ABCMdb

ABCC7 p.Ser737Ala

ClinVar:	c.2210C>T , p.Ser737Phe ? , not provided
CF databases:	c.2210C>T , p.Ser737Phe (CFTR1) ? , This nucleotide change was identified in two Italian patients.
Predicted by SNAP2:	A: N (61%), C: D (59%), D: D (75%), E: D (66%), F: N (61%), G: N (66%), H: D (63%), I: N (57%), K: N (57%), L: N (53%), M: N (53%), N: N (72%), P: D (71%), Q: N (66%), R: D (66%), T: N (78%), V: N (61%), W: D (75%), Y: D (53%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: D, N: N, P: N, Q: N, R: N, T: N, V: D, W: D, Y: D,

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[hide] Contribution of R domain phosphoserines to the fun... Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L835-41. Baldursson O, Berger HA, Welsh MJ
Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia.
Am J Physiol Lung Cell Mol Physiol. 2000 Nov;279(5):L835-41., [PMID:11053017]

Abstract [show]
The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl(-) current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Ser(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone increased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser(737) may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.

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14 Changing Ser737 to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser737 may inhibit current in wild-type CFTR. Login to comment
56 In each of the serine to alanine mutants, S660A, S737A, S795A, and S813A, alanine replaced serine at the designated residue. Login to comment
108 The S795A channels generated more current, but it was still less than that produced by wild-type CFTR. Interestingly, current from the S737A variant tended to be greater than that of wild-type CFTR, although the difference was not significant. Login to comment
114 Current from the S795A variant was not different from that in wild-type CFTR. Interestingly, current generated by the S737A variant was more than twice that generated by wild-type CFTR. Login to comment
134 The S737A variant generated more current than the wild type, and the S-Quad-A mutant generated less. Login to comment
137 The data show that the S-Quad-A variant was less sensitive to forskolin than the wild type and that the S737A variant was more sensitive than wild-type CFTR. Login to comment
140 The results show that current increased more rapidly with S737A than with wild-type and S-Quad-A CFTR. Login to comment
152 The increase in current and the more rapid activation observed when Ser737 was mutated to alanine suggested that phosphorylation of Ser737 may inhibit current. Login to comment
157 Activation by increasing concentrations of forskolin of Cl- current in epithelia expressing wild-type CFTR, S737A, and SQuad-A. Login to comment
161 C: current in epithelia expressing wild-type CFTR, S737A, and S-Quad-A in response to forskolin. Login to comment
187 Time course of Cl- current activation in epithelia expressing wild-type CFTR, S737A, and S-Quad-A. Login to comment
225 A particularly striking example is the different function of S737A in cells and in excised membrane patches. Login to comment
226 In FRT cells and in Xenopus oocytes (28), Ser737 generated more current than wild-type CFTR, but, in patches, S737A showed the same open state probability as the wild type, and the sensitivity to ATP was reduced (29). Login to comment

[hide] Regulation of the cystic fibrosis transmembrane co... J Biol Chem. 2001 Mar 16;276(11):7689-92. Epub 2001 Jan 23. Ostedgaard LS, Baldursson O, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by its R domain.
J Biol Chem. 2001 Mar 16;276(11):7689-92. Epub 2001 Jan 23., 2001-03-16 [PMID:11244086]

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35 In excised, cell-free patches, the S737A mutant reduced Po (20), but in Xenopus oocytes and epithelia, the S737A mutant increased cAMP-stimulated current (21, 23). Login to comment

[hide] Capsaicin potentiates wild-type and mutant cystic ... Mol Pharmacol. 2004 Jun;65(6):1415-26. Ai T, Bompadre SG, Wang X, Hu S, Li M, Hwang TC
Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents.
Mol Pharmacol. 2004 Jun;65(6):1415-26., [PMID:15155835]

Abstract [show]
To examine the effects of capsaicin on cystic fibrosis transmembrane conductance regulator (CFTR), we recorded wild-type and mutant CFTR chloride-channel currents using patch-clamp methods. The effects of capsaicin were compared with those of genistein, a well-characterized CFTR activator. In whole-cell experiments, capsaicin potentiates cAMP-stimulated wild-type CFTR currents expressed in NIH 3T3 cells or Chinese hamster ovary cells in a dose-dependent manner with a maximal response approximately 60% of that with genistein and an apparent Kd of 48.4 +/- 6.8 microM. In cell-attached recordings, capsaicin alone fails to activate CFTR in cells that show negligible basal CFTR activity, indicating that capsaicin does not stimulate the cAMP cascade. The magnitude of potentiation with capsaicin depends on the channel activity before drug application; the lower the prestimulated Po, the higher the potentiation. Single-channel kinetic analysis shows that capsaicin potentiates CFTR by increasing the opening rate and decreasing the closing rate of the channel. Capsaicin may act as a partial agonist of genistein because the maximally enhanced wild-type CFTR currents with genistein are partially inhibited by capsaicin. Capsaicin increases DeltaR-CFTR, a protein kinase A (PKA)-independent, constitutively active channel, in cell-attached patches. In excised inside-out patches, capsaicin potentiates the PKA-phosphorylated, ATP-dependent CFTR activity. Both capsaicin and genistein potentiate the cAMP-stimulated G551D-CFTR, DeltaF508-CFTR, and 8SA mutant channel currents. The binding site for capsaicin is probably located at the cytoplasmic domain of CFTR, because pipette application of capsaicin fails to potentiate CFTR activity. In conclusion, capsaicin is a partial agonist of genistein in activation of the CFTR chloride channel. Both compounds affect ATP-dependent gating of CFTR.

