ABCMdb

ABCC7 p.Ser686Ala

CF databases:	c.2057C>A , p.Ser686Tyr (CFTR1) ? ,
Predicted by SNAP2:	A: N (72%), C: N (57%), D: N (66%), E: N (78%), F: D (59%), G: N (78%), H: N (57%), I: D (59%), K: N (66%), L: D (59%), M: D (53%), N: N (82%), P: D (59%), Q: N (82%), R: D (53%), T: N (82%), V: D (53%), W: D (63%), Y: N (61%),
Predicted by PROVEAN:	A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: N,

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[hide] PKC-mediated stimulation of amphibian CFTR depends... J Gen Physiol. 2001 May;117(5):457-68. Button B, Reuss L, Altenberg GA
PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR.
J Gen Physiol. 2001 May;117(5):457-68., [PMID:11331356]

Abstract [show]
Mutations of the CFTR, a phosphorylation-regulated Cl(-) channel, cause cystic fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in the regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PKC is required for the effect of PKA on CFTR, stimulation of PKC by itself has only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagenesis experiments indicate that a single site (Thr665) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus site that includes Thr665, but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the presence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylation site and is unrelated to the net charge of the R domain.

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73 plus addition of HaeIII site), 5Ј-GTCAAGAATAAAGCTTTTAAG- CAGG-3Ј (Ser686 to Ala, plus addition of HindIII site), 5Ј-TGGG- GATTTCGCTGAGAAAAGAAAGAG-3Ј (Ser694 to Ala, plus addition of DdeI site), and 5Ј-CAAGAAAAACTGCAGTTCG- TAAAATG-3Ј (Ser790 to Ala, plus addition of PstI site). Login to comment
202 Substitution of the two conserved serine residues known to be phosphorylated (Ser686 and Ser790; Picciotto et al., 1992) with alanine residues (S686A/ S790A-XCFTR; Fig. 7, A and C) did not affect the activation by PMA. Login to comment
214 (A) Representative I-V relationships from an oocyte expressing the double knockout of conserved PKC consensus phosphorylation sites (S686A/S790A-XCFTR). Login to comment
224 The data in Fig. 9 indicate that this is not the case because the cAMP-activated currents are not different among oocytes expressing wild-type XCFTR, S686A/ S790A-XCFTR, or T665A/S694A-XCFTR. Login to comment
246 Time course of the currents after stimulation with the cAMP cocktail in oocytes injected with wild-type XCFTR, S686A/S790A-XCFTR, or T665A/S694A-XCFTR cRNAs. Login to comment

[hide] Phosphorylation of protein kinase C sites in NBD1 ... J Physiol. 2003 Apr 1;548(Pt 1):39-52. Epub 2003 Feb 14. Chappe V, Hinkson DA, Zhu T, Chang XB, Riordan JR, Hanrahan JW
Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA.
J Physiol. 2003 Apr 1;548(Pt 1):39-52. Epub 2003 Feb 14., 2003-04-01 [PMID:12588899]

Abstract [show]
Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA' mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5-10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA.

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14 To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA` mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). Login to comment
145 To distinguish these possible mechanisms we constructed a mutant (9CA) in which all PKC consensus sequences between the Walker B motif of NBD1 and second transmembrane domain (TMD2; i.e. the seventh membrane-spanning segment; T582A, T604A, S641A, T682A, S686A, S707A, S790A, T791A and S809A) were eliminated (Fig. 4A and B). Login to comment

[hide] Stimulatory and inhibitory protein kinase C consen... Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):390-5. Epub 2003 Dec 26. Chappe V, Hinkson DA, Howell LD, Evagelidis A, Liao J, Chang XB, Riordan JR, Hanrahan JW
Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator.
Proc Natl Acad Sci U S A. 2004 Jan 6;101(1):390-5. Epub 2003 Dec 26., 2004-01-06 [PMID:14695900]

Abstract [show]
Protein kinase C (PKC) phosphorylation stimulates the cystic fibrosis transmembrane conductance regulator (CFTR) channel and enhances its activation by protein kinase A (PKA) through mechanisms that remain poorly understood. We have examined the effects of mutating consensus sequences for PKC phosphorylation and report here evidence for both stimulatory and inhibitory sites. Sequences were mutated in subsets and the mutants characterized by patch clamping. Activation of a 4CA mutant (S707A/S790A/T791A/S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A/T604A/S641A) and 2CA (T682A/S686A) channels to PKA were both drastically reduced (>90%). When each mutation in the 3CA and 2CA constructs was studied individually in a wild-type background, T582, T604, and S686 were found to be essential for PKA activation. Responses were restored when these three residues were reintroduced simultaneously into a 9CA mutant lacking all nine PKC consensus sequences (R6CA revertant); however, PKC phosphorylation was not required for this rescue. Nevertheless, two of the sites (T604 and S686) were phosphorylated in vitro, and PKC alone partially activated wild-type CFTR, the 4CA mutant, and the point mutants T582A and T604A, but not S686A channels, indicating that PKC does act at S686. The region encompassing S641 and T682 is inhibitory, because S641A enhanced activation by PKA, and T682A channels had 4-fold larger responses to PKC compared to wild-type channels. These results identify functionally important PKC consensus sequences on CFTR and will facilitate studies of its convergent regulation by PKC and PKA.

