title: Isotype-specific activation of cystic fibrosis transmembrane conductance regulator-chloride channels by cGMP-dependent protein kinase II.
abstract: Type II cGMP-dependent protein kinase (cGKII) isolated from pig intestinal brush borders and type I alpha cGK (cGKI) purified from bovine lung were compared for their ability to activate the cystic fibrosis transmembrane conductance regulator (CFTR)-Cl- channel in excised, inside-out membrane patches from NIH-3T3 fibroblasts and from a rat intestinal cell line (IEC-CF7) stably expressing recombinant CFTR. In both cell models, in the presence of cGMP and ATP, cGKII was found to mimic the effect of the catalytic subunit of cAMP-dependent protein kinase (cAK) on opening CFTR-Cl-channels, albeit with different kinetics (2-3-min lag time, reduced rate of activation). By contrast, cGKI or a monomeric cGKI catalytic fragment was incapable of opening CFTR-Cl- channels and also failed to potentiate cGKII activation of the channels. The cAK activation but not the cGKII activation was blocked by a cAK inhibitor peptide. The slow activation by cGKII could not be ascribed to counteracting protein phosphatases, since neither calyculin A, a potent inhibitor of phosphatase 1 and 2A, nor ATP gamma S (adenosine 5'-O-(thiotriphosphate)), producing stable thiophosphorylation, was able to enhance the activation kinetics. Channels preactivated by cGKII closed instantaneously upon removal of ATP and kinase but reopened in the presence of ATP alone. Paradoxically, immunoprecipitated CFTR or CF-2, a cloned R domain fragment of CFTR (amino acids 645-835) could be phosphorylated to a similar extent with only minor kinetic differences by both isotypes of cGK. Phosphopeptide maps of CF-2 and CFTR, however, revealed very subtle differences in site-specificity between the cGK isoforms. These results indicate that cGKII, in contrast to cGKI alpha, is a potential activator of chloride transport in CFTR-expressing cell types.
authors.affiliation: Department of Cell Biology, Faculty of Medicine and Health Sciences, 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: M
authors.initials: M
authors.lastname: Edixhoven
authors.forename: A B
authors.initials: AB
authors.lastname: Vaandrager
authors.forename: B J
authors.initials: BJ
authors.lastname: Scholte
authors.forename: S M
authors.initials: SM
authors.lastname: Lohmann
authors.forename: A C
authors.initials: AC
authors.lastname: Nairn
authors.forename: H R
authors.initials: HR
authors.lastname: de Jonge
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D018118:Chloride Channels / Q000096:biosynthesis / Q000378:metabolism*; D017869:Cyclic GMP-Dependent Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007422:Intestines / Q000201:enzymology; D007527:Isoenzymes / Q000096:biosynthesis / Q000378:metabolism; D007700:Kinetics; D008168:Lung / Q000201:enzymology; D046911:Macromolecular Substances; D008387:Marine Toxins; D008564:Membrane Potentials; D008871:Microvilli / Q000201:enzymology; D010080:Oxazoles / Q000494:pharmacology; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D013552:Swine; D014162:Transfection
chemical_list: D018118:Chloride Channels; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D046911:Macromolecular Substances; D008387:Marine Toxins; D010080:Oxazoles; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C022571:adenosine 5'-O-(3-thiotriphosphate); C059041:calyculin A; D000255:Adenosine Triphosphate; D017869:Cyclic GMP-Dependent Protein Kinases; D054645:Protein Phosphatase 1; D017027:Protein Tyrosine Phosphatases