Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the anion channel mutated
in cystic fibrosis (CF) patients, is activated by the catalytic subunit of protein
kinase A (PKA-C). PKA-C activates CFTR both noncatalytically, through binding, and
catalytically, through phosphorylation of multiple serines in CFTR's regulatory (R)
domain. Here, we identify key molecular determinants of the CFTR/PKA-C interaction
essential for these processes. By comparing CFTR current activation in the presence
of ATP or an ATP analog unsuitable for phosphotransfer, as well as pseudosubstrate
peptides of various lengths, we identify two distinct specific regions of the PKA-C
surface which interact with CFTR to cause noncatalytic and catalytic CFTR stimulation,
respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct
hydrophobic patch (the "docking site") is responsible for noncatalytic CFTR activation,
achieved by stabilizing the R domain in a "released" conformation permissive to channel
gating. Furthermore, by comparing PKA-C variants with different posttranslational
modification patterns, we find that direct membrane tethering of the kinase through
its N-terminal myristoyl group is an unappreciated fundamental requirement for CFTR
activation: PKA-C demyristoylation abolishes noncatalytic, and profoundly slows catalytic,
CFTR stimulation. For the F508del CFTR mutant, present in ~90% of CF patients, maximal
activation by demyristoylated PKA-C is reduced by ~10-fold compared to that by myristoylated
PKA-C. Finally, in bacterial genera that contain common CF pathogens, we identify
virulence factors that demyristoylate PKA-C in vitro, raising the possibility that
during recurrent bacterial infections in CF patients, PKA-C demyristoylation may contribute
to the exacerbation of lung disease.
