title: CFTR chloride channel activation by genistein: the role of serine/threonine protein phosphatases.
abstract: We have previously shown [B. Illek, H. Fischer, G. F. Santos, J. H. Widdicombe, T. E. Machen, and W. W. Reenstra, Am. J. Physiol. 268 (Cell Physiol. 37): C886-C893, 1995] that genistein, a tyrosine kinase inhibitor, activates the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel in NIH/3T3 cells that have been stably transfected with an expression vector for the CFTR (NIH-CFTR cells). In this study, we present evidence suggesting that both genistein and the serine/threonine protein phosphatase (PPase) inhibitor calyculin A activate the CFTR by inhibiting PPase activity. As measured by 125I efflux, genistein and calyculin A stimulate the CFTR to approximately 50% of the maximal activity with forskolin. Neither agonist increases CFTR activity at saturating forskolin concentrations, but genistein and calyculin A have an additive effect on CFTR activity. Forskolin, but neither genistein nor calyculin A, stimulates protein kinase A(PKA) activity. The PKA inhibitor H-89 inhibits CFTR activation and in vivo phosphorylation by all three agonists. Proteolytic digestion of in vivo phosphorylated CFTR suggests that the CFTR is phosphorylated on the same sites during stimulation with genistein and forskolin but on different sites stimulation with calyculin A. The data suggest that genistein and calyculin A inhibit different PPase activities, allowing CFTR phosphorylation and partial stimulation, by a basal PKA activity.
authors.affiliation: Department of Clinical Science, Alfred I. duPont Institute, Wilmington, Delaware 19899, USA.
authors.forename: W W
authors.initials: WW
authors.lastname: Reenstra
authors.forename: K
authors.initials: K
authors.lastname: Yurko-Mauro
authors.forename: A
authors.initials: A
authors.lastname: Dam
authors.forename: S
authors.initials: S
authors.lastname: Raman
authors.forename: S
authors.initials: S
authors.lastname: Shorten
mesh_terms: D016475:3T3 Cells; D000818:Animals; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D004357:Drug Synergism; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology*; D007546:Isoquinolines / Q000494:pharmacology; D008387:Marine Toxins; D051379:Mice; D010080:Oxazoles / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D014162:Transfection
chemical_list: D007529:Isoflavones; D007546:Isoquinolines; D008387:Marine Toxins; D010080:Oxazoles; D047428:Protein Kinase Inhibitors; D013449:Sulfonamides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D005576:Colforsin; C059041:calyculin A; D019833:Genistein; D010749:Phosphoprotein Phosphatases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide