Stimulatory and inhibitory protein kinase C consensus sequences regulate the cystic fibrosis transmembrane conductance regulator.
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 (S707A/S790A/T791A/S809A) by PKA was similar to that of wild-type CFTR and was enhanced by PKC, whereas responses of 3CA (T582A/T604A/S641A) and 2CA (T682A/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.
Chappe
Valerie
V
Department of Physiology, McGill University, Montréal, QC, Canada H3G 1Y6. valerie.chappe1@mcgill.ca
Hinkson
Deborah A
DA
Howell
L Daniel
LD
Evagelidis
Alexandra
A
Liao
Jie
J
Chang
Xiu-Bao
XB
Riordan
John R
JR
Hanrahan
John W
JW
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002417:Cattle; D016384:Consensus Sequence; D006224:Cricetinae; D017868:Cyclic AMP-Dependent Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D018408:Patch-Clamp Techniques; D010449:Peptide Mapping; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
C505032:CFTR protein, human; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C