ABCC7 p.Thr1471Ala
| Predicted by SNAP2: | A: N (87%), C: N (66%), D: N (53%), E: N (61%), F: N (53%), G: N (82%), H: N (61%), I: N (78%), K: N (72%), L: N (66%), M: N (78%), N: N (72%), P: N (72%), Q: N (72%), R: N (57%), S: N (87%), V: N (78%), W: D (53%), Y: N (53%), |
| Predicted by PROVEAN: | A: N, C: N, D: N, E: N, F: N, G: N, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, V: N, W: N, Y: N, |
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[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
6 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.Thr1471Ala 23067305:6:254
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.Thr1471Ala 23067305:44:190
status: NEW215 wt-CFTR with mutated T1471 T1471A fragmentation is very similar to the wt.
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ABCC7 p.Thr1471Ala 23067305:215:27
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.Thr1471Ala 23067305:220:36
status: NEW[hide] Detection of phospho-sites generated by protein ki... PLoS One. 2013 Sep 18;8(9):e74232. doi: 10.1371/journal.pone.0074232. eCollection 2013. Venerando A, Franchin C, Cant N, Cozza G, Pagano MA, Tosoni K, Al-Zahrani A, Arrigoni G, Ford RC, Mehta A, Pinna LA
Detection of phospho-sites generated by protein kinase CK2 in CFTR: mechanistic aspects of Thr1471 phosphorylation.
PLoS One. 2013 Sep 18;8(9):e74232. doi: 10.1371/journal.pone.0074232. eCollection 2013., [PMID:24058532]
Abstract [show]
By mass spectrometry analysis of mouse Cystic Fibrosis Transmembrane-conductance Regulator (mCFTR) expressed in yeast we have detected 21 phosphopeptides accounting for 22 potential phospho-residues, 12 of which could be unambiguously assigned. Most are conserved in human CFTR (hCFTR) and the majority cluster in the Regulatory Domain, lying within consensus sequences for PKA, as identified in previous mammalian studies. This validates our yeast expression model. A number of phospho-residues were novel and human conserved, notably mouse Ser670, Ser723, Ser737, and Thr1467, that all lie in acidic sequences, compatible with their phosphorylation by protein kinase CK2. Thr1467 is localized in the C-terminal tail, embedded in a functionally important and very acidic sequence (EETEEE) which displays an optimal consensus for protein kinase CK2. Herein, we show that Thr1467, homologous to human Thr1471 is readily phosphorylated by CK2. Indeed a 42 amino acid peptide encompassing the C-terminal segment of human CFTR is readily phosphorylated at Thr1471 with favorable kinetics (Km 1.7 microM) by CK2 holoenzyme, but neither by its isolated catalytic subunit nor by other acidophilic Ser/Thr kinases (CK1, PLK2/3, GCK/FAM20C). Our finding that by treating CFTR expressing BHK cells with the very specific CK2 inhibitor CX4945, newly synthesized wild type CFTR (and even more its Phe508del mutant) accumulates more abundantly than in the absence of CK2 inhibitor, supports the conclusion that phosphorylation of CFTR by CK2 correlates with decreased stability of the protein.
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No. Sentence Comment
91 Cell Culture Baby hamster kidney cells (BHK) stably expressing human CFTR (hCFTR) either wild type (WT), or Phe508del, or WT T1471A were growth to reach 70-80% confluency [13].
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ABCC7 p.Thr1471Ala 24058532:91:125
status: NEW147 Accordingly a hCFTR mutant no longer susceptible to such a phosphorylation (T1471A) was overexpressed in BHK cells as compared to hCFTR wild type.
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ABCC7 p.Thr1471Ala 24058532:147:76
status: NEW162 Once blockade is removed de novo synthesis of CFTR wild type is significantly stimulated if endogenous CK2 is inhibited by cell treatment with CX4945 (Panel A), a similar effect of CX4945 being also detectable if wild type CFTR is replaced by its T1471A mutant, which is no longer susceptible to phosphorylation at Thr1471 (Panel B).
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ABCC7 p.Thr1471Ala 24058532:162:247
status: NEW172 BHK cells expressing human CFTR wild type (A), T1471A (B) and Phe508del (C) were treated for 2 h with CHX to stop protein synthesis (compare first and second lanes).
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ABCC7 p.Thr1471Ala 24058532:172:47
status: NEW193 To get clues about the possible consequences of CFTR phosphorylation by CK2, advantage has been taken of the most selective CK2 inhibitor available to date, CX-4945 [43] and of a CFTR mutant in which Thr1471 has been replaced by alanine (T1471A) [13].
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ABCC7 p.Thr1471Ala 24058532:193:200
status: NEWX
ABCC7 p.Thr1471Ala 24058532:193:238
status: NEW197 On the other hand the effect of CK2 on CFTR stability seems not to be mediated by Thr1471, at least in a wild type background, since a similar effect of CX4945 was observed with CFTR wild type and its T1471A mutant (Figure 5, compare Panels A and B).
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ABCC7 p.Thr1471Ala 24058532:197:201
status: NEW206 (TIF) Figure S4 BHK cells expressing WT, WT/T1471A or Phe508delCFTR were exposed to either cycloheximide (CHX, 100 mg/ml) or Brefeldin A (BFA, 200 ng/ml).
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ABCC7 p.Thr1471Ala 24058532:206:45
status: NEW