Phosphorylation of protein kinase C sites in NBD1 and the R domain control