title: Genistein activates CFTR Cl- channels via a tyrosine kinase- and protein phosphatase-independent mechanism.
abstract: Previous studies have revealed an adenosine 3',5'-cyclic monophosphate (cAMP)-independent activation of cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels by the tyrosine kinase inhibitor genistein. To further explore its mechanism of action, we have reconstituted genistein activation of CFTR in excised inside-out membrane patches. In the presence or absence of ATP, genistein appeared unable to open silent CFTR Cl- channels. However, on CFTR prephosphorylation by cAMP-dependent protein kinase (cAK), genistein enhanced CFTR activity by twofold, resulting from a prolonged burst duration. Genistein could also hyperactivate partially phosphorylated CFTR in the absence of cAK and therefore is different from 5'-adenylylimidodiphosphate, which required fully phosphorylated CFTR. Phosphatase-resistant thiophosphorylation likewise primed the CFTR Cl- channel for hyperactivation by genistein in the absence of cAK. Replacement of ATP by GTP as a hydrolyzable nucleotide triphosphate for CFTR did not impair the ability of genistein to activate thiophosphorylated CFTR, despite the fact that GTP is a poor substrate for tyrosine kinases. These findings argue against a role of protein phosphatases or tyrosine kinases but suggest a more direct interaction of genistein with CFTR, possibly at the level of the second nucleotide-binding domain.
authors.affiliation: Department of Cell Biology, Erasmus University, Rotterdam, The Netherlands.
authors.forename: P J
authors.initials: PJ
authors.lastname: French
authors.forename: J
authors.initials: J
authors.lastname: Bijman
authors.forename: A G
authors.initials: AG
authors.lastname: Bot
authors.forename: W E
authors.initials: WE
authors.lastname: Boomaars
authors.forename: B J
authors.initials: BJ
authors.lastname: Scholte
authors.forename: H R
authors.initials: HR
authors.lastname: de Jonge
mesh_terms: D016475:3T3 Cells; D000255:Adenosine Triphosphate / Q000502:physiology; D000818:Animals; D018118:Chloride Channels / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000378:metabolism* / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D019833:Genistein; D007529:Isoflavones / Q000494:pharmacology*; D051379:Mice; D010744:Phosphoric Monoester Hydrolases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000502:physiology*
chemical_list: D018118:Chloride Channels; D004791:Enzyme Inhibitors; D007529:Isoflavones; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000255:Adenosine Triphosphate; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D010744:Phosphoric Monoester Hydrolases