ABCC7 p.Ser641Ala
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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).
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ABCC7 p.Ser641Ala 12588899:14:237
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.Ser641Ala 12588899:145:240
status: NEW
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
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.Ser641Ala 14695900:7:61
status: NEW41 3CA (T582A͞T604A͞S641A) was made by PCR mutagenesis by using wild-type CFTR cDNA as the template and the following forward (F) and reverse (R) primers: T582A (F, TACCTAGATGTTTTAGCAGAAAAAGAAATATTT- GAA; R, TTCAAATATTTCTTTTTCTGCTAAAACAT- CTAGGTA), T604A (F, ACTAGGATTTTGGTCGCTTCTA- AAATGGAACAT; R, ATGTTCCATTTTAGAAGCGAC- CAAAATCCTAGT), S641A (F, CTACAGCCAGACTTT- GCCTCAAAACTCATGGGA; R, TCCCATGAGTTTTG- AGGCAAAGTCTGGCTGTAG).
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ABCC7 p.Ser641Ala 14695900:41:346
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.Ser641Ala 14695900:45:41
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.Ser641Ala 14695900:59:160
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.Ser641Ala 14695900:112:146
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.Ser641Ala 14695900:113:40
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.Ser641Ala 14695900:114:20
status: NEW133 On the other hand, replacing serine 641 with alanine enhanced channel activation at all PKA concentrations and eliminated the dependence on PKC (Fig. 3B).
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ABCC7 p.Ser641Ala 14695900:133:29
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.Ser641Ala 14695900:135:0
status: NEW155 Activation of T682A and S641A channels.
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ABCC7 p.Ser641Ala 14695900:155:24
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.Ser641Ala 14695900:156:85
status: NEW159 (C) Activation by PKC alone. NPo of wild-type and S641A channels before (gray bars) and after (dark bars) addition of PKC for 15 min.
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ABCC7 p.Ser641Ala 14695900:159:50
status: NEW
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
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.Ser641Ala 23760269:130:232
status: NEW