ABCC7 p.Ser511Ala
| CF databases: |
c.1532C>G
,
p.Ser511Cys
(CFTR1)
?
,
|
| Predicted by SNAP2: | A: N (87%), C: N (72%), D: N (82%), E: N (93%), F: D (53%), G: N (87%), H: N (61%), I: N (78%), K: N (87%), L: N (78%), M: N (57%), N: N (93%), P: N (82%), Q: N (87%), R: N (82%), T: N (97%), V: N (82%), W: D (59%), Y: D (53%), |
| Predicted by PROVEAN: | A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, T: N, V: N, W: D, Y: D, |
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[hide] Protein kinase CK2, cystic fibrosis transmembrane ... J Biol Chem. 2007 Apr 6;282(14):10804-13. Epub 2007 Feb 8. Treharne KJ, Crawford RM, Xu Z, Chen JH, Best OG, Schulte EA, Gruenert DC, Wilson SM, Sheppard DN, Kunzelmann K, Mehta A
Protein kinase CK2, cystic fibrosis transmembrane conductance regulator, and the deltaF508 mutation: F508 deletion disrupts a kinase-binding site.
J Biol Chem. 2007 Apr 6;282(14):10804-13. Epub 2007 Feb 8., 2007-04-06 [PMID:17289674]
Abstract [show]
Deletion of phenylalanine 508 (DeltaF508) from the first nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most common mutation in cystic fibrosis. The F508 region lies within a surface-exposed loop that has not been assigned any interaction with associated proteins. Here we demonstrate that the pleiotropic protein kinase CK2 that controls protein trafficking, cell proliferation, and development binds wild-type CFTR near F508 and phosphorylates NBD1 at Ser-511 in vivo and that mutation of Ser-511 disrupts CFTR channel gating. Importantly, the interaction of CK2 with NBD1 is selectively abrogated by the DeltaF508 mutation without disrupting four established CFTR-associated kinases and two phosphatases. Loss of CK2 association is functionally corroborated by the insensitivity of DeltaF508-CFTR to CK2 inhibition, the absence of CK2 activity in DeltaF508 CFTR-expressing cell membranes, and inhibition of CFTR channel activity by a peptide that mimics the F508 region of CFTR (but not the equivalent DeltaF508 peptide). Disruption of this CK2-CFTR association is the first described DeltaF508-dependent protein-protein interaction that provides a new molecular paradigm in the most frequent form of cystic fibrosis.
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No. Sentence Comment
94 Generation of the S511A Mutant in the NBD1 Region of CFTR-The cftr gene-specific primers, forward, 5Ј-tcatctttggtgttgcctat- gatgaatat-3Ј, and reverse, 5Ј-atattcatcataggcaacaccaaagatga-3Ј, were used to introduce a Ser to Ala point mutation at the 511 site in the CFTR NBD1 region using PfuTurbo DNA polymerase in a PCR-based method (Quickchange SDM kit, Invitrogen).
X
ABCC7 p.Ser511Ala 17289674:94:18
status: NEW102 Co-immunoprecipitation of CK2 with CFTR Mutants-BHK cells were transfected with vectors encoding wild-type, ⌬F508, S511A, or S511D CFTR.
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ABCC7 p.Ser511Ala 17289674:102:122
status: NEW189 Phosphorylation assays using NBD1/ NBD1-S511A protein demonstrated that PKA-dependent phosphorylation remained intact.
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ABCC7 p.Ser511Ala 17289674:189:40
status: NEW190 In contrast, CK2 failed to phosphorylate the S511A mutant (Fig. 5D, open columns).
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ABCC7 p.Ser511Ala 17289674:190:45
status: NEW211 These results suggest that neither ⌬F508-NBD1 nor S511A-NBD1 domains interact with CK2 in vitro; we therefore investigated whether this defect is mirrored in full-length CFTR.
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ABCC7 p.Ser511Ala 17289674:211:57
status: NEW212 Wild-type, but Not ⌬F508 or S511A CFTR, Induces CK2 Membrane Localization-Having shown that CK2 membrane localization is dependent on the presence of wild-type CFTR in bronchial cell lines, we investigated whether this phenomenon could be reproduced by expression of CFTR in a CFTR-naive cell line.
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ABCC7 p.Ser511Ala 17289674:212:35
status: NEW216 No CK2 protein was found associated with S511A or ⌬F508 mutant CFTR, whereas wild-type, S511D, and the ⌬F508-S511D double mutant CFTR all bound CK2 (Fig. 6A).
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ABCC7 p.Ser511Ala 17289674:216:41
status: NEW218 CK2 activity was present in wild-type, S511D, and ⌬F508-S511D-CFTR-transfected membranes but was absent from those of S511A and ⌬F508 (Fig. 6B).
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ABCC7 p.Ser511Ala 17289674:218:125
status: NEW228 S511D (ϮF508) and S511A decreased the Po of CFTR 3-fold, without altering current flow through individual chan- FIGURE 5.
