ABCMdb

ABCC7 p.Thr582Ala

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Publications

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

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
41 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). 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
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
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
121 T582A and T604A resembled wild-type channels Fig. 2. 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
125 (B) Maximum current [current (I)], pA, measured at the plateau) for T582A mutant recorded with (F) or without (Œ) PKC pretreatment. Login to comment
148 This result, when combined with the activation of T582A and T604A mutants by PKC alone, argues that S686 and S641 (Fig. 3C) are both necessary for PKC activation. 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

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