Contribution of R domain phosphoserines to the function of CFTR studied in Fischer rat thyroid epithelia.
The regulatory domain of cystic fibrosis transmembrane conductance regulator (CFTR) regulates channel activity when several serines are phosphorylated by cAMP-dependent protein kinase. To further define the functional role of individual phosphoserines, we studied CFTR containing previously studied and new serine to alanine mutations. We expressed these constructs in Fischer rat thyroid epithelia and measured transepithelial Cl(-) current. Mutation of four in vivo phosphorylation sites, Ser(660), Ser(737), Ser(795), and Ser(813) (S-Quad-A), substantially decreased cAMP-stimulated current, suggesting that these four sites account for most of the phosphorylation-dependent response. Mutation of either Ser(660) or Ser(813) alone significantly decreased current, indicating that these residues play a key role in phosphorylation-dependent stimulation. However, neither Ser(660) nor Ser(813) alone increased current to wild-type levels; both residues were required. Changing Ser(737) to alanine increased current above wild-type levels, suggesting that phosphorylation of Ser(737) may inhibit current in wild-type CFTR. These data help define the functional role of regulatory domain phosphoserines and suggest interactions between individual phosphoserines.
Baldursson
O
O
Howard Hughes Medical Institute, University of Iowa College of Medicine, Iowa City, Iowa 52242, USA.
Berger
H A
HA
Welsh
M J
MJ
D000409:Alanine; D019943:Amino Acid Substitution; D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000502:physiology*; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D010768:Phosphoserine; D051381:Rats; D011916:Rats, Inbred F344; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012694:Serine; D013961:Thyroid Gland / Q000166:cytology / Q000502:physiology*; D014162:Transfection
D011994:Recombinant Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D010768:Phosphoserine; D005576:Colforsin; D012694:Serine; D000409:Alanine