ABCC7 p.Thr791Ala
| Predicted by SNAP2: | A: N (93%), C: N (93%), D: N (82%), E: N (78%), F: N (93%), G: N (87%), H: N (87%), I: N (87%), K: N (82%), L: N (93%), M: N (87%), N: N (87%), P: N (82%), Q: N (87%), R: N (87%), S: N (97%), V: N (93%), W: N (66%), Y: N (93%), |
| 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, S: N, V: N, W: N, Y: N, |
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[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).
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ABCC7 p.Thr791Ala 12588899:14:267
status: NEW145 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).
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ABCC7 p.Thr791Ala 12588899:145:275
status: NEW[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).
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ABCC7 p.Thr791Ala 18799655:256:306
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
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).
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ABCC7 p.Thr791Ala 23760269:130:262
status: NEW