title: The major cystic fibrosis causing mutation exhibits defective propensity for phosphorylation.
abstract: The major cystic fibrosis causing mutation, F508del-CFTR (where CFTR is cystic fibrosis transmembrane conductance regulator), impairs biosynthetic maturation of the CFTR protein, limiting its expression as a phosphorylation-dependent channel on the cell surface. The maturation defect can be partially rescued by low-temperature (27°C) cell culture conditions or small-molecule corrector compounds. Following its partial rescue, the open probability of F508del-CFTR is enhanced by the potentiator compound, VX-770. However, the channel activity of rescued F508del-CFTR remains less than that of the Wt-CFTR protein in the presence of VX-770. In this study, we asked if there are allosteric effects of F508del on the phosphorylation-regulated R domain. To identify defects in the R domain, we compared the phosphorylation status at protein kinase A sites in the R domain of Wt and F508del-CFTR. Here we show that phosphorylation of Ser-660, quantified by SRM-MS, is reduced in F508del-CFTR. Although the generation of a phosphomimic at this site (substituting aspartic acid for serine) did not modify the maturation defect, it did enhance F508del-CFTR channel function after pharmacological rescue with corrector VX-809, and treatment with the potentiator, VX-770. These findings support the concept that defective phosphorylation of F508del-CFTR partially accounts for its altered channel activity at the cell surface.
authors.affiliation: Programme in Molecular Structure and Function, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada; Department of Biochemistry, The University of Toronto, Toronto, ON, Canada.
authors.forename: Stan
authors.initials: S
authors.lastname: Pasyk
authors.forename: Steven
authors.initials: S
authors.lastname: Molinski
authors.forename: Saumel
authors.initials: S
authors.lastname: Ahmadi
authors.forename: Mohabir
authors.initials: M
authors.lastname: Ramjeesingh
authors.forename: Ling-Jun
authors.initials: LJ
authors.lastname: Huan
authors.forename: Stephanie
authors.initials: S
authors.lastname: Chin
authors.forename: Kai
authors.initials: K
authors.lastname: Du
authors.forename: Herman
authors.initials: H
authors.lastname: Yeger
authors.forename: Paul
authors.initials: P
authors.lastname: Taylor
authors.forename: Michael F
authors.initials: MF
authors.lastname: Moran
authors.forename: Christine E
authors.initials: CE
authors.lastname: Bear
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003550:Cystic Fibrosis / Q000235:genetics* / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D017384:Sequence Deletion
chemical_list: D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Cell biology; Cystic fibrosis; Mass spectrometry; Phosphorylation; Protein kinase A (PKA); Protein misfolding