title: Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.
authors.affiliation: Department of Pharmacology, University of Washington, Seattle 98195, USA.
authors.forename: R A
authors.initials: RA
authors.lastname: McDonald
authors.forename: R P
authors.initials: RP
authors.lastname: Matthews
authors.forename: R L
authors.initials: RL
authors.lastname: Idzerda
authors.forename: G S
authors.initials: GS
authors.lastname: McKnight
mesh_terms: D001483:Base Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003550:Cystic Fibrosis / Q000201:enzymology* / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic; D014407:Tumor Cells, Cultured
chemical_list: C505032:CFTR protein, human; D008565:Membrane Proteins; D012333:RNA, Messenger; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D004247:DNA; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases