ABCMdb

PMID: 14695900

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 [PubMed]

Sentences

No. Mutations Sentence Comment

3 ABCC7 p.Ser686Ala Activation of a 4CA mutant (S707A͞S790A͞T791A͞S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A͞T604A͞S641A) and 2CA (T682A͞ S686A) channels to PKA were both drastically reduced (>90%). Login to comment 6 ABCC7 p.Ser686Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala 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 7 ABCC7 p.Thr682Ala ABCC7 p.Ser641Ala 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. Login to comment 41 ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala ABCC7 p.Ser641Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Thr682Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala ABCC7 p.Ser641Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Thr682Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala ABCC7 p.Ser641Ala (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 86 ABCC7 p.Ser686Ala ABCC7 p.Thr682Ala 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. Login to comment 112 ABCC7 p.Ser686Ala ABCC7 p.Thr682Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala ABCC7 p.Ser641Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Thr682Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala ABCC7 p.Ser641Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Ser686Ala ABCC7 p.Thr682Ala ABCC7 p.Thr604Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala ABCC7 p.Thr582Ala ABCC7 p.Ser641Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Thr604Ala Maximum currents mediated by T604A and S686A channels are summarized in Fig. 2C for comparison with wild-type channels. Login to comment 117 ABCC7 p.Thr604Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Thr604Ala 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 ABCC7 p.Ser686Ala ABCC7 p.Ser686Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala 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 ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala T582A and T604A resembled wild-type channels Fig. 2. Login to comment 123 ABCC7 p.Ser686Ala ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala (A) Recordings of T582A, T604A, and S686A channels by using the inside-out configuration [pipette potential (Vp) ϭ ϩ30 mV]. Login to comment 125 ABCC7 p.Thr582Ala (B) Maximum current [current (I)], pA, measured at the plateau) for T582A mutant recorded with (F) or without (Œ) PKC pretreatment. Login to comment 127 ABCC7 p.Ser686Ala ABCC7 p.Thr604Ala (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 132 ABCC7 p.Thr682Ala Responsiveness of T682A to PKA was similar to wild-type (Fig. 3A). Login to comment 133 ABCC7 p.Ser641Ala On the other hand, replacing serine 641 with alanine enhanced channel activation at all PKA concentrations and eliminated the dependence on PKC (Fig. 3B). Login to comment 135 ABCC7 p.Thr682Ala ABCC7 p.Ser641Ala S641A dramatically reduced activation by PKC alone (Fig. 3C), whereas T682A enhanced PKC activation by Ͼ4-fold (Fig. 3 D and E). Login to comment 148 ABCC7 p.Thr604Ala ABCC7 p.Thr582Ala 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 155 ABCC7 p.Thr682Ala ABCC7 p.Ser641Ala Activation of T682A and S641A channels. Login to comment 156 ABCC7 p.Thr682Ala ABCC7 p.Ser641Ala Maximum current (measured at the plateau) vs. PKA concentration for (A) T682A or (B) S641A channels pretreated (circles) or not pretreated (triangles) with PKC. Login to comment 159 ABCC7 p.Ser641Ala (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. Login to comment 161 ABCC7 p.Thr682Ala (D) NPo of wild-type and T682A channels measured before (gray bars) and after (dark bars) addition of PKC ϩ ATP for 15 min. Login to comment 162 ABCC7 p.Thr682Ala (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. Login to comment