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No. Sentence Comment
3 Activation of a 4CA mutant (S707A͞S790A͞T791A͞S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A͞T604A͞S641A) and 2CA (T682A͞ S686A) channels to PKA were both drastically reduced (>90%). Login to comment
6 Nevertheless, two of the sites (T604 and S686) were phosphorylated in vitro, and PKC alone partially activated wild-type CFTR, the 4CA mutant, and the point mutants T582A and T604A, but not S686A channels, indicating that PKC does act at S686. Login to comment
45 Individual point mutations T582A, T604A, S641A, T682A, and S686A were subsequently introduced into wild-type CFTR by using the QuickChange (Stratagene) mutagenesis kit, which was also used to create a revertant mutant R6CA (A582T͞A604T͞A686S-9CA) in which three wild-type PKC consensus sequences were restored in the 9CA mutant described (18). Login to comment
59 (C) Western blot showing BHK cells stably transfected with wild-type CFTR (lane 1), 9CA-CFTR (lane 2), R6CA-CFTR (lane 3), T582A-CFTR (lane 4), T604A (lane 5), S641A (lane 6), T682A (lane 7), or S686A (lane 8). Login to comment
86 The peptides (Ͼ85% purity) used in this study were as follows (͞denotes trypsin cut site, radiolabeled peptide sequences are shown in capital letters, and predicted PKC-phosphorylated residues are underlined): T604,͞lmank͞tr͞ILVTSK͞mehlk͞; T682-(S686A), FSLEGDAPVSWTETK͞k͞qafk͞; S686- (T682A), teak͞k͞QSFK͞qtgefgek͞; S707,͞NSILNPINSIR͞k͞ fsivqk͞; S790, ihr͞k͞TTASTR͞k͞vsla. Login to comment
112 Den- sitometry of Western blots (Fig. 1C) revealed that most mutants had moderately reduced expression compared to wild-type CFTR (T682A 80.2%, S641A 79.2%, T582A 70.5%, S686A 45.4%, and T604A 24.6%; P Ͻ 0.05, n ϭ four to six blots of each mutant). Login to comment
113 PKA and PKC activation of T582A, T604A, S641A, T682A, and S686A channels was assessed by using the same protocol as in Fig. 1. Login to comment
114 Two single mutants (S641A and T682A) were activated by PKA, whereas the other three (T582A, T604A, and S686A) had only small responses (reduced by Ͼ90%; Fig. 2 A-C; note logarithmic scale of the ordinate in Fig. 2C), indicating that among the mutations in 2CA and 3CA, T582A, T604A, and S686A are essential for CFTR activation. Login to comment
115 T582A was partially responsive to 200-400 units͞ml PKA (Ϸ26% compared to wild-type CFTR) (Fig. 2 A and B), whereas activations of T604A and S686A were nearly abolished at all PKA concentrations tested. Login to comment
116 Maximum currents mediated by T604A and S686A channels are summarized in Fig. 2C for comparison with wild-type channels. Login to comment
118 S686A channels appeared more active than T604A channels, but this difference was small enough to be explained by their higher expression (compare Fig. 1C). Login to comment
120 PKA responses of T582A, T604A, and S686A were not enhanced by PKC pretreatment, therefore we expected their stimulation by PKC alone to be similarly impaired; however, this was observed only for S686A (NPo ϭ 0.53 Ϯ 0.2 vs. 1.2 Ϯ 0.38; n ϭ 6, P ϭ 0.015). Login to comment
123 (A) Recordings of T582A, T604A, and S686A channels by using the inside-out configuration [pipette potential (Vp) ϭ ϩ30 mV]. Login to comment
127 (C) Maximum current plotted logarithmically for T604A (dark bars) and S686A (gray bars), for comparison with wild-type CFTR (white and dotted bars represent PKA alone and PKC ϩ PKA activity, respectively). 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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No. Sentence Comment
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] PKC phosphorylation modulates PKA-dependent bindin... Am J Physiol Cell Physiol. 2008 Nov;295(5):C1366-75. Epub 2008 Sep 17. Seavilleklein G, Amer N, Evagelidis A, Chappe F, Irvine T, Hanrahan JW, Chappe V
PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR.
Am J Physiol Cell Physiol. 2008 Nov;295(5):C1366-75. Epub 2008 Sep 17., [PMID:18799655]

Abstract [show]
Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called DeltaR-Split CFTR, which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Coexpression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with DeltaR-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of DeltaR-Split/7CA-RD channels was similar to that of DeltaR-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.

