title: Phosphorylation-dependent 14-3-3 protein interactions regulate CFTR biogenesis.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP/protein kinase A (PKA)-regulated chloride channel whose phosphorylation controls anion secretion across epithelial cell apical membranes. We examined the hypothesis that cAMP/PKA stimulation regulates CFTR biogenesis posttranslationally, based on predicted 14-3-3 binding motifs within CFTR and forskolin-induced CFTR expression. The 14-3-3β, γ, and ε isoforms were expressed in airway cells and interacted with CFTR in coimmunoprecipitation assays. Forskolin stimulation (15 min) increased 14-3-3β and ε binding to immature and mature CFTR (bands B and C), and 14-3-3 overexpression increased CFTR bands B and C and cell surface band C. In pulse-chase experiments, 14-3-3β increased the synthesis of immature CFTR, reduced its degradation rate, and increased conversion of immature to mature CFTR. Conversely, 14-3-3β knockdown decreased CFTR B and C bands (70 and 55%) and elicited parallel reductions in cell surface CFTR and forskolin-stimulated anion efflux. In vitro, 14-3-3β interacted with the CFTR regulatory region, and by nuclear magnetic resonance analysis, this interaction occurred at known PKA phosphorylated sites. In coimmunoprecipitation assays, forskolin stimulated the CFTR/14-3-3β interaction while reducing CFTR's interaction with coat protein complex 1 (COP1). Thus 14-3-3 binding to phosphorylated CFTR augments its biogenesis by reducing retrograde retrieval of CFTR to the endoplasmic reticulum. This mechanism permits cAMP/PKA stimulation to make more CFTR available for anion secretion.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, PA 15261, USA.
authors.forename: Xiubin
authors.initials: X
authors.lastname: Liang
authors.forename: Ana Carina
authors.initials: AC
authors.lastname: Da Paula
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Bozóky
authors.forename: Hui
authors.initials: H
authors.lastname: Zhang
authors.forename: Carol A
authors.initials: CA
authors.lastname: Bertrand
authors.forename: Kathryn W
authors.initials: KW
authors.lastname: Peters
authors.forename: Julie D
authors.initials: JD
authors.lastname: Forman-Kay
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005576:Colforsin / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D006801:Humans; D010766:Phosphorylation; D020033:Protein Isoforms / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; C505032:CFTR protein, human; D020033:Protein Isoforms; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases