Protein kinase A associates with cystic fibrosis transmembrane conductanceregulator via an interaction with ezrin

The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithe lial Cl- channel whose activity is controlled by cAMP-dependent protein kin ase (PKA)mediated phosphorylation. We found that CFTR immunoprecipitates fr om Calu-3 airway cells contain endogenous PKA, which is capable of phosphor ylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by t he PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFT R could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-L eu-Gly (kemptide), Both the catalytic and type II regulatory subunits of PK A are identified by immunoblotting CFTR immunoprecipitates, demonstrating t hat the endogenous kinase associated with CFTR is PKA, type II (PKA II). Ph osphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a prote in kinase A anchoring protein (AKAP) is responsible for the association bet ween PKA and CFTR, Ezrin may function as this AKAP, since it is expressed i n Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is i mmunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of C alu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl- curren t, but Ht31P does not. Taken together, these data demonstrate that PRA II i s linked physically and functionally to CFTR by an AKAP interaction, and th ey suggest that ezrin serves as an AKAP for PHA-mediated phosphorylation of CFTR,