ABCMdb

ABCC7 p.Thr682Ala

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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

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. 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
86 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 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
132 Responsiveness of T682A to PKA was similar to wild-type (Fig. 3A). Login to comment
135 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
155 Activation of T682A and S641A channels. Login to comment
156 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
161 (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 (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