ABCC7 p.Thr682Ala
| Predicted by SNAP2: | A: N (78%), C: N (72%), D: N (61%), E: N (72%), F: N (57%), G: N (78%), H: N (66%), I: N (72%), K: N (78%), L: N (61%), M: N (78%), N: N (78%), P: N (78%), Q: N (82%), R: N (66%), S: N (87%), V: N (78%), W: D (66%), Y: N (57%), |
| 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.Thr682Ala 12588899:14:243
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.Thr682Ala 12588899:145:247
status: NEW[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
7 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.
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ABCC7 p.Thr682Ala 14695900:7:99
status: NEW45 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).
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ABCC7 p.Thr682Ala 14695900:45:48
status: NEW59 (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).
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ABCC7 p.Thr682Ala 14695900:59:176
status: NEW86 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.
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ABCC7 p.Thr682Ala 14695900:86:346
status: NEW112 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).
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ABCC7 p.Thr682Ala 14695900:112:133
status: NEW113 PKA and PKC activation of T582A, T604A, S641A, T682A, and S686A channels was assessed by using the same protocol as in Fig. 1.
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ABCC7 p.Thr682Ala 14695900:113:47
status: NEW114 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.
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ABCC7 p.Thr682Ala 14695900:114:30
status: NEW132 Responsiveness of T682A to PKA was similar to wild-type (Fig. 3A).
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ABCC7 p.Thr682Ala 14695900:132:18
status: NEW135 S641A dramatically reduced activation by PKC alone (Fig. 3C), whereas T682A enhanced PKC activation by Ͼ4-fold (Fig. 3 D and E).
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ABCC7 p.Thr682Ala 14695900:135:70
status: NEW155 Activation of T682A and S641A channels.
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ABCC7 p.Thr682Ala 14695900:155:14
status: NEW156 Maximum current (measured at the plateau) vs. PKA concentration for (A) T682A or (B) S641A channels pretreated (circles) or not pretreated (triangles) with PKC.
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ABCC7 p.Thr682Ala 14695900:156:72
status: NEW161 (D) NPo of wild-type and T682A channels measured before (gray bars) and after (dark bars) addition of PKC ϩ ATP for 15 min.
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ABCC7 p.Thr682Ala 14695900:161:25
status: NEW162 (E) Patch-clamp recording of T682A channels by using the inside-out configuration [pipette potential (Vp) ϭ ϩ30 mV] activated by PKC ϩ ATP after preincubation with phosphatases as described in Materials and Methods.
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ABCC7 p.Thr682Ala 14695900:162:29
status: NEW