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No. Sentence Comment
256 S686 is a likely candidate to mediate stimulated PKC-dependent interactions since mutation of Ser686 to alanine in the full-length CFTR channel dramatically reduced CFTR activation to the level of 9CA-CFTR (5), whereas mutation of more distal PKC sites in the RD had no effect on the function (S707A/S790A/T791A/S809A). Login to comment

[hide] Control of CFTR channel gating by phosphorylation ... Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107. Gadsby DC, Nairn AC
Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis.
Physiol Rev. 1999 Jan;79(1 Suppl):S77-S107., [PMID:9922377]

Abstract [show]
Control of CTFR Channel Gating by Phosphorylation and Nucleotide Hydrolysis. Physiol. Rev. 79, Suppl.: S77-S107, 1999. - The cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is the protein product of the gene defective in cystic fibrosis, the most common lethal genetic disease among Caucasians. Unlike any other known ion channel, CFTR belongs to the ATP-binding cassette superfamily of transporters and, like all other family members, CFTR includes two cytoplasmic nucleotide-binding domains (NBDs), both of which bind and hydrolyze ATP. It appears that in a single open-close gating cycle, an individual CFTR channel hydrolyzes one ATP molecule at the NH2-terminal NBD to open the channel, and then binds and hydrolyzes a second ATP molecule at the COOH-terminal NBD to close the channel. This complex coordinated behavior of the two NBDs is orchestrated by multiple protein kinase A-dependent phosphorylation events, at least some of which occur within the third large cytoplasmic domain, called the regulatory domain. Two or more kinds of protein phosphatases selectively dephosphorylate distinct sites. Under appropriately controlled conditions of progressive phosphorylation or dephosphorylation, three functionally different phosphoforms of a single CFTR channel can be distinguished on the basis of channel opening and closing kinetics. Recording single CFTR channel currents affords an unprecedented opportunity to reproducibly examine, and manipulate, individual ATP hydrolysis cycles in a single molecule, in its natural environment, in real time.

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No. Sentence Comment
202 although not examined at the single-channel level, the sensitivity of mutant S686A CFTR channels in oocytes to 4. Login to comment

[hide] Regulation of recombinant cardiac cystic fibrosis ... Biophys J. 1999 Apr;76(4):1972-87. Yamazaki J, Britton F, Collier ML, Horowitz B, Hume JR
Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C.
Biophys J. 1999 Apr;76(4):1972-87., [PMID:10096895]

Abstract [show]
We investigated the regulation of cardiac cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by protein kinase C (PKC) in Xenopus oocytes injected with cRNA encoding the cardiac (exon 5-) CFTR Cl- channel isoform. Membrane currents were recorded using a two-electrode voltage clamp technique. Activators of PKC or a cAMP cocktail elicited robust time-independent Cl- currents in cardiac CFTR-injected oocytes, but not in control water-injected oocytes. The effects of costimulation of both pathways were additive; however, maximum protein kinase A (PKA) activation occluded further activation by PKC. In oocytes expressing either the cardiac (exon 5-) or epithelial (exon 5+) CFTR isoform, Cl- currents activated by PKA were sustained, whereas PKC-activated currents were transient, with initial activation followed by slow current decay in the continued presence of phorbol esters, the latter effect likely due to down-regulation of endogenous PKC activity. The specific PKA inhibitor, adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS), and various protein phosphatase inhibitors were used to determine whether the stimulatory effects of PKC are dependent upon the PKA phosphorylation state of cardiac CFTR channels. Intraoocyte injection of 1,2-bis(2-aminophenoxy)ethane-N,N, N,N-tetraacetic acid (BAPTA) or pretreatment of oocytes with BAPTA-acetoxymethyl-ester (BAPTA-AM) nearly completely prevented dephosphorylation of CFTR currents activated by cAMP, an effect consistent with inhibition of protein phosphatase 2C (PP2C) by chelation of intracellular Mg2+. PKC-induced stimulation of CFTR channels was prevented by inhibition of basal endogenous PKA activity, and phorbol esters failed to stimulate CFTR channels trapped into either the partially PKA phosphorylated (P1) or the fully PKA phosphorylated (P1P2) channel states. Site-directed mutagenesis of serines (S686 and S790) within two consensus PKC phosphorylation sites on the cardiac CFTR regulatory domain attentuated, but did not eliminate, the stimulatory effects of phorbol esters on mutant CFTR channels. The effects of PKC on cardiac CFTR Cl- channels are consistent with a simple model in which PKC phosphorylation of the R domain facilitates PKA-induced transitions from dephosphorylated (D) to partially (P1) phosphorylated and fully (P1P2) phosphorylated channel states.

