Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator

Abstract

Protein kinase C (PKC) phosphorylation stimulates the cystic fibrosis transmembrane conductance regulator (CFTR) channel and enhances its activation by protein kinase A (PKA) through mechanisms that remain poorly understood. We have examined the effects of mutating consensus sequences for PKC phosphorylation and report here evidence for both stimulatory and inhibitory sites. Sequences were mutated in subsets and the mutants characterized by patch clamping. Activation of a 4CA mutant (S707A͞S790A͞T791A͞S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A͞T604A͞S641A) and 2CA (T682A͞ S686A) channels to PKA were both drastically reduced (>90%). When each mutation in the 3CA and 2CA constructs was studied individually in a wild-type background, T582, T604, and S686 were found to be essential for PKA activation. Responses were restored when these three residues were reintroduced simultaneously into a 9CA mutant lacking all nine PKC consensus sequences (R6CA revertant); however, PKC phosphorylation was not required for this rescue. Nevertheless, two of the sites (T604 and S686) were phosphorylated in vitro, and PKC alone partially activated wild-type CFTR, the 4CA mutant, and the point mutants T582A and T604A, but not S686A channels, indicating that PKC does act at S686. The region encompassing S641 and T682 is inhibitory, because S641A enhanced activation by PKA, and T682A channels had 4-fold larger responses to PKC compared to wild-type channels. These results identify functionally important PKC consensus sequences on CFTR and will facilitate studies of its convergent regulation by PKC and PKA.

CFTR (ABCC7) is a hydrolyzable-ligand-gated channel

“…Protein kinase C also plays a role in CFTR activation [13], primarily in the case of the human protein, by enhancing the response to PKA [14]. This seems to occur without elevating or accelerating phosphorylation by PKA [14].…”

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…Here we discuss the effects of PKA phosphorylation on channel gating, which is the best characterized mode of CFTR regulation by phosphorylation. Protein kinase C (Chappe et al 2004) and AMP kinase (King et al 2009) stimulate and inhibit CFTR activity, respectively, by less well understood mechanisms.…”

Search for proteins with similarity to the CFTR R domain using an optimized RDBMS solution, mBioSQL

“…The R domain provides this control; phosphorylation of its multiple sites by PKA is obligatory for channel gating. Despite extensive investigations [4,5,10,[18][19][20] the mechanism whereby the unphosphorylated R domain maintains the non-gating state is not understood. As a supplement to the various experimental approaches that have been applied, we have attempted to obtain functional clues by searching for similarities with other proteins.…”