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ABCC7 p.Ser511Ala 17289674:228:24
status: NEW237 D, CK2-dependent but not PKA-dependent phosphorylation is abolished in S511A-NBD1; CK2 and PKA phos- phorylatewild-typeNBD1additivelyatdistinctsites,andtheirrespectiveinhibitorsarespecific(nϭ3Ϯrange).
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ABCC7 p.Ser511Ala 17289674:237:71
status: NEW258 The Po observed in S511A/S511D was similar to that of ⌬F508 CFTR when it is induced to traffic to the membrane by a low temperature correction.
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ABCC7 p.Ser511Ala 17289674:258:19
status: NEW261 Thus we find that S511A and ⌬F508 share an inability to bind CK2, but S511D restores CK2 binding with or without F508.
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ABCC7 p.Ser511Ala 17289674:261:18
status: NEW268 CK2 association with CFTR is abrogated by ⌬F508 or S511A mutation; CK2 phosphorylates CFTR at Ser-511 in vivo.
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ABCC7 p.Ser511Ala 17289674:268:58
status: NEW[hide] Inhibition of protein kinase CK2 closes the CFTR C... Cell Physiol Biochem. 2009;24(5-6):347-60. Epub 2009 Nov 4. Treharne KJ, Xu Z, Chen JH, Best OG, Cassidy DM, Gruenert DC, Hegyi P, Gray MA, Sheppard DN, Kunzelmann K, Mehta A
Inhibition of protein kinase CK2 closes the CFTR Cl channel, but has no effect on the cystic fibrosis mutant deltaF508-CFTR.
Cell Physiol Biochem. 2009;24(5-6):347-60. Epub 2009 Nov 4., [PMID:19910675]
Abstract [show]
BACKGROUND: Deletion of phenylalanine-508 (DeltaF508) from the first nucleotide-binding domain (NBD1) in the wild-type cystic fibrosis (CF) transmembrane-conductance regulator (wtCFTR) causes CF. However, the mechanistic relationship between DeltaF508-CFTR and the diversity of CF disease is unexplained. The surface location of F508 on NBD1 creates the potential for protein-protein interactions and nearby, lies a consensus sequence (SYDE) reported to control the pleiotropic protein kinase CK2. METHODS: Electrophysiology, immunofluorescence and biochemistry applied to CFTR-expressing cells, Xenopus oocytes, pancreatic ducts and patient biopsies. RESULTS: Irrespective of PKA activation, CK2 inhibition (ducts, oocytes, cells) attenuates CFTR-dependent Cl(-) transport, closing wtCFTR in cell-attached membrane patches. CK2 and wtCFTR co-precipitate and CK2 co-localized with wtCFTR (but not DeltaF508-CFTR) in apical membranes of human airway biopsies. Comparing wild-type and DeltaF508CFTR expressing oocytes, only DeltaF508-CFTR Cl(-) currents were insensitive to two CK2 inhibitors. Furthermore, wtCFTR was inhibited by injecting a peptide mimicking the F508 region, whereas the DeltaF508-equivalent peptide had no effect. CONCLUSIONS: CK2 controls wtCFTR, but not DeltaF508-CFTR. Others find that peptides from the F508 region of NBD1 allosterically control CK2, acting through F508. Hence, disruption of CK2-CFTR interaction by DeltaF508-CFTR might disrupt multiple, membrane-associated, CK2-dependent pathways, creating a new molecular disease paradigm for deleted F508 in CFTR.
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No. Sentence Comment
216 Figure 7a demonstrates that mutation of S511 in wild-type CFTR is without effect on the single-channel activity of CFTR; neither the i nor the Po of S511A- and S511D-CFTR differed from those of wild-type CFTR when measured in excised inside-out membrane patches from BHK cells stably expressing CFTR constructs.
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ABCC7 p.Ser511Ala 19910675:216:149
status: NEW248 Values are means + SEM (n = 6, except S511A where n = 4).
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ABCC7 p.Ser511Ala 19910675:248:38
status: NEW255 It was not possible to study the construct S511A-CFTR in Xenopus oocytes because for unknown reasons oocytes expressing this construct all died immediately after microelectrode impalement.
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ABCC7 p.Ser511Ala 19910675:255:43
status: NEW[hide] CFTR mutations altering CFTR fragmentation. Biochem J. 2013 Jan 1;449(1):295-305. doi: 10.1042/BJ20121240. Tosoni K, Stobbart M, Cassidy DM, Venerando A, Pagano MA, Luz S, Amaral MD, Kunzelmann K, Pinna LA, Farinha CM, Mehta A
CFTR mutations altering CFTR fragmentation.