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No. Sentence Comment
34 Site-directed mutagenesis The serine at position 686 and/or 790 was modified by polymerase chain reaction-based site-directed mutagenesis (Jones and Howard, 1991) to alanine to create S686A, S790A, and S686 ϩ 790A cardiac CFTR cDNA. Login to comment
283 We examined cAMP and PDBu regulation of cardiac (exon 5-) CFTR channels in oocytes injected with mRNA encoding three mutant constructs: S686A, S790A, and the double mutant S686A-S790A. Login to comment
284 Fig. 10, A and B, shows representative currents at 70 mV for the S686A and S686A-S790A mutants during exposure to the cAMP cocktail, PDBu, and then a subsequent cAMP cocktail in the continued presence of PDBu. Login to comment
287 Fig. 10 C shows a comparison of the absolute current amplitudes activated by cAMP in Xenopus oocytes injected with 47 ng of mRNA encoding S686A(card), S790A(card), S686A-S790A(card), wild-type cardiac (card), or wild-type epithelial CFTR. Login to comment
289 This is consistent with a previous report that showed no significant differences in PKA activation sensitivity for the S686A mutant in epithelial CFTR channels (Wilkinson et al., 1996). Login to comment
291 Normalized PDBu-induced current amplitudes were 55.2 Ϯ 8.8% (n ϭ 4) for wild-type channels, 27.6 Ϯ 5.5% (n ϭ 5) for S686A, 29.0 Ϯ 3.6% (n ϭ 4) for S790A, and 25.1 Ϯ 2.4% (n ϭ 4) for the S686A-S790A double mutant. Login to comment
315 However, in oo- FIGURE 10 Effects of cAMP and PDBu on cardiac S686A and S790A mutant CFTR channels. Login to comment
316 (A and B) The effects of cAMP cocktail (1ϫ) and PDBu (100 nM) on cardiac CFTR-S686A and CFTR-S686A, S790A were examined using the same protocol as in Fig. 5 A. Login to comment
339 PDBu-induced CFTR current amplitudes were reduced by approximately half in the S686A, S790A, and the double mutant S686A-S790A constructs examined. Login to comment
281 We examined cAMP and PDBu regulation of cardiac (exon 5afa;) CFTR channels in oocytes injected with mRNA encoding three mutant constructs: S686A, S790A, and the double mutant S686A-S790A. Login to comment
282 Fig. 10, A and B, shows representative currents at 70 mV for the S686A and S686A-S790A mutants during exposure to the cAMP cocktail, PDBu, and then a subsequent cAMP cocktail in the continued presence of PDBu. Login to comment
285 Fig. 10 C shows a comparison of the absolute current amplitudes activated by cAMP in Xenopus oocytes injected with 47 ng of mRNA encoding S686A(card), S790A(card), S686A-S790A(card), wild-type cardiac (card), or wild-type epithelial CFTR. Login to comment
313 However, in oo- FIGURE 10 Effects of cAMP and PDBu on cardiac S686A and S790A mutant CFTR channels. Login to comment
314 (A and B) The effects of cAMP cocktail (1afb;) and PDBu (100 nM) on cardiac CFTR-S686A and CFTR-S686A, S790A were examined using the same protocol as in Fig. 5 A. Login to comment
337 PDBu-induced CFTR current amplitudes were reduced by approximately half in the S686A, S790A, and the double mutant S686A-S790A constructs examined. 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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No. Sentence 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

[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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No. Sentence 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
124 CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 -813 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. 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
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
126 CFTR S-Oct-A (S660A,S686A,S700A, CFTR A 795 737 4+B5 - 8 1 3 B," -02 -B3 sion was high, we expected the mutant channels to be less active than wild-type CFTR C1-channels. 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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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

[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
130 To study PKC regulation without using inhibitors that could perturb other signaling pathways, we used BHK cells expressing 9CA-CFTR, a mutant that lacks all 9 PKC consensus sites in the RD and NBD1 regulatory extension (T582A/T604A/S641A/T682/S686A/S707A/ S790A/T791A/S809A) (13). Login to comment