Gα_q (47, 50). Another potential nonreceptor signal may be infection because Pseudomonas aeruginosa invasion requires activation of SFKs (51, 52). Whether tyrosine kinases mediate the regulation of CFTR in airway epithelial host defense, whereas PKA mediates hormonal regulation of CFTR in other organs such as the pancreas, remains to be explored. Regardless, the present results demonstrate phosphorylation and robust activation of CFTR by the tyrosine kinases Src and Pyk2 and raise interesting questions regarding the sites and structural consequences of tyrosine phosphorylation.

Glossary

AMP-PNP

adenylyl-imidodiphosphate

BHK

baby hamster kidney

Cch

carbachol

CFTR

cystic fibrosis transmembrane conductance regulator

FAK

focal adhesion kinase

IP

immunoprecipitation

i/V

current-voltage

NBD

nucleotide-binding domain

P₀

open probability

PP2A

protein phosphatase 2

PTPase

protein tyrosine phosphatase

Pyk2

proline-rich tyrosine kinase 2

SFK

src family kinase

Src

proto-oncogene tyrosine-protein kinase

SrcInh

Src inhibitor