ABCC7 p.Tyr512Phe
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PMID: 21807898
[PubMed]
Mendes AI et al: "Antagonistic Regulation of Cystic Fibrosis Transmembrane Conductance Regulator Cell Surface Expression by Protein Kinases WNK4 and Spleen Tyrosine Kinase."
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
PMID: 24178769
[PubMed]
Cesaro L et al: "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."
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