ABCC7 p.Ser700Ala
| Predicted by SNAP2: | A: N (78%), C: N (61%), D: N (66%), E: N (66%), F: N (57%), G: N (82%), H: N (78%), I: N (61%), K: N (72%), L: N (61%), M: D (53%), N: N (82%), P: N (61%), Q: N (82%), R: N (66%), T: N (82%), V: N (66%), W: D (71%), Y: N (66%), |
| 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] 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.
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ABCC7 p.Ser700Ala 15155835:142:147
status: NEW[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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No. Sentence Comment
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.
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ABCC7 p.Ser700Ala 19328185:132:117
status: NEW152 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.
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ABCC7 p.Ser700Ala 19328185:152:45
status: NEW131 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.
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ABCC7 p.Ser700Ala 19328185:131:117
status: NEW151 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.
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ABCC7 p.Ser700Ala 19328185:151:45
status: NEW[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.
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ABCC7 p.Ser700Ala 9252549:87:32
status: NEW[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.
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ABCC7 p.Ser700Ala 7690753:66:121
status: NEW124 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.
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ABCC7 p.Ser700Ala 7690753:124:26
status: NEW174 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.
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ABCC7 p.Ser700Ala 7690753:174:449
status: NEW176 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A)(C).
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ABCC7 p.Ser700Ala 7690753:176:36
status: NEW65 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.
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ABCC7 p.Ser700Ala 7690753:65:121
status: NEW126 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.
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ABCC7 p.Ser700Ala 7690753:126:26
status: NEW178 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.
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ABCC7 p.Ser700Ala 7690753:178:451
status: NEW180 COS-7 cells were mock- (S660A,S686A,S700A,S712A,S737A,S768A,S795A,S813A) (C).
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ABCC7 p.Ser700Ala 7690753:180:36
status: NEW[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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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).
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ABCC7 p.Ser700Ala 7684377:37:111
status: NEW44 S700A (mutation at 2230 bp destroys the EcoRI site) and S712Awere introduced into pUCF2.5/4SA by replacing the DraIIIIHpaI fragment (Fig. lA)containing S660/700/712/737A (the construct was named pUCF2.5/6SA).
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ABCC7 p.Ser700Ala 7684377:44:0
status: NEW[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).
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ABCC7 p.Ser700Ala 23760269:102:265
status: NEW