PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR.
Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called DeltaR-Split CFTR, which encodes the front and back halves of CFTR as separate polypeptides without the RD. These fragments assemble to form a constitutively active CFTR channel. Coexpression of the third fragment corresponding to the missing RD restores regulation by PKA, and this is associated with dramatically enhanced binding of the phosphorylated RD. In the present study, we examined the effect of PKC phosphorylation on this PKA-induced interaction. We report here that PKC alone enhanced association of the RD with DeltaR-Split CFTR and that binding was further enhanced when the RD was phosphorylated by both kinases. Mutation of all seven PKC consensus sequences on the RD (7CA-RD) did not affect its association under basal (unphosphorylated) conditions but abolished phosphorylation-induced binding by both kinases. Iodide efflux responses provided further support for the essential role of RD binding in channel regulation. The basal activity of DeltaR-Split/7CA-RD channels was similar to that of DeltaR-Split/wild type (WT)-RD channels, whereas cAMP-stimulated iodide efflux was greatly diminished by removal of the PKC sites, indicating that 7CA-RD binding maintains channels in an inactive state that is unresponsive to PKA. These results suggest a novel mechanism for CFTR regulation in which PKC modulates PKA-induced domain-domain interactions.
Seavilleklein
Gage
G
Dept. of Physiology and Biophysics, 5850 College St., Halifax, NS, Canada B3H 1X5.
Amer
Noha
N
Evagelidis
Alexandra
A
Chappe
Frédéric
F
Irvine
Thomas
T
Hanrahan
John W
JW
Chappe
Valérie
V
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002460:Cell Line; D016384:Consensus Sequence; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits / Q000235:genetics / Q000378:metabolism*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D007454:Iodides / Q000378:metabolism; D015640:Ion Channel Gating; D009154:Mutation; D010766:Phosphorylation; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
D007454:Iodides; D011993:Recombinant Fusion Proteins; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D000242:Cyclic AMP; D054751:Cyclic AMP-Dependent Protein Kinase Catalytic Subunits; D011493:Protein Kinase C