Current insights into the role of PKA phosphorylation in CFTR channel activity and the pharmacological rescue of cystic fibrosis disease-causing mutants

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) channel gating is predominantly regulated by protein kinase A (PKA)-dependent phosphorylation. In addition to regulating CFTR channel activity, PKA phosphorylation is also involved in enhancing CFTR trafficking and mediating conformational changes at the interdomain interfaces of the protein. The major cystic fibrosis (CF)-causing mutation is the deletion of phenylalanine at position 508 (F508del); it causes many defects that affect CFTR trafficking, stability, and gating at the cell surface. Due to the multiple roles of PKA phosphorylation, there is growing interest in targeting PKA-dependent signaling for rescuing the trafficking and functional defects of F508del-CFTR. This review will discuss the effects of PKA phosphorylation on wild-type CFTR, the consequences of CF mutations on PKA phosphorylation, and the development of therapies that target PKA-mediated signaling.

Keywords: Channel gating; Cytoskeleton; Interdomain interactions; Modulators; Phosphorylation; Trafficking.

Fig. 1

The deletion of phenylalanine at position 508 (F508del) alters phosphoregulation of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking. Left panel: protein kinase A (PKA) phosphorylation promotes CFTR gene expression via the cyclic adenosine monophosphate (cAMP) response element. Phosphorylation also enhances interactions of wild-type CFTR (Wt-CFTR) protein with 14-3-3, which facilitates CFTR exit from the endoplasmic reticulum (ER) and promotes its forward trafficking to the cell surface. Zoom in of the cell surface: phosphorylation enhances the interaction between CFTR and the Na⁺/H⁺ exchanger regulatory factor (NHERF1)–ezrin–actin complex which stabilizes the protein at the cell surface. Ezrin also brings PKA closer to CFTR which is essential for regulating channel gating. At the cell surface, CFTR can be internalized by endocytosis into early endosomes which can then recycle back to the cell surface or undergo lysosomal degradation. PKA phosphorylation can also enhance cell surface expression of CFTR by promoting recycling. Right panel: the major population of F508del-CFTR is retained in the ER and undergoes ER-associated degradation via the proteasome. A limited number of F508del-CFTR protein can escape the ER and reach the Golgi apparatus; however, aberrant exposure of “retention motifs” redirects the protein back to the ER. F508del-CFTR may also have impaired interactions with 14-3-3 due to defective phosphorylation of the mutant, resulting in decreased forward trafficking. Zoom in of the cell surface: the small population of F508del-CFTR that reaches the cell surface is unstable and is targeted for degradation via the peripheral protein quality control, possibly due to defective interactions with the NHERF1–ezrin–actin complex. The phosphorylation defect also results in reduced recycling leading to decreased cell surface expression of F508del-CFTR. Not depicted for clarity: CFTR trafficks within vesicles in the cell

Fig. 2

F508del-CFTR exhibits defective phosphorylation-dependent conformational changes essential for channel gating. Top panel: In Wt-CFTR, PKA increases phosphorylation at multiple sites which include S422 in the regulatory insertion (RI) of NBD1, S660 in the regulatory extension (RE) located at the amino-terminus of the R domain and other sites in the R domain. CFTR has two adenosine triphosphate (ATP)-binding sites at the NBD dimers: site 1 is non-canonical and does not hydrolyze ATP, whereas site 2 is canonical and exhibits ATPase activity. PKA phosphorylation decreases the alpha helical content of the RE and RI which removes their steric hindrances on nucleotide-binding domain 1 (NBD1). This enhances interactions at the NBD1:NBD2 interface (necessary for ATP binding) as well as the intracellular loop 1 (ICL1):NBD1 and ICL4:NBD1 interfaces (necessary for conveying conformational changes from the NBDs to the membrane spanning domains for channel gating). Bottom panel: F508del-CFTR exhibits defective phosphorylation at S660 in the RE. F508del-CFTR may also exhibit defective phosphorylation at S422 in the RI and/or other phosphorylation sites in the R domain, but this could not be confirmed with our mass spectrometry methods (sites depicted as question marks). F508del-NBD1 retains aberrant interactions with the RE and RI upon PKA phosphorylation; this prevents NBD1 from interacting with NBD2, ICL1 and ICL4 which results in impaired ATP binding/hydrolysis and defective conformational changes necessary for channel gating