ABCMdb

ABCC7 p.Ser790Ala

Predicted by SNAP2:	A: N (87%), C: N (78%), D: N (57%), E: N (61%), F: N (72%), G: N (78%), H: N (82%), I: N (72%), K: N (66%), L: N (78%), M: N (78%), N: N (82%), P: N (72%), Q: N (78%), R: N (78%), T: N (93%), V: N (82%), W: N (57%), Y: N (82%),
Predicted by PROVEAN:	A: N, C: D, 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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No. Sentence Comment
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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No. Sentence Comment
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] 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] 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
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
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] 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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No. Sentence Comment
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