Abstract
The cystic fibrosis transmembrane conductance regulator (CFTR) is an epithelial Cl(-) channel whose activity is controlled by cAMP-dependent protein kinase (PKA)-mediated phosphorylation. We found that CFTR immunoprecipitates from Calu-3 airway cells contain endogenous PKA, which is capable of phosphorylating CFTR. This phosphorylation is stimulated by cAMP and inhibited by the PKA inhibitory peptide. The endogenous PKA that co-precipitates with CFTR could also phosphorylate the PKA substrate peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly (kemptide). Both the catalytic and type II regulatory subunits of PKA are identified by immunoblotting CFTR immunoprecipitates, demonstrating that the endogenous kinase associated with CFTR is PKA, type II (PKA II). Phosphorylation reactions mediated by CFTR-associated PKA II are inhibited by Ht31 peptide but not by the control peptide Ht31P, indicating that a protein kinase A anchoring protein (AKAP) is responsible for the association between PKA and CFTR. Ezrin may function as this AKAP, since it is expressed in Calu-3 and T84 epithelia, ezrin binds RII in overlay assays, and RII is immunoprecipitated with ezrin from Calu-3 cells. Whole-cell patch clamp of Calu-3 cells shows that Ht31 peptide reduces cAMP-stimulated CFTR Cl(-) current, but Ht31P does not. Taken together, these data demonstrate that PKA II is linked physically and functionally to CFTR by an AKAP interaction, and they suggest that ezrin serves as an AKAP for PKA-mediated phosphorylation of CFTR.
Highlights
The cystic fibrosis transmembrane conductance regulator (CFTR)1 is the basis of the cAMP-activated anion conductance pathway at the apical membranes of epithelial cells [1]
In the presence of cAMP, a major diffuse band with a molecular mass of ϳ180 kDa was phosphorylated by a protein kinase that was co-immunoprecipitated with CFTR (Fig. 1, lane 3)
We examined the effect of cAMP on the endogenous kinase activity by incubating the CFTR immunoprecipitate with [␥-32P]ATP and 10 M cAMP
Summary
The cystic fibrosis transmembrane conductance regulator (CFTR) is the basis of the cAMP-activated anion conductance pathway at the apical membranes of epithelial cells [1]. A model ClϪ secretory epithelium, T84, was found to express both RI and RII isoforms of PKA; and in these cells, about 2⁄3 of total PKA activity was due to RII that was localized on cellular structures [4] These findings raise the possibility that PKA may regulate CFTR via compartmental restrictions that are based on protein interactions and that this arrangement may lead to phosphorylation of CFTR at specific sites within the protein. In patch clamp experiments, the functional activation of CFTR by PKA could be disrupted by conditions that interfere with ezrin binding of RII These findings provide physical and functional evidence that PKA regulation of CFTR is AKAP-mediated, and they suggest that ezrin is a CFTR-associated AKAP in secretory epithelial cells
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