Biochem J. 2013 Jan 1;449(1):295-305. doi: 10.1042/BJ20121240., [PMID:23067305]
Abstract [show]
Most CF (cystic fibrosis) results from deletion of a phenylalanine (F508) in the CFTR {CF transmembrane-conductance regulator; ABCC7 [ABC (ATP-binding cassette) sub-family C member 7]} which causes ER (endoplasmic reticulum) degradation of the mutant. Using stably CFTR-expressing BHK (baby-hamster kidney) cell lines we demonstrated that wild-type CTFR and the F508delCFTR mutant are cleaved into differently sized N- and C-terminal-bearing fragments, with each hemi-CFTR carrying its nearest NBD (nucleotide-binding domain), reflecting differential cleavage through the central CFTR R-domain. Similar NBD1-bearing fragments are present in the natively expressing HBE (human bronchial epithelial) cell line. We also observe multiple smaller fragments of different sizes in BHK cells, particularly after F508del mutation (ladder pattern). Trapping wild-type CFTR in the ER did not generate a F508del fragmentation fingerprint. Fragments change their size/pattern again post-mutation at sites involved in CFTR's in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1). The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D). We conclude that the F508delCFTR mutant is not degraded completely and there exists a relationship between CFTR's fragmentation fingerprint and the CFTR sequence through putative CK2-interactive sites that lie near F508.
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No. Sentence Comment
5 Fragments change their size/pattern again post-mutation at sites involved in CFTR`s in vitro interaction with the pleiotropic protein kinase CK2 (S511A in NBD1).
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ABCC7 p.Ser511Ala 23067305:5:146
status: NEW6 The F508del and S511A mutations generate different fragmentation fingerprints that are each unlike the wild-type; yet, both mutants generate new N-terminal-bearing CFTR fragments that are not observed with other CK2-related mutations (S511D, S422A/D and T1471A/D).
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ABCC7 p.Ser511Ala 23067305:6:16
status: NEW44 Cell culture, lysis, protein solubilization and Western blotting The cell culture methods to create the stable CFTR-expressing cell lines (WT, F, WT S422A, WT S422D, WT S511A, WT S511D, WT T1471A and WT T1471D) and their culture protocols have been described recently [25].
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ABCC7 p.Ser511Ala 23067305:44:170
status: NEW197 S511A-CFTR and its cognate NBD2 The diffuse 80 kDa NBD2 and the C-terminal-bearing fragment so characteristic of the wt-CFTR fingerprint is now grossly attenuated (Figure 7C, compare lanes 2 and 5).
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ABCC7 p.Ser511Ala 23067305:197:0
status: NEW199 First, the fragment bearing NBD2 and the C-terminus of CFTR may be more easily degraded when the N-terminal 110 kDa (NDB1/N-terminal) fragment (Figures 7A and 7B) is generated by the S511A mutation.
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ABCC7 p.Ser511Ala 23067305:199:183
status: NEW200 Secondly, the epitope of NBD2 recognized by this monoclonal antibody (WPSGGQMT) may be masked after S511A mutation by some post-translational event.
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ABCC7 p.Ser511Ala 23067305:200:100
status: NEW201 Thirdly, the CFTR fracture point induced by this S511A mutant lies near this region as opposed to the R-domain region seen with wt-CFTR in the Figures above.
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ABCC7 p.Ser511Ala 23067305:201:49
status: NEW202 S511A-CFTR and its cognate NBD1 Consistent with the idea of an altered fracture point when S511A-CFTR is present, a new faint ~100-110 kDa band appears.
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ABCC7 p.Ser511Ala 23067305:202:0
status: NEWX
ABCC7 p.Ser511Ala 23067305:202:91
status: NEW204 Note the N-terminal- and NBD1- (but not NBD2) bearing ~120 kDa fragment seen after S511A (but not S511D) mutation in lanes 5 and 6 in (A) and (B), but not in (C) and (D), the latter probing the C-terminus.
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ABCC7 p.Ser511Ala 23067305:204:83
status: NEW211 Thus it is probable that the S511A mutation creates a band at 100-110 kDa-bearing NBD1 and the N-terminus, but we cannot be sure about the relationship between this band and the relative amounts of the Q1-Q4 NBD1-bearing fragments.
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ABCC7 p.Ser511Ala 23067305:211:29
status: NEW214 The combined S511 site mutant data suggest the existence of a new S511A-dependent CFTR cleavage whose generation is either dependent on the non-availability of the expected wt hydroxy group on S511 or is induced by a structural change after substitution with alanine (but not the negatively charged aspartate).
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ABCC7 p.Ser511Ala 23067305:214:66
status: NEW220 In summary, the two S511A/S511D and T1471A/T1471D pairs manifested very different CFTR cleavage patterns and abundance of full-length CFTR when present on a wt background.
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ABCC7 p.Ser511Ala 23067305:220:20
status: NEW223 Only F508delCFTR and S511A CFTR generated a new CFTR band at about 110 kDa.
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ABCC7 p.Ser511Ala 23067305:223:21
status: NEW233 It is also unlikely that CK2-relevant CFTR mutants such as S511A- and S511D-CFTR generate an ER-trapped unfolded CFTR, as they have wt patterns of maturation and turnover during pulse-chase experiments in the same cell line [25]; yet, they manifest very different fragmentation.
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ABCC7 p.Ser511Ala 23067305:233:59
status: NEW