ABCC7 p.Tyr512Phe
| Predicted by SNAP2: | A: D (80%), C: D (71%), D: D (91%), E: D (91%), F: D (59%), G: D (91%), H: N (53%), I: D (75%), K: D (91%), L: D (53%), M: N (61%), N: D (85%), P: D (91%), Q: D (80%), R: D (91%), S: D (85%), T: D (85%), V: D (80%), W: D (66%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: N, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: N, |
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[hide] Antagonistic Regulation of Cystic Fibrosis Transme... Mol Cell Biol. 2011 Oct;31(19):4076-86. Epub 2011 Aug 1. Mendes AI, Matos P, Moniz S, Luz S, Amaral MD, Farinha CM, Jordan P
Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase.
Mol Cell Biol. 2011 Oct;31(19):4076-86. Epub 2011 Aug 1., [PMID:21807898]
Abstract [show]
Members of the WNK (with-no-lysine [K]) subfamily of protein kinases regulate various ion channels involved in sodium, potassium, and chloride homeostasis by either inducing their phosphorylation or regulating the number of channel proteins expressed at the cell surface. Here, we describe findings demonstrating that the cell surface expression of the cystic fibrosis transmembrane conductance regulator (CFTR) is also regulated by WNK4 in mammalian cells. This effect of WNK4 is independent of the presence of kinase and involves interaction with and inhibition of spleen tyrosine kinase (Syk), which phosphorylates Tyr512 in the first nucleotide-binding domain 1 (NBD1) of CFTR. Transfection of catalytically active Syk into CFTR-expressing baby hamster kidney cells reduces the cell surface expression of CFTR, whereas that of WNK4 promotes it. This is shown by biotinylation of cell surface proteins, immunofluorescence microscopy, and functional efflux assays. Mutation of Tyr512 to either glutamic acid or phenylalanine is sufficient to alter CFTR surface levels. In human airway epithelial cells, downregulation of endogenous Syk and WNK4 confirms their roles as physiologic regulators of CFTR surface expression. Together, our results show that Tyr512 phosphorylation is a novel signal regulating the prevalence of CFTR at the cell surface and that WNK4 and Syk perform an antagonistic role in this process.
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No. Sentence Comment
41 pET-167 Sumo-NBD1 (first nucleotide binding domain of CFTR, kindly provided by168 Philip J. Thomas, Dallas) was mutated at CFTR codon 512 from TAT to TTT to169 obtain pET-Sumo-NBD1-Y512F.
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ABCC7 p.Tyr512Phe 21807898:41:181
status: NEW42 For subcloning of Myc-tagged NBD1 and170 NBD1-Y512F, their cDNAs which correspond to CFTR codons 388-677 were171 PCR-amplified from pET-Sumo-NBD1 with primers NBD1-F (5` ACG ACT172 ACA GAA GTA GTG ATG) and NBD1-R (5` TTA GAC AGG AGC ATC173 TCC TTC), cloned into pCR2.1 TOPO-TA vector (Invitrogen) and transferred174 into a pcDNA3-Myc expression vector.
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ABCC7 p.Tyr512Phe 21807898:42:46
status: NEW43 CFTR-Y512F and CFTR-Y512E175 mutants were generated by changing codon 512 of human pNUT-CFTR (26)176 from TAT to TTT or GAG, respectively.
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ABCC7 p.Tyr512Phe 21807898:43:5
status: NEW63 For the production of recombinant human OSR1, a reported259 WNK4 substrate (44), or of recombinant human NBD1 domain, pET-OSR1-260 D164A, pET-Sumo-NBD1 or pET-Sumo-NBD1-Y512F were expressed in the261 E. coli BL21 strain under isopropylthiogalactosidase (IPTG, 0.1 mM) induction262 and the bacterial pellets harvested at 1400 x g, 20 min and frozen.
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ABCC7 p.Tyr512Phe 21807898:63:169
status: NEW116 Whereas immunoprecipitated Myc-443 NBD1 domain was found to be phosphorylated by Syk in vitro, the444 corresponding NBD1-Y512F mutant was not recognized as substrate (FIG. 4 -445 right panels).
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ABCC7 p.Tyr512Phe 21807898:116:121
status: NEW117 Although this approach does not guarantee that Syk recognizes the446 isolated NBD1 with the same efficiency as full-length CFTR, the results447 confirmed qualitatively that Syk can only phosphorylate wt but not NBD1-448 Y512F.449 450 WNK4 inhibits NBD1 phosphorylation by Syk.
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ABCC7 p.Tyr512Phe 21807898:117:220
status: NEW140 These510 mutants thus mimic the effects on CFTR of transfecting either Syk-wt or Syk-511 kd. To further confirm that Tyr512 mediates the observed effects of Syk and512 WNK4 in vivo, we first co-transfected the cells with non-phosphorylatable513 CFTR-Y512F and YFP-Syk-wt.
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ABCC7 p.Tyr512Phe 21807898:140:250
status: NEW141 Surface biotinylation revealed that Syk-wt514 could no longer reduce CFTR-Y512F surface expression (FIG. 8B, two middle515 lanes).
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ABCC7 p.Tyr512Phe 21807898:141:74
status: NEW[hide] Phosphorylation of cystic fibrosis transmembrane c... Amino Acids. 2013 Dec;45(6):1423-9. doi: 10.1007/s00726-013-1613-y. Epub 2013 Nov 1. Cesaro L, Marin O, Venerando A, Donella-Deana A, Pinna LA
Phosphorylation of cystic fibrosis transmembrane conductance regulator (CFTR) serine-511 by the combined action of tyrosine kinases and CK2: the implication of tyrosine-512 and phenylalanine-508.
Amino Acids. 2013 Dec;45(6):1423-9. doi: 10.1007/s00726-013-1613-y. Epub 2013 Nov 1., [PMID:24178769]
Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) harbors, close to Phe-508, whose deletion is the commonest cause of cystic fibrosis, a conserved potential CK2 phospho-acceptor site (Ser511), which however is not susceptible to phosphorylation by CK2. To shed light on this apparent paradox, a series of systematically substituted peptides encompassing Ser511 were assayed for their ability to be phosphorylated. The main outcomes of our study are the following: (a) Tyr512 plays a prominent role as a negative determinant as its replacement by Ala restores Ser511 phosphorylation by CK2; (b) an even more pronounced phosphorylation of Ser511 is promoted if Tyr512 is replaced by phospho-tyrosine instead of alanine; (c) Tyr512 and, to a lesser extent, Tyr515 are readily phosphorylated by Lyn, a protein tyrosine kinase of the Src family, in a manner which is enhanced by the concomitant Phe508 deletion. Collectively taken, our data, in conjunction with the notion that Tyr515 is phosphorylated in vivo, disclose the possibility that CFTR Ser511 can be phosphorylated by the combined action of tyrosine kinases and CK2 and disclose a new mechanism of hierarchical phosphorylation where the role of the priming kinase is that of removing negative determinant(s).
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No. Sentence Comment
51 To note that phosphorylation is not restored if Tyr512 is replaced by Phe instead of Ala (peptide 8), while it is dramatically enhanced if Tyr512 is replaced by aspartic acid (peptide 9).
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ABCC7 p.Tyr512Phe 24178769:51:48
status: NEW