ABCMdb

PMID: 24727471

Luz S, Cihil KM, Brautigan DL, Amaral MD, Farinha CM, Swiatecka-Urban A
LMTK2-mediated phosphorylation regulates CFTR endocytosis in human airway epithelial cells.
J Biol Chem. 2014 May 23;289(21):15080-93. doi: 10.1074/jbc.M114.563742. Epub 2014 Apr 11., [PubMed]

Sentences

No. Mutations Sentence Comment

8 ABCC7 p.Ser737Ala Compared with the wild-type CFTR, the phosphorylation-deficient mutant CFTR-S737A shows increased cell surface density and decreased endocytosis. Login to comment 203 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala To demonstrate specificity of the phosphosite antibody, parental CFBE41o-cells were transiently transfected with WT-CFTR or CFTR with the S737A substitution (CFTR-S737A) inactivating the phosphorylation site (31). Login to comment 204 ABCC7 p.Ser737Ala Even with calyculin A treatment of the cells, the CFTR-S737A mutant was not detected by antibody Ab-737, unlike the WT-CFTR (Fig. 5B). Login to comment 233 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala B, parental CFBE41o-cells were transfected with the WT-CFTR (WT) or the mutant CFTR-S737A (S737A). Login to comment 234 ABCC7 p.Ser737Ala Unlike the WT-CFTR, the CFTR-S737A was not detected by the antibody Ab-737 in WCL. Login to comment 287 ABCC7 p.Ser737Ala The Phosphorylation-deficient CFTR-S737A Mutant is More Abundant at the Plasma Membrane Due to Decreased Endocytosis-If the LMTK2 mediated phosphorylation of CFTR-Ser737 induces CFTR endocytosis, eliminating the phosphorylation site by the S737A substitution should reduce CFTR endocytosis. Login to comment 288 ABCC7 p.Ser737Ala Parental CFBE41o-cells were transfected with WT-CFTR or the CFTR-S737A mutant. Login to comment 308 ABCC7 p.Ser737Ala The S737A mutation attenuated CFTR endocytosis and increased the plasma membrane abundance of CFTR (Fig. 8). Login to comment 331 ABCC7 p.Ser737Ala Experiments demonstrating that compared with WT-CFTR the phosphorylation-deficient mutant CFTR S737A has increased plasma membrane abundance at steady-state and decreased endocytosis in CFBE41o-cells. Login to comment 332 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala Parental CFBE41o-cells were transfected with the WT-CFTR (WT) or the CFTR-S737A mutant (S737A), and cells were cultured on collagen-coated tissue culture plates for 48 h to form monolayers. Login to comment 334 ABCC7 p.Ser737Ala Immunoblots (A) and summaryofdata(B)demonstratingthattheS737AmutationdecreasedCFTRendocytosis.TheamountofbiotinylatedWT-CFTRorCFTR-S737Aremainingafter the GSH treatment at 4 &#b0;C without warming to 37 &#b0;C was considered background and was subtracted from the amount of biotinylated WT-CFTR or CFTR-S737A remaining after warming to 37 &#b0;C. Login to comment 335 ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala ABCC7 p.Ser737Ala Endocytosis of WT-CFTR or CFTR-S737A was calculated after subtracting the background (see above) and was expressed as the percent of WT-CFTR or CFTR-S737A remaining biotinylated before and after warming to 37 &#b0;C. WT-CFTR and CFTR-S737A was detected with antibody CFF596. Login to comment 338 ABCC7 p.Ser737Ala The whole cell lysate (WCL) expression of WT-CFTR or CFTR-S737A mutant detected with antibody CFF596 was used as a loading control to account for small differences in the expression levels of the transiently transfected proteins. Login to comment