ABCMdb

ABCC7 p.Ser686Ala

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PMID: 11331356 [PubMed] Button B et al: "PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. insertion of this site confers PKC sensitivity to human CFTR."

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

PMID: 12588899 [PubMed] Chappe V et al: "Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA."

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

PMID: 14695900 [PubMed] Chappe V et al: "Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator."

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

PMID: 15155835 [PubMed] Ai T et al: "Capsaicin potentiates wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride-channel currents."

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

PMID: 18799655 [PubMed] Seavilleklein G et al: "PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR."

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

PMID: 9922377 [PubMed] Gadsby DC et al: "Control of CFTR channel gating by phosphorylation and nucleotide hydrolysis."

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

PMID: 10096895 [PubMed] Yamazaki J et al: "Regulation of recombinant cardiac cystic fibrosis transmembrane conductance regulator chloride channels by protein kinase C."

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

PMID: 9252549 [PubMed] Wilkinson DJ et al: "CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain."

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

PMID: 7690753 [PubMed] Rich DP et al: "Regulation of the cystic fibrosis transmembrane conductance regulator Cl- channel by negative charge in the R domain."

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

PMID: 7684377 [PubMed] Chang XB et al: "Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites."

No. Sentence 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

PMID: 23760269 [PubMed] Billet A et al: "Role of tyrosine phosphorylation in the muscarinic activation of the cystic fibrosis transmembrane conductance regulator (CFTR)."

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