ABCMdb

ABCC7 p.Thr604Ala

ClinVar:	c.1811C>G , p.Thr604Ser ? , not provided c.1811C>T , p.Thr604Ile ? , not provided
CF databases:	c.1811C>T , p.Thr604Ile (CFTR1) D , The residue 604, conserved among species, is involved here in a polar to non polar substitution. T604I was found in a male patient having a moderate form of CF with PS, and carrying [delta]F508 on the other chromosome. c.1811C>G , p.Thr604Ser (CFTR1) ? ,
Predicted by SNAP2:	A: D (53%), C: D (80%), D: D (91%), E: D (91%), F: D (91%), G: D (85%), H: D (91%), I: D (91%), K: D (91%), L: D (91%), M: D (91%), N: D (80%), P: D (91%), Q: D (85%), R: D (91%), S: D (71%), V: D (85%), W: D (91%), Y: D (91%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, V: D, W: D, Y: D,

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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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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

[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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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
116 Maximum currents mediated by T604A and S686A channels are summarized in Fig. 2C for comparison with wild-type channels. Login to comment
117 The current carried by T604A channels was only 4% that of wild-type channels (3.12 Ϯ 1.1 pA; n ϭ 12 patches). Login to comment
118 S686A channels appeared more active than T604A channels, but this difference was small enough to be explained by their higher expression (compare Fig. 1C). 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
127 (C) Maximum current plotted logarithmically for T604A (dark bars) and S686A (gray bars), for comparison with wild-type CFTR (white and dotted bars represent PKA alone and PKC ϩ PKA activity, respectively). 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

[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). Login to comment