title: Role of individual R domain phosphorylation sites in CFTR regulation by protein kinase A.
abstract: The cystic fibrosis transmembrane conductance regulator (CFTR) plays a critical role in transcellular ion transport and when defective, results in the genetic disease cystic fibrosis. CFTR is novel in the ATP-binding cassette superfamily as an ion channel that is enabled by a unique unstructured regulatory domain. This R domain contains multiple protein kinase A sites, which when phosphorylated allow channel gating. Most of the sites have been indicated to stimulate channel activity, while two of them have been suggested to be inhibitory. It is unknown whether individual sites act coordinately or distinctly. To address this issue, we raised monoclonal antibodies recognizing the unphosphorylated, but not the phosphorylated states of four functionally relevant sites (700, 737, 768, and 813). This enabled simultaneous monitoring of their phosphorylation and dephosphorylation and revealed that both processes occurred rapidly at the first three sites, but more slowly at the fourth. The parallel phosphorylation rates of the stimulatory 700 and the putative inhibitory 737 and 768 sites prompted us to reexamine the role of the latter two. With serines 737 and 768 reintroduced individually into a PKA insensitive variant, in which serines at 15 sites had been replaced by alanines, a level of channel activation by PKA was restored, showing that these sites can mediate stimulation. Thus, we have provided new tools to study the CFTR regulation by phosphorylation and found that sites proposed to inhibit channel activity can also participate in stimulation.
authors.affiliation: Department of Biochemistry, University of North Carolina-Chapel Hill, Chapel Hill, NC 27599, USA.
authors.forename: Tamás
authors.initials: T
authors.lastname: Hegedus
authors.forename: Andrei
authors.initials: A
authors.lastname: Aleksandrov
authors.forename: April
authors.initials: A
authors.lastname: Mengos
authors.forename: Liying
authors.initials: L
authors.lastname: Cui
authors.forename: Timothy J
authors.initials: TJ
authors.lastname: Jensen
authors.forename: John R
authors.initials: JR
authors.lastname: Riordan