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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

PKC phosphorylation modulates PKA-dependent binding of the R domain to other domains of CFTR

¹Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia; and ²Department of Physiology, McGill University, Montréal, Quebec, Canada

Submitted 24 January 2008 ; accepted in final form 13 September 2008

Activity of the CFTR channel is regulated by phosphorylation of its regulatory domain (RD). In a previous study, we developed a bicistronic construct called R-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 R-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 R-Split/7CA-RD channels was similar to that of R-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.

cystic fibrosis; the cystic fibrosis transmembrane conductance regulator chloride channel; domain-domain interactions; protein kinase A and protein kinase C phosphorylation

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