title: Mutation of potential phosphorylation sites in the recombinant R domain of the cystic fibrosis transmembrane conductance regulator has significant effects on domain conformation.
abstract: Mutation of potential cAMP dependent protein kinase sites in the R domain of the cystic fibrosis transmembrane conductance regulator has significant effects on protein function. Mutation of the potential phosphorylation sites from serine to alanine, to abolish the site, reduced sensitivity to activation, or to glutamic acid, to mimic phosphorylation, caused some constitutive activity. To explore the structural effects of these mutations, recombinant R domain peptides were studied: the wild type, a mutant with nine serine residues changed to alanine, and a mutant with eight serine residues changed to glutamic acid. As assessed by C.D. spectroscopy, the mutants have substantially different secondary structure than the wild type, in agreement with the predictive algorithm of Gascuel and Golmard. The results show that mutagenesis of residues alters the polypeptide structurally as well as preventing it from serving as a phosphorylation substrate. Hence, the functional consequences of the mutations may not be entirely due to effects on phosphorylation.
authors.affiliation: Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada.
authors.forename: A M
authors.initials: AM
authors.lastname: Dulhanty
authors.forename: X B
authors.initials: XB
authors.lastname: Chang
authors.forename: J R
authors.initials: JR
authors.lastname: Riordan
mesh_terms: D000595:Amino Acid Sequence; D018118:Chloride Channels / Q000737:chemistry*; D002942:Circular Dichroism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D008565:Membrane Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010766:Phosphorylation; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013379:Substrate Specificity; D013912:Threonine
chemical_list: D018118:Chloride Channels; D008565:Membrane Proteins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D013912:Threonine; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases