title: Demonstration of phosphoryl group transfer indicates that the ATP-binding cassette (ABC) transporter cystic fibrosis transmembrane conductance regulator (CFTR) exhibits adenylate kinase activity.
abstract: Cystic fibrosis transmembrane conductance regulator (CFTR) is a membrane-spanning adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter. ABC transporters and other nuclear and cytoplasmic ABC proteins have ATPase activity that is coupled to their biological function. Recent studies with CFTR and two nonmembrane-bound ABC proteins, the DNA repair enzyme Rad50 and a structural maintenance of chromosome (SMC) protein, challenge the model that the function of all ABC proteins depends solely on their associated ATPase activity. Patch clamp studies indicated that in the presence of physiologically relevant concentrations of adenosine 5'-monophosphate (AMP), CFTR Cl(-) channel function is coupled to adenylate kinase activity (ATP+AMP <==> 2 ADP). Work with Rad50 and SMC showed that these enzymes catalyze both ATPase and adenylate kinase reactions. However, despite the supportive electrophysiological results with CFTR, there are no biochemical data demonstrating intrinsic adenylate kinase activity of a membrane-bound ABC transporter. We developed a biochemical assay for adenylate kinase activity, in which the radioactive γ-phosphate of a nucleotide triphosphate could transfer to a photoactivatable AMP analog. UV irradiation could then trap the (32)P on the adenylate kinase. With this assay, we discovered phosphoryl group transfer that labeled CFTR, thereby demonstrating its adenylate kinase activity. Our results also suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for adenylate kinase activity. These biochemical data complement earlier biophysical studies of CFTR and indicate that the ABC transporter CFTR can function as an adenylate kinase.
authors.affiliation: Department of Pediatrics, University of Iowa, Iowa City, Iowa 52242, USA. christoph-randak@uiowa.edu
authors.forename: Christoph O
authors.initials: CO
authors.lastname: Randak
authors.forename: Amanda R
authors.initials: AR
authors.lastname: Ver Heul
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Welsh
mesh_terms: D017766:Acid Anhydride Hydrolases; D000244:Adenosine Diphosphate / Q000235:genetics / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000235:genetics / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D010766:Phosphorylation / Q000502:physiology
chemical_list: C505032:CFTR protein, human; D004268:DNA-Binding Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D000262:Adenylyl Cyclases; D045643:DNA Repair Enzymes