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142 We further explored the action of capsaicin using CFTR mutants whose eight major PKA consensus serines are substituted with alanine (S660A, S686A, S700A, S712A, S737A, S768A, S795A, and S813A), so called S-oct-A or 8SA. Login to comment

[hide] Preferential phosphorylation of R-domain Serine 76... J Gen Physiol. 2005 Feb;125(2):171-86. Epub 2005 Jan 18. Csanady L, Seto-Young D, Chan KW, Cenciarelli C, Angel BB, Qin J, McLachlin DT, Krutchinsky AN, Chait BT, Nairn AC, Gadsby DC
Preferential phosphorylation of R-domain Serine 768 dampens activation of CFTR channels by PKA.
J Gen Physiol. 2005 Feb;125(2):171-86. Epub 2005 Jan 18., [PMID:15657296]

Abstract [show]
CFTR (cystic fibrosis transmembrane conductance regulator), the protein whose dysfunction causes cystic fibrosis, is a chloride ion channel whose gating is controlled by interactions of MgATP with CFTR's two cytoplasmic nucleotide binding domains, but only after several serines in CFTR's regulatory (R) domain have been phosphorylated by cAMP-dependent protein kinase (PKA). Whereas eight R-domain serines have previously been shown to be phosphorylated in purified CFTR, it is not known how individual phosphoserines regulate channel gating, although two of them, at positions 737 and 768, have been suggested to be inhibitory. Here we show, using mass spectrometric analysis, that Ser 768 is the first site phosphorylated in purified R-domain protein, and that it and five other R-domain sites are already phosphorylated in resting Xenopus oocytes expressing wild-type (WT) human epithelial CFTR. The WT channels have lower activity than S768A channels (with Ser 768 mutated to Ala) in resting oocytes, confirming the inhibitory influence of phosphoserine 768. In excised patches exposed to a range of PKA concentrations, the open probability (P(o)) of mutant S768A channels exceeded that of WT CFTR channels at all [PKA], and the half-maximally activating [PKA] for WT channels was twice that for S768A channels. As the open burst duration of S768A CFTR channels was almost double that of WT channels, at both low (55 nM) and high (550 nM) [PKA], we conclude that the principal mechanism by which phosphoserine 768 inhibits WT CFTR is by hastening the termination of open channel bursts. The right-shifted P(o)-[PKA] curve of WT channels might explain their slower activation, compared with S768A channels, at low [PKA]. The finding that phosphorylation kinetics of WT or S768A R-domain peptides were similar provides no support for an alternative explanation, that early phosphorylation of Ser 768 in WT CFTR might also impair subsequent phosphorylation of stimulatory R-domain serines. The observed reduced sensitivity to activation by [PKA] imparted by Ser 768 might serve to ensure activation of WT CFTR by strong stimuli while dampening responses to weak signals.

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208 In addition, to verify the inferred link between the major mobility shift of the R domain and phosphorylation of Ser 737 (Fig. 2), the other candidate inhibitory serine (Wilkinson et al., 1997), we studied phosphorylation of His-tagged R-domain proteins containing the single mutation S737A, or the double mutation S737A-S768A. Login to comment
209 The incremental mobility shifts already seen in Figs. 1 and 2 as phosphorylation of the R domain progressed were recapitulated in the His-tagged WT peptide (Fig. 8 A), and were not substantially altered by the S768A mutation in either the WT or S737A background (Fig. 8, B and D vs. A and C). Login to comment
210 As anticipated, however, the S737A mutation abolished the large mobility shift of both WT and S768A R-domain peptides (Fig. 8, C and D vs. A and B). Login to comment
217 WT (A) and mutant R-domain peptides, with Ser768 replaced by alanine (S768A; B), Ser737 replaced by alanine (S737A; C), or Ser737 and Ser768 both replaced by alanine (S737A-S768A; D), were phosphorylated with 10 nM PKA and 5 ␮M ␥32P-MgATP for 0.5-60 min as indicated. Login to comment
219 The major mobility shift (to band 3; Figs. 1 and 2) was seen in the WT and S768A peptides, but not in the S737A or S737A-S768A peptides. Login to comment
296 Mass spectrometry and site-directed mutagenesis revealed that the major mobility shift was linked to phosphorylation of Ser 737 both at low and high [MgATP] (Fig. 2), and this requirement was confirmed by the absence of the large mobility shift after mutation of Ser 737 to Ala in either WT or S768A R-domain peptide (Fig. 8, A-D; see also Borchardt et al., 1996; Kole et al., 1998). Login to comment
299 Though we did not examine its consequences for channel gating, the S737A mutation has been reported to leave burst duration unchanged from that of WT CFTR (Winter and Welsh, 1997), whereas we found the S768A mutation to roughly double burst duration (Fig. 6). Login to comment

[hide] Too much salt, too little soda: cystic fibrosis. Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415. Quinton PM
Too much salt, too little soda: cystic fibrosis.
Sheng Li Xue Bao. 2007 Aug 25;59(4):397-415., 2007-08-25 [PMID:17700961]

Abstract [show]
Cystic fibrosis (CF) of the pancreas is the most widely accepted name of the most common fatal inherited single gene defect disease among Caucasians. Its incidence among other races is thought to be significantly less, but mutations in the gene have been reported in most, if not all, major populations. This review is intended to give general concepts of the molecular as well as physiological basis of the pathology that develops in the disease. First, an overview of the organ pathology and genetics is presented, followed by the molecular structure of the gene product (cystic fibrosis transmembrane conductance regulator, CFTR), its properties, functions, and controls as currently understood. Second, since mutations appear to be expressed primarily as a defect in electrolyte transport, effects and mechanisms of pathology are presented for two characteristically affected organs where the etiology is best described: the sweat gland, which excretes far too much NaCl ("salt") and the pancreas, which excretes far too little HCO3(- )("soda"). Unfortunately, morbidity and mortality in CF develop principally from refractory airway infections, the basis of which remains controversial. Consequently, we conclude by considering possible mechanisms by which defects in anion transport might predispose the CF lung to chronic infections.

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78 Substitutionofalanine for phosphorylatable S737A or S768A enhanced the channel activity, suggesting that phosphorylation at either of these sites may inhibit phosphorylation of other stimulatory sites[72] . Login to comment

[hide] Mechanistic insight into control of CFTR by AMPK. J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18. Kongsuphol P, Cassidy D, Hieke B, Treharne KJ, Schreiber R, Mehta A, Kunzelmann K
Mechanistic insight into control of CFTR by AMPK.
J Biol Chem. 2009 Feb 27;284(9):5645-53. Epub 2008 Dec 18., 2009-02-27 [PMID:19095655]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP and protein kinase A (PKA)-regulated Cl(-) channel in the apical membrane of epithelial cells. The metabolically regulated and adenosine monophosphate-stimulated kinase (AMPK) is colocalized with CFTR and attenuates its function. However, the sites for CFTR phosphorylation and the precise mechanism of inhibition of CFTR by AMPK remain obscure. We demonstrate that CFTR normally remains closed at baseline, but nevertheless, opens after inhibition of AMPK. AMPK phosphorylates CFTR in vitro at two essential serines (Ser(737) and Ser(768)) in the R domain, formerly identified as "inhibitory" PKA sites. Replacement of both serines by alanines (i) reduced phosphorylation of the R domain, with Ser(768) having dramatically greater impact, (ii) produced CFTR channels that were partially open in the absence of any stimulation, (iii) significantly augmented their activation by IBMX/forskolin, and (iv) eliminated CFTR inhibition post AMPK activation. Attenuation of CFTR by AMPK activation was detectable in the absence of cAMP-dependent stimulation but disappeared in maximally stimulated oocytes. Our data also suggest that AMP is produced by local phosphodiesterases in close proximity to CFTR. Thus we propose that CFTR channels are kept closed in nonstimulated epithelia with high baseline AMPK activity but CFTR may be basally active in tissues with lowered endogenous AMPK activity.

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43 EXPERIMENTAL PROCEDURES cRNAs for CFTR and Double Electrode Voltage Clamp-Oocytes were injected with cRNA (10 ng, 47 nl of double-distilled water) encoding wtCFTR, L1430A/L1431A, S573A, S1248A, F508del-CFTR, G551D-CFTR, S768A, S737A, S768D, S737D, E1474X, and AMPK␣1. Login to comment
93 Fig. 2B demonstrates that AMPK indeed phosphorylates the R domain in vitro and this AMPK phosphorylation is largely reduced in R domain mutants S737A and S768A (compare lanes 2 and 3 in Fig. 2B, lower panel). Login to comment
94 The data also suggest that serine 768 has a much greater reductive impact because S768A almost abolished all the phosphorylation, whereas some were preserved with S737A. Login to comment
96 We quantified data (supplemental Fig. S2) from three independent experiments and found that the S737A mutation leads to a small 23 Ϯ 8% reduction in counts, whereas S768A leads to a major 81 Ϯ 5% (mean Ϯ range) reduction similar to the double mutant (87% Ϯ 4%) when compared with the wild type (100%). Login to comment
125 It is clear that S768A (lower left panel) has a more profound inhibitory effect on R domain phosphorylation compared with S737A (upper right) given that all these experiments were run simultaneously with similar concentrations of R domain protein and kinase, and each imaged for identical lengths of time. Login to comment
126 Broadly, prolonged incubation that previously created two major spots found with AMPK alone was now supplemented by multiple small spots that almost disappeared after S768A mutation, but much less so after S737A mutation (Fig. 2D, compare lower two panels with upper). Login to comment
128 When expressed in oocytes, CFTR bearing the R domain mutants S737A and S768A as well as the double mutant S737A/ S768A (Fig. 3A) produced dramatically enhanced conductances (S768A Ͼ S737A; Fig. 3B) upon stimulation with forskolin (2 ␮M) and IBMX (1 mM). Login to comment
130 The large Cl-conductances generated by S737A/S768A were inhibited by 5 ␮M of the specific chloride channel blocker CFTRinh-172 (Fig. 3D), or alternatively, the PKA inhibitor KT5720 and Cl- replacement by gluconate (not shown). Login to comment
131 Once again the differential roles of these two serines were observed because the conductance observed with S737A was almost 100 ␮S lower than that found with S768A. Login to comment
135 B, phosphoryl- ationoftheRdomainwithPKAandAMPK.AMPKphosphorylationwaslargely reduced or abolished in S737A and S768A, respectively (lower panel). Login to comment
146 Overall the data suggest that S737A, S768A, and the double mutant S737A/ S768A were no longer sensitive to stimulation or inhibition of AMPK indicating that phosphorylation at both serines is required (Fig. 3C, middle panels). Login to comment
151 In contrast to wtCFTR the CFTR mutants S737A (not shown), S768A, and S737A/S768A produced a high Cl- con- ductance under basal conditions, i.e. in the absence of IBMX and forskolin (Fig. 4, A and B). Login to comment
155 A, whole cell currents activated by IBMX (1 mM) and forskolin (2 ␮M) in wtCFTR and S737A/ S768A-CFTRexpressingoocytes.B,summaryofwholecellconductancesgen- erated by wtCFTR and different CFTR mutants. Login to comment
156 C, summary of CFTR whole cell conductances generated by wtCFTR and different CFTR mutants, and effects of phenformin and compound C. D, summary of the whole cell conductance activated by S737A/S768A-CFTR and inhibition by the CFTR blocker CFTRinh-172. Login to comment
161 S737A/S768A-CFTR generates a baseline conductance. Login to comment
162 A, effect of extracellular Cl- replacement by gluconate (glcn) on whole cell currents generated by wtCFTR, S768A-CFTR, and S737A/S768A-CFTR in the absence of IBMX and forskolin. Login to comment
164 C, summary of the whole cell conductances generated by wtCFTR and S737A/S768A-CFTRintheabsenceofstimulationwithIBMXandforskolinand effectsofphenformin,compoundC,andCFTRinh-172.DandE,baselinewhole cell conductances generated by wild type CFTR (D) and S737A/S768A-CFTR (E) and effects of compound C and the PKA inhibitor KT520. Login to comment
170 Moreover, the PKA inhibitor KT5720 (50 ␮M) inhibited this enhanced baseline CFTR conductance generated by S737A/ S768A irrespective of the presence of compound C (Fig. 4E). Login to comment
172 We interpret this finding to suggest that the sensitivity of S737A/ S768A-CFTR toward PKA inhibition is retained, which implies that PKA is now active after the loss of AMPK sensitivity. Login to comment
178 The initial recovery from PKA stimulation was equal in S737A/ S768A (1.1 Ϯ 0.3 ␮S/min) and wtCFTR (1.1 Ϯ 0.1 ␮S/min) (Fig. 5C), but was enhanced in ooctyes coexpressing kinase-dead AMPK␣1-K45R (2.8 Ϯ 0.4 ␮S/min), whereas overexpression of wtAMPK␣1beta1␥1 literally eliminated CFTR currents (Fig. 5B). Login to comment
180 Although AMPK largely antagonizes activation of CFTR, the mutated serines S737A and S768A do not seem to influence the recovery time from forskolin stimulation. Login to comment
210 C, inactivation of conductances generated by wtCFTR and S737A/S768A-CFTR. Login to comment
225 Even maximal stimulation of wtCFTR with a mixture of 8-Br- - cAMP, IBMX, and forskolin does not produce the same high level of conductance as S737A-CFTR or S768A-CFTR. Login to comment

[hide] State-dependent regulation of cystic fibrosis tran... J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15. Wang G
State-dependent regulation of cystic fibrosis transmembrane conductance regulator (CFTR) gating by a high affinity Fe3+ bridge between the regulatory domain and cytoplasmic loop 3.
J Biol Chem. 2010 Dec 24;285(52):40438-47. Epub 2010 Oct 15., 2010-12-24 [PMID:20952391]

