Structural determinants of protein kinase A essential for CFTR channel activation
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 reversibly, through binding, and irreversibly, through phosphorylation of multiple serines in CFTRs 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 reversible and irreversible CFTR stimulation, respectively. Whereas the "substrate site" mediates CFTR phosphorylation, a distinct hydrophobic patch (the "docking site") is responsible for reversible 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 reversible, and profoundly slows irreversible, CFTR stimulation. For the F508del CFTR mutant, present in [~]90% of CF patients, maximal activation by de-myristoylated 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. Significance StatementCFTR is an anion channel crucial for salt-water transport across epithelia, and is activated by the catalytic subunit of protein kinase A (PKA-C). Reduced activity of mutant CFTR causes cystic fibrosis and CFTR hyperstimulation by sustained PKA-C activity causes diarrhea. PKA-C activates CFTR reversibly through simple binding, and irreversibly by phosphorylating the channel. We uncover here important structural requirements for these two processes. First, two distinct PKA-C surface areas mediate reversible and irreversible CFTR activation. Second, membrane anchoring of PKA-C through a covalently linked fatty (myristic) acid is required for both effects. Finally, we identify bacterial enzymes that cleave the myristic acid from PKA-C, thereby reducing activation of mutant CFTR channels, present in cystic fibrosis patients, by up to tenfold.