ABCMdb

PMID: 17289674

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 [PubMed]

Sentences

No. Mutations Sentence Comment

94 ABCC7 p.Ser511Ala 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). Login to comment 102 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp Co-immunoprecipitation of CK2 with CFTR Mutants-BHK cells were transfected with vectors encoding wild-type, ⌬F508, S511A, or S511D CFTR. Login to comment 189 ABCC7 p.Ser511Ala Phosphorylation assays using NBD1/ NBD1-S511A protein demonstrated that PKA-dependent phosphorylation remained intact. Login to comment 190 ABCC7 p.Ser511Ala In contrast, CK2 failed to phosphorylate the S511A mutant (Fig. 5D, open columns). Login to comment 211 ABCC7 p.Ser511Ala 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. Login to comment 212 ABCC7 p.Ser511Ala 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. Login to comment 216 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp ABCC7 p.Ser511Asp 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). Login to comment 218 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp ABCC7 p.Ser511Asp 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). Login to comment 219 ABCC7 p.Ser511Asp Thus, the S511D mutation rescues the CFTR-CK2 association abolished by ⌬F508. Login to comment 221 ABCC7 p.Ser511Asp Hep-G2 (human liver) cells were transfected with either wild-type or S511D CFTR (eliminating the putative CK2 target serine at Ser-511) using vectors encoding either NBD1 alone or full-length CFTR. Login to comment 224 ABCC7 p.Ser511Asp In cells expressing full-length CFTR, either adding TBB or transfecting S511D CFTR reduces the phosphoserine content; however, the effect of CK2 inhibition is more pronounced than Ser-511 mutation (Fig. 6C, right histogram). Login to comment 228 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp S511D (ϮF508) and S511A decreased the Po of CFTR 3-fold, without altering current flow through individual chan- FIGURE 5. Login to comment 237 ABCC7 p.Ser511Ala D, CK2-dependent but not PKA-dependent phosphorylation is abolished in S511A-NBD1; CK2 and PKA phos- phorylatewild-typeNBD1additivelyatdistinctsites,andtheirrespectiveinhibitorsarespecific(nϭ3Ϯrange). Login to comment 242 ABCC7 p.Ser511Asp ABCC7 p.Ser511Asp TBB failed to inhibit the cAMP-dependent chloride currents generated by S511D CFTR or the doubly mutated ⌬F508-S511D CFTR (Fig. 7B, compare with wild type, Fig. 3A), even though these mutants both bind CK2. Login to comment 258 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp 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. Login to comment 261 ABCC7 p.Ser511Ala ABCC7 p.Ser511Asp Thus we find that S511A and ⌬F508 share an inability to bind CK2, but S511D restores CK2 binding with or without F508. Login to comment 267 ABCC7 p.Ser511Asp ABCC7 p.Ser511Asp Interestingly, unlike ⌬F508-CFTR (40), neither S511D nor ⌬F508-S511D-CFTR required incubation at low temperature to detect channel activity at the plasma membrane in CHO cells FIGURE 6. Login to comment 268 ABCC7 p.Ser511Ala CK2 association with CFTR is abrogated by ⌬F508 or S511A mutation; CK2 phosphorylates CFTR at Ser-511 in vivo. Login to comment