Abstract [show]
The unique regulatory (R) domain differentiates the human CFTR channel from other ATP-binding cassette transporters and exerts multiple effects on channel function. However, the underlying mechanisms are unclear. Here, an intracellular high affinity (2.3 x 10(-19) M) Fe(3+) bridge is reported as a novel approach to regulating channel gating. It inhibited CFTR activity by primarily reducing an open probability and an opening rate, and inhibition was reversed by EDTA and phenanthroline. His-950, His-954, Cys-832, His-775, and Asp-836 were found essential for inhibition and phosphorylated Ser-768 may enhance Fe(3+) binding. More importantly, inhibition by Fe(3+) was state-dependent. Sensitivity to Fe(3+) was reduced when the channel was locked in an open state by AMP-PNP. Similarly, a K978C mutation from cytoplasmic loop 3 (CL3), which promotes ATP-independent channel opening, greatly weakened inhibition by Fe(3+) no matter whether NBD2 was present or not. Therefore, although ATP binding-induced dimerization of NBD1-NBD2 is required for channel gating, regulation of CFTR activity by Fe(3+) may involve an interaction between the R domain and CL3. These findings may support proximity of the R domain to the cytoplasmic loops. They also suggest that Fe(3+) homeostasis may play a critical role in regulating pathophysiological CFTR activity because dysregulation of this protein causes cystic fibrosis, secretary diarrhea, and infertility.

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162 Although Fig. 4E indicates that both S768A and S737A mutants were much inhibited by Fe3ϩ , these sites may not be excluded as Fe3ϩ ligands. Login to comment
180 Unlike S768A, S737A exhibited a weak DTT effect (Fig. 6D), suggesting that phosphorylated Ser-737 may not participate in Fe3ϩ binding. Login to comment

[hide] The inhibition mechanism of non-phosphorylated Ser... J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8. Wang G
The inhibition mechanism of non-phosphorylated Ser768 in the regulatory domain of cystic fibrosis transmembrane conductance regulator.
J Biol Chem. 2011 Jan 21;286(3):2171-82. Epub 2010 Nov 8., 2011-01-21 [PMID:21059651]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a member of the ATP-binding cassette transporters but serves as a chloride channel dysfunctional in cystic fibrosis. The activity of CFTR is tightly controlled not only by ATP-driven dimerization of its nucleotide-binding domains but also by phosphorylation of a unique regulatory (R) domain by protein kinase A (PKA). The R domain has multiple excitatory phosphorylation sites, but Ser(737) and Ser(768) are inhibitory. The underlying mechanism is unclear. Here, sulfhydryl-specific cross-linking strategy was employed to demonstrate that Ser(768) or Ser(737) could interact with outwardly facing hydrophilic residues of cytoplasmic loop 3 regulating channel gating. Furthermore, mutation of these residues to alanines promoted channel opening by curcumin in an ATP-dependent manner even in the absence of PKA. However, mutation of Ser(768) and His(950) with different hydrogen bond donors or acceptors clearly changed ATP- and PKA-dependent channel activity no matter whether curcumin was present or not. More importantly, significant activation of a double mutant H950R/S768R needed only ATP. Finally, in vitro and in vivo single channel recordings suggest that Ser(768) may form a putative hydrogen bond with His(950) of cytoplasmic loop 3 to prevent channel opening by ATP in the non-phosphorylated state and by subsequent cAMP-dependent phosphorylation. These observations support an electron cryomicroscopy-based structural model on which the R domain is closed to cytoplasmic loops regulating channel gating.

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143 In contrast, curcumin had no such effect on S737A and H954A mutants, suggesting that they may be weak inhibitory residues, although disulfide cross-linking of S737C to H954C strongly inhibited channel activity (Figs. 2E and 4E). Login to comment
144 Because curcumin increased initial channel activity of most mutants at the R-CL3 interface after ATP was present, it is reasonable that subsequent PKA dependence was greatly reduced except for S737A and H954A. Login to comment
234 Error bars, S.E. TABLE 1 Potential roles in hydrogen bonding at the CL3-R domain interface Note that mutants whose channel activity was increased by curcumin in the presence of ATP are highlighted in boldface type. Residues Role in H-bond Mutants Arg Strong donor H950R, S768R, H950R/S768R, H950R/S768D, H950D/S768R Asp Strong acceptor H950D, S768D, H950D/S768D, H950R/S768D, H950D/S768R Thr, Gln, Ser, His Donor/Acceptor H950Q, S768T, WT Ala Negative control K946A, H950A, K951A, H954A, S955A, Q958A, S737A, S768A, ⌬R Inhibition of CFTR by Ser768 JANUARY 21, 2011•VOLUME 286•NUMBER 3 JOURNAL OF BIOLOGICAL CHEMISTRY 2177 matter whether cAMP was present or not in the extracellular perfusate. Login to comment
311 In contrast, the S737A mutation failed to promote channel opening by ATP and curcumin, although it was also closed to CL3 (Figs. 2-4). Login to comment

[hide] Structural models of CFTR-AMPK and CFTR-PKA intera... J Mol Model. 2011 Apr 1. Siwiak M, Edelman A, Zielenkiewicz P
Structural models of CFTR-AMPK and CFTR-PKA interactions: R-domain flexibility is a key factor in CFTR regulation.
J Mol Model. 2011 Apr 1., 2011-04-01 [PMID:21455600]

Abstract [show]
Cystic fibrosis (CF), the most common lethal genetic disease among Caucasians, is caused by mutations in cystic fibrosis transmembrane conductance regulator (CFTR). CFTR's main role is to transport chloride ions across epithelial cell membranes. It also regulates many cell functions. However, the exact role of CFTR in cellular processes is not yet fully understood. It is recognized that a key factor in CFTR-related regulation is its phosphorylation state. The important kinases regulating CFTR are cAMP-dependent protein kinase A (PKA) and 5'-AMP-activated protein kinase (AMPK). PKA and AMPK have opposite effects on CFTR activity despite their highly similar structures and recognition motifs. Utilizing homology modeling, in silico mutagenesis and literature mining, we supplement available information regarding the atomic-resolution structures of PKA, AMPK and CFTR, and the complexes CFTR-PKA and CFTR-AMPK. The atomic-resolution structural predictions reveal an unexpected availability of CFTR Ser813 for phosphorylation by both PKA and AMPK. These results indicate the key role of the structural flexibility of the serine-rich R-domain in CFTR regulation by phosphorylation.

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121 However, experiments [7] have shown an 80% decrease in AMPK phosphorylation for the CFTR double mutant S737A-S768A. Login to comment
156 This shows that, at the molecular level, it is possible that 20% of the currently unexplained phosphorylation signal observed in the CFTR double mutant S737A-S768A incubated with AMPK may come from "activator" serines such as S813. Login to comment
155 This shows that, at the molecular level, it is possible that 20% of the currently unexplained phosphorylation signal observed in the CFTR double mutant S737A-S768A incubated with AMPK may come from "activator" serines such as S813. Login to comment

[hide] Role of individual R domain phosphorylation sites ... Biochim Biophys Acta. 2009 Jun;1788(6):1341-9. Epub 2009 Mar 26. Hegedus T, Aleksandrov A, Mengos A, Cui L, Jensen TJ, Riordan JR
Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A.
Biochim Biophys Acta. 2009 Jun;1788(6):1341-9. Epub 2009 Mar 26., [PMID:19328185]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.

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132 On the other hand, the much lower rate of S813 phosphorylation appeared to be reduced somewhat further by either the S700A or S737A substitutions, but not by the S768A substitution. Login to comment
152 Membranes isolated from BHK cells expressing S700A (A), S737A (B), S768A (C), or S813A (D) were subjected to phosphorylation for 0, 2, 4, 8, 16, 32, and 64 min. Login to comment
242 The S737A and S768A constructs exhibit slightly higher basal open probability (Po =0.06) compared to wild type (Po ≪0.01) in good agreement with data of Csanady et al. [24]. Login to comment
245 The channel properties of the S737A mutant were previously reported to be very similar to those of the wild type by Wilkinson et al. [10]. Login to comment
131 On the other hand, the much lower rate of S813 phosphorylation appeared to be reduced somewhat further by either the S700A or S737A substitutions, but not by the S768A substitution. Login to comment
151 Membranes isolated from BHK cells expressing S700A (A), S737A (B), S768A (C), or S813A (D) were subjected to phosphorylation for 0, 2, 4, 8, 16, 32, and 64 min. Login to comment
241 The S737A and S768A constructs exhibit slightly higher basal open probability (Po =0.06) compared to wild type (Po âa;0.01) in good agreement with data of Csanady et al. [24]. Login to comment
244 The channel properties of the S737A mutant were previously reported to be very similar to those of the wild type by Wilkinson et al. [10]. Login to comment

[hide] Computational studies reveal phosphorylation-depen... J Mol Biol. 2008 May 16;378(5):1052-63. Epub 2008 Mar 26. Hegedus T, Serohijos AW, Dokholyan NV, He L, Riordan JR
Computational studies reveal phosphorylation-dependent changes in the unstructured R domain of CFTR.
J Mol Biol. 2008 May 16;378(5):1052-63. Epub 2008 Mar 26., [PMID:18423665]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent chloride channel that is mutated in cystic fibrosis, an inherited disease of high morbidity and mortality. The phosphorylation of its approximately 200 amino acid R domain by protein kinase A is obligatory for channel gating under normal conditions. The R domain contains more than ten PKA phosphorylation sites. No individual site is essential but phosphorylation of increasing numbers of sites enables progressively greater channel activity. In spite of numerous studies of the role of the R domain in CFTR regulation, its mechanism of action remains largely unknown. This is because neither its structure nor its interactions with other parts of CFTR have been completely elucidated. Studies have shown that the R domain lacks well-defined secondary structural elements and is an intrinsically disordered region of the channel protein. Here, we have analyzed the disorder pattern and employed computational methods to explore low-energy conformations of the R domain. The specific disorder and secondary structure patterns detected suggest the presence of molecular recognition elements (MoREs) that may mediate phosphorylation-regulated intra- and inter-domain interactions. Simulations were performed to generate an ensemble of accessible R domain conformations. Although the calculated structures may represent more compact conformers than occur in vivo, their secondary structure propensities are consistent with predictions and published experimental data. Equilibrium simulations of a mimic of a phosphorylated R domain showed that it exhibited an increased radius of gyration. In one possible interpretation of these findings, by changing its size, the globally unstructured R domain may act as an entropic spring to perturb the packing of membrane-spanning sequences that constitute the ion permeability pathway and thereby activate channel gating.

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28 Replacement of serine with alanine at position 737 or 768 was reported to produce a channel with a higher level of activity in both the non-phosphorylated and phosphorylated state.16,19-21 These studies led to the indirect conclusion that these two sites are inhibitory in their phosphorylated state. Login to comment
27 Replacement of serine with alanine at position 737 or 768 was reported to produce a channel with a higher level of activity in both the non-phosphorylated and phosphorylated state.16,19-21 These studies led to the indirect conclusion that these two sites are inhibitory in their phosphorylated state. Login to comment

[hide] Stimulation of CFTR activity by its phosphorylated... Nature. 1997 Sep 18;389(6648):294-6. Winter MC, Welsh MJ
Stimulation of CFTR activity by its phosphorylated R domain.
Nature. 1997 Sep 18;389(6648):294-6., [PMID:9305845]

Abstract [show]
Phosphorylation controls the activity of ion channels in many tissues. In epithelia, the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by phosphorylation of serine residues in its regulatory (R) domain and then gated by binding and hydrolysis of ATP by the nucleotide-binding domains. Current models propose that the unphosphorylated R domain serves as an inhibitory particle that occludes the pore, much like the inhibitory 'ball' in Shaker K+ channels; presumably, phosphorylation relieves this inhibition. Here we test this by adding an R-domain peptide to a CFTR variant in which much of the R domain had been deleted (CFTR-deltaR/S660A): in contrast to predictions, we found that adding an unphosphorylated R domain to CFTR-deltaR/S660A did not inhibit activity, whereas a phosphorylated R-domain peptide stimulated activity. To investigate how phosphorylation controls activity, we studied channel gating and found that phosphorylation of the R domain increases the rate of channel opening by enhancing the sensitivity to ATP. Our results indicate that CFTR is regulated by a new mechanism in which phosphorylation of one domain stimulates the interaction of ATP with another domain, thereby increasing activity.

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162 Mutation of the individual serines reduced Po, but to different extents: the S795A mutation had the largest effect and S737A had little or no effect in the presence of 1 mM ATP (Fig. 3a, b). Login to comment
185 Number of experiments for b/c and d were: 17/6 for wild-type (WT), 8 for wild-type (-PKA), 8/7 for S660A, 5/5 for S737A, 8/6 for S795A, 9/7 for S813A, and 11/4 for S-Quad-A. Login to comment
188 0.0 0.1 0.2 0.3 0.4 0.5 0.6 Po 0 0.2 0.4 0.6 0.8 1 10 [ATP] (mM) S813A S795A S737A S660A WT (-PKA) WT Figure 4 Effect of ATP concentration on Po of CFTR containing phosphorylation site mutations. Login to comment

[hide] CFTR activation: additive effects of stimulatory a... Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33. Wilkinson DJ, Strong TV, Mansoura MK, Wood DL, Smith SS, Collins FS, Dawson DC
CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain.
Am J Physiol. 1997 Jul;273(1 Pt 1):L127-33., [PMID:9252549]

Abstract [show]
To investigate the functional significance of individual consensus phosphorylation sites within the R domain of cystic fibrosis transmembrane conductance regulator (CFTR), serines were eliminated by substituting them with alanine. Included in this analysis were serine-660, -670, -686, -700, -712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimination of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -670, -700, -795, or -813 significantly increased the half-maximal activation constant (KA) for activation by 3-isobutyl-1-methylxanthine, which is consistent with the hypothesis that phosphorylation at any of these sites promotes CFTR activation. The effect of substitution at serine-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Substitution at serine-641, -686, and -712 had no significant effect on activation sensitivity. The effects of multiple serine to alanine substitutions were consistent with the notion that phosphorylation at individual sites produced roughly additive effects, suggesting that the effect produced by phosphorylation of any one serine was not dependent on the phosphorylation state of other serines. These results are consistent with the notion that, although none of the phosphorylation sites studied here are absolutely necessary for activation of CFTR, individual sites contribute differently to the gating of the channel.

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15 In contrast, alanine substitution at serine-737 or -768 actually decreased the KA for activation, suggesting that phosphorylation at either of these sites is inhibitory. Login to comment
87 S686A 0.75 ?I 0.07 9 N PKC site S700A 0.86 t 0.07* 12 S ++ +++ S712A 0.67 t 0.12 9 N - ++ S737A 0.35 5 0.05* 8 I +++ +++ S768A 0.09 IT 0.03* 8 I - ++++ S795A 1.24 + 0.22* 9 S +++ ++++ S813A 3.18-+0.36* 6 S ++++ ++ Values of half-maximal inhibition constant for activation (KA) are means + SE by 3-isobutyl-1-methylxanthine (IBMX) obtained for wild-type cystic fibrosis transmembrane conductance regulator (CFTR) and variants with single serine-to-alanine substitutions. Login to comment
107 Symbols show averaged IBMX dose-response data for wild-type CFTR and several single-site serine-to-alanine mutants (S660A, S737A, S768A, S795A, and S813A). Login to comment

[hide] Regulation of the cystic fibrosis transmembrane co... J Biol Chem. 1993 Sep 25;268(27):20259-67. Rich DP, Berger HA, Cheng SH, Travis SM, Saxena M, Smith AE, Welsh MJ
Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain.
J Biol Chem. 1993 Sep 25;268(27):20259-67., [PMID:7690753]

Abstract [show]
Phosphorylation by cAMP-dependent protein kinase (PKA) regulates the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel. We previously showed that in vivo PKA phosphorylated 4 serines (Ser-660, Ser-737, Ser-795, and Ser-813) within the R domain. Here we show that a mutant CFTR lacking all 4 serines can still be phosphorylated by PKA to yield an activated Cl- channel, but channel open-state probability was substantially reduced. We also observed phosphorylation and Cl- channel activity in another mutant lacking all 8 consensus PKA serines in the R domain. We were unable to identify the residual phosphorylation sites by tryptic phosphopeptide mapping. These data suggest two possible interpretations: (a) additional, as yet unidentified, phosphorylation sites within CFTR may also open the channel, or (b) the 4 serines, previously identified as in vivo PKA phosphorylation sites, are the primary regulatory sites within CFTR, but in their absence, other sites can be phosphorylated to open the channel. The additional sites are likely located within the R domain: CFTR delta R-S660A, which lacks much of the R domain (residues 708-835) and replaces Ser-660 with an alanine, was no longer regulated by PKA. Substitution of aspartate for consensus PKA phosphorylation sites in the R domain mimicked the effect of phosphorylation. Mutants containing six or more serine-to-aspartate substitutions generated Cl- channels that opened without PKA phosphorylation. These results suggest that the R domain keeps the channel closed and that phosphorylation of the R domain or insertion of the negatively charged aspartate opens the channel, perhaps by electrostatic interactions.

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48 Mutants are named by the number of the amino acid residue preceded by the wild-type amino acid and followed bythe aminoacid to which the residue was changed using the single-letter aminoacid code; for example,inthemutant CFTR S66OA,S737A,S795A,S813A (also called CFTR S-Quad-A for convenience), alanines are substituted for serines at amino acid positions 660, 737, 795, and 813. Login to comment
66 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) weretran- siently expressed in HeLa cells. Login to comment
89 Functional assay of CFTR S-Quad-Aby SPQ fluorescence.The changein SPQfluorescenceis shown for HeLa cellsexpress- ing wild-type CFTR (n = 53, where n = number of cells) or CFTR S-Quad-A tS660A,S737A,S795A,S813A)(n = 53) and for virus only- infectedcells(no plasmid) as a negative control (n = 47). Login to comment
94 Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment
109 RESULTS Serine-to-Alanine Substitutionsin the R Domain Do NotAbolish CFTR Cl- Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still bephosphorylated in vivofollowing CAMPstimulation. Login to comment
121 The single-channel open-stateprobabil- ity for wild-type CFTR (n = 14), CFTR S-Quad-A (S600A,S737A, S712A,S737A,S768A,S795A,S813A) (n = 7), or CFTR S-Oct-D (S660D,S686D,S700D,S712D,S737D,S768D,S795D,S813D)(n = 7in ATP alone (-PKA);n = 10 inPKA and ATP (+PkX))C1-channels was determined as described under "Experimental Procedures." Login to comment
123 S795A,S813A) (n = 5). Login to comment
139 CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A)( B )were transiently expressed inCOS-7 cells. Login to comment
174 Fig. 1lA shows that simultaneous substitutionof the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A MockS-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B)or pMT-CFl`R S-Oct-A FIG.9. Login to comment
176 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A)(C). Login to comment
47 Mutants are named by the number of the amino acid residue preceded by the wild-type amino acid and followed bythe aminoacid to which the residue was changed using the single-letter amino acid code; for example,inthemutant CFTR S66OA,S737A,S795A,S813A (also called CFTR S-Quad-A for convenience), alanines are substituted for serines at amino acid positions 660, 737, 795, and 813. Login to comment
65 In vivo phosphorylation ofwild-typeand mutant CFTR CFTR S-Quad-A (S66OA,S737A,S795A,S813A), or CFTR S-Oct-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) were transiently expressed in HeLa cells. Login to comment
95 Single-channelanalysis of gle-channel traces from an excised, in-cell expressing CFTR S-Quad-A side-out membrane patch from a HeLa (S660A,S737A,S795A,S813A).Each trace is about 14s long. Login to comment
111 RESULTS Serine-to-Alanine Substitutions in the R Domain Do NotAbolish CFTR Cl-Channel Activity-& a firststep to address the possibility that additional PKA phosphorylation sites might regulate CFTR, we asked whether the S-Quad-A (S660A,S737A,S795A,S813A) mutant could still be phosphorylated in vivofollowing CAMPstimulation. Login to comment
142 CFTR (A) or CFTR S-Quad-A (S660A,S737A,S795A,S813A) ( B )were transiently expressed inCOS-7 cells. Login to comment
178 Fig. 1lA shows that simultaneous substitution of the four in vivo PKA sites with aspartates (in the S-Quad-D mutant) did not generate constitutively active CFTR C1-channels as assessed by the SPQ fluorescence assay: the mutant channels opened only after stimulation by CAMP.We observed similar results withCFTR S-Quint-D which contained substitutionsof A Mock S-Hept-A C S-OCt-A transfected (A) or were transfected with pMT-CFTR S-Hept-A (S660A,S686A,S700A,S712A,S737A,S768A,S795A) (B) or pMT-CFl`R S-Oct-A FIG. 9. Login to comment
180 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) (C). Login to comment

[hide] Protein kinase A (PKA) still activates CFTR chlori... J Biol Chem. 1993 May 25;268(15):11304-11. Chang XB, Tabcharani JA, Hou YX, Jensen TJ, Kartner N, Alon N, Hanrahan JW, Riordan JR
Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites.
J Biol Chem. 1993 May 25;268(15):11304-11., [PMID:7684377]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) plays a central role in transepithelial ion transport by acting as a tightly regulated apical chloride channel. Regulation is achieved by the concerted action of ATP at conserved nucleotide binding folds and serine phosphorylation at multiple sites by protein kinases A (PKA) and C (PKC). A previous investigation concluded that activation by PKA is critically dependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A, S813A), because a "Quad" mutant lacking these sites could not be activated. We show in the present work that not only can this mutant be phosphorylated and activated, but a mutant in which all 10 predicted PKA sites have been altered still retains significant PKA-activated function. Potentiation of the PKA response by PKC is also preserved in this mutant. Thus CFTR may be regulated by cryptic PKA sites which also mediate interactions between different kinases. Such hierarchical phosphorylation of CFTR by obvious and cryptic PKA sites could provide a metered response to secretagogues.

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7 A previous investigationconcluded thatactivationby PKA is crit- icallydependent on phosphorylation at four of the nine predicted PKA sites in the R domain (S660A, S737A, S795A,SS13A), becausea "Quad"mutant lacking these sites couldnotbeactivated. Login to comment
37 The following mutations were introduced into CFTR, S422A (TCT to GCT), S660A (TCA to GCA), S686A (TCT to GCT), S700A (TCT toGCT), S712A (TCC to GCC), S737A (TCC to GCC), S768A (TCT toGCT), T788A (ACAto GCA),S795A (TCA to GCA), S813A (TCA to GCA), S660E (TCA to GAA),S737E (TCC to GAG), S795E (TCA to GAA), and S813E (TCA to GAA). Login to comment
39 The counterparts of CFTR cDNA in pUCF2.5 were replaced by PCR- mutated versions by interchange of the following fragments, S660A, DraIIIIEcoRI fragment, S737A, EcoRIIHpaIfragment, S795A and S813A,StyIIStyI fragment(Fig. 1A). Login to comment
40 A mutant containing S660/737/ 795/813A (pUCF2.5/4SA) was assembled by replacing the counterparts of a plasmid containing S795/813A with the DraIII/EcoRI fragment (S660A) and EcoRI/HpaI fragment (S737A). Login to comment

[hide] Role of tyrosine phosphorylation in the muscarinic... J Biol Chem. 2013 Jul 26;288(30):21815-23. doi: 10.1074/jbc.M113.479360. Epub 2013 Jun 11. Billet A, Luo Y, Balghi H, Hanrahan JW
Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR).
J Biol Chem. 2013 Jul 26;288(30):21815-23. doi: 10.1074/jbc.M113.479360. Epub 2013 Jun 11., [PMID:23760269]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride (Cl(-)) channel, which plays an important role in physiological anion and fluid secretion, and is defective in several diseases. Although its activation by PKA and PKC has been studied extensively, its regulation by receptors is less well understood. To study signaling involved in CFTR activation, we measured whole-cell Cl(-) currents in BHK cells cotransfected with GPCRs and CFTR. In cells expressing the M3 muscarinic acetylcholine receptor, the agonist carbachol (Cch) caused strong activation of CFTR through two pathways; the canonical PKA-dependent mechanism and a second mechanism that involves tyrosine phosphorylation. The role of PKA was suggested by partial inhibition of cholinergic stimulation by the specific PKA inhibitor Rp-cAMPS. The role of tyrosine kinases was suggested by Cch stimulation of 15SA-CFTR and 9CA-CFTR, mutants that lack 15 PKA or 9 PKC consensus sequences and are unresponsive to PKA or PKC stimulation, respectively. Moreover the residual Cch response was sensitive to inhibitors of the Pyk2 and Src tyrosine kinase family. Our results suggest that tyrosine phosphorylation acts on CFTR directly and through inhibition of the phosphatase PP2A. Results suggest that PKA and tyrosine kinases contribute to CFTR regulation by GPCRs that are expressed at the apical membrane of intestinal and airway epithelia.

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102 Carbachol Stimulates CFTR through PKA and Non-PKA Signaling Pathways-To explore PKA-independent regulation of CFTR without using inhibitors that might have confounding effects on other pathways, we studied the activation of 15SA-CFTR (S422A/S660A/S670A/S686A/T690A/S700A/S712A/ S737A/S753A/S768A/T787A/T788A/S790A/S795A/S813A). Login to comment

[hide] LMTK2-mediated phosphorylation regulates CFTR endo... J Biol Chem. 2014 May 23;289(21):15080-93. doi: 10.1074/jbc.M114.563742. Epub 2014 Apr 11. Luz S, Cihil KM, Brautigan DL, Amaral MD, Farinha CM, Swiatecka-Urban A
LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.
J Biol Chem. 2014 May 23;289(21):15080-93. doi: 10.1074/jbc.M114.563742. Epub 2014 Apr 11., [PMID:24727471]

Abstract [show]
Cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl(-)-selective ion channel expressed in fluid-transporting epithelia. Lemur tyrosine kinase 2 (LMTK2) is a transmembrane protein with serine and threonine but not tyrosine kinase activity. Previous work identified CFTR as an in vitro substrate of LMTK2, suggesting a functional link. Here we demonstrate that LMTK2 co-immunoprecipitates with CFTR and phosphorylates CFTR-Ser(737) in human airway epithelial cells. LMTK2 knockdown or expression of inactive LMTK2 kinase domain increases cell surface density of CFTR by attenuating its endocytosis in human airway epithelial cells. Moreover, LMTK2 knockdown increases Cl(-) secretion mediated by the wild-type and rescued DeltaF508-CFTR. Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. These results demonstrate a novel mechanism of the phospho-dependent inhibitory effect of CFTR-Ser(737) mediated by LMTK2 via endocytosis and inhibition of the cell surface density of CFTR Cl(-) channels. These data indicate that targeting LMTK2 may increase the cell surface density of CFTR Cl(-) channels and improve stability of pharmacologically rescued DeltaF508-CFTR in patients with cystic fibrosis.

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8 Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. Login to comment
203 To demonstrate specificity of the phosphosite antibody, parental CFBE41o-cells were transiently transfected with WT-CFTR or CFTR with the S737A substitution (CFTR-S737A) inactivating the phosphorylation site (31). Login to comment
204 Even with calyculin A treatment of the cells, the CFTR-S737A mutant was not detected by antibody Ab-737, unlike the WT-CFTR (Fig. 5B). Login to comment
233 B, parental CFBE41o-cells were transfected with the WT-CFTR (WT) or the mutant CFTR-S737A (S737A). Login to comment
234 Unlike the WT-CFTR, the CFTR-S737A was not detected by the antibody Ab-737 in WCL. Login to comment
287 The Phosphorylation-deficient CFTR-S737A Mutant is More Abundant at the Plasma Membrane Due to Decreased Endocytosis-If the LMTK2 mediated phosphorylation of CFTR-Ser737 induces CFTR endocytosis, eliminating the phosphorylation site by the S737A substitution should reduce CFTR endocytosis. Login to comment
288 Parental CFBE41o-cells were transfected with WT-CFTR or the CFTR-S737A mutant. Login to comment
308 The S737A mutation attenuated CFTR endocytosis and increased the plasma membrane abundance of CFTR (Fig. 8). Login to comment
331 Experiments demonstrating that compared with WT-CFTR the phosphorylation-deficient mutant CFTR S737A has increased plasma membrane abundance at steady-state and decreased endocytosis in CFBE41o-cells. Login to comment
332 Parental CFBE41o-cells were transfected with the WT-CFTR (WT) or the CFTR-S737A mutant (S737A), and cells were cultured on collagen-coated tissue culture plates for 48 h to form monolayers. Login to comment
334 Immunoblots (A) and summaryofdata(B)demonstratingthattheS737AmutationdecreasedCFTRendocytosis.TheamountofbiotinylatedWT-CFTRorCFTR-S737Aremainingafter the GSH treatment at 4 &#b0;C without warming to 37 &#b0;C was considered background and was subtracted from the amount of biotinylated WT-CFTR or CFTR-S737A remaining after warming to 37 &#b0;C. Login to comment
335 Endocytosis of WT-CFTR or CFTR-S737A was calculated after subtracting the background (see above) and was expressed as the percent of WT-CFTR or CFTR-S737A remaining biotinylated before and after warming to 37 &#b0;C. WT-CFTR and CFTR-S737A was detected with antibody CFF596. Login to comment
338 The whole cell lysate (WCL) expression of WT-CFTR or CFTR-S737A mutant detected with antibody CFF596 was used as a loading control to account for small differences in the expression levels of the transiently transfected proteins. Login to comment

[hide] Sphingosine-1-Phosphate Is a Novel Regulator of Cy... PLoS One. 2015 Jun 16;10(6):e0130313. doi: 10.1371/journal.pone.0130313. eCollection 2015. Malik FA, Meissner A, Semenkov I, Molinski S, Pasyk S, Ahmadi S, Bui HH, Bear CE, Lidington D, Bolz SS
Sphingosine-1-Phosphate Is a Novel Regulator of Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Activity.
PLoS One. 2015 Jun 16;10(6):e0130313. doi: 10.1371/journal.pone.0130313. eCollection 2015., [PMID:26079370]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) attenuates sphingosine-1-phosphate (S1P) signaling in resistance arteries and has emerged as a prominent regulator of myogenic vasoconstriction. This investigation demonstrates that S1P inhibits CFTR activity via adenosine monophosphate-activated kinase (AMPK), establishing a potential feedback link. In Baby Hamster Kidney (BHK) cells expressing wild-type human CFTR, S1P (1mumol/L) attenuates forskolin-stimulated, CFTR-dependent iodide efflux. S1P's inhibitory effect is rapid (within 30 seconds), transient and correlates with CFTR serine residue 737 (S737) phosphorylation. Both S1P receptor antagonism (4mumol/L VPC 23019) and AMPK inhibition (80mumol/L Compound C or AMPK siRNA) attenuate S1P-stimluated (i) AMPK phosphorylation, (ii) CFTR S737 phosphorylation and (iii) CFTR activity inhibition. In BHK cells expressing the DeltaF508 CFTR mutant (CFTRDeltaF508), the most common mutation causing cystic fibrosis, both S1P receptor antagonism and AMPK inhibition enhance CFTR activity, without instigating discernable correction. In summary, we demonstrate that S1P/AMPK signaling transiently attenuates CFTR activity. Since our previous work positions CFTR as a negative S1P signaling regulator, this signaling link may positively reinforce S1P signals. This discovery has clinical ramifications for the treatment of disease states associated with enhanced S1P signaling and/or deficient CFTR activity (e.g. cystic fibrosis, heart failure). S1P receptor/AMPK inhibition could synergistically enhance the efficacy of therapeutic strategies aiming to correct aberrant CFTR trafficking.

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123 (F) S1P fails to inhibit iodide efflux in na&#ef;ve BHK cells transiently transfected with a plasmid encoding a mutated CFTR containing a serine-to-alanine substitution at amino acid 737 (CFTRS737A ). Login to comment