title: The cystic fibrosis transmembrane conductance regulator is regulated by a direct interaction with the protein phosphatase 2A.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated chloride channel expressed at the apical surface of epithelia. Although the regulation of CFTR by protein kinases is well documented, channel deactivation by phosphatases is not well understood. We find that the serine/threonine phosphatase PP2A can physically associate with the CFTR COOH terminus. PP2A is a heterotrimeric phosphatase composed of a catalytic subunit and two divergent regulatory subunits (A and B). The cellular localization and substrate specificity of PP2A is determined by the unique combination of A and B regulatory subunits, which can give rise to at least 75 different enzymes. By mass spectrometry, we identified the exact PP2A regulatory subunits associated with CFTR as Aalpha and B'epsilon and find that the B'epsilon subunit binds CFTR directly. PP2A subunits localize to the apical surface of airway epithelia and PP2A phosphatase activity co-purifies with CFTR in Calu-3 cells. In functional assays, inhibitors of PP2A block rundown of basal CFTR currents and increase channel activity in excised patches of airway epithelia and in intact mouse jejunum. Moreover, PP2A inhibition in well differentiated human bronchial epithelial cells results in a CFTR-dependent increase in the airway surface liquid. Our data demonstrate that PP2A is a relevant CFTR phosphatase in epithelial tissues. Our results may help reconcile differences in phosphatase-mediated channel regulation observed for different tissues and cells. Furthermore, PP2A may be a clinically relevant drug target for CF, which should be considered in future studies.
authors.affiliation: Department of Cell and Developmental Biology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, USA.
authors.forename: William R
authors.initials: WR
authors.lastname: Thelin
authors.forename: Mehmet
authors.initials: M
authors.lastname: Kesimer
authors.forename: Robert
authors.initials: R
authors.lastname: Tarran
authors.forename: Silvia M
authors.initials: SM
authors.lastname: Kreda
authors.forename: Barbara R
authors.initials: BR
authors.lastname: Grubb
authors.forename: John K
authors.initials: JK
authors.lastname: Sheehan
authors.forename: M Jackson
authors.initials: MJ
authors.lastname: Stutts
authors.forename: Sharon L
authors.initials: SL
authors.lastname: Milgram
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D001980:Bronchi / Q000378:metabolism; D020134:Catalytic Domain; D002460:Cell Line; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism / Q000502:physiology*; D019281:Dimerization; D004848:Epithelium / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D013058:Mass Spectrometry; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000737:chemistry; D011485:Protein Binding; D054648:Protein Phosphatase 2; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid
chemical_list: C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D010744:Phosphoric Monoester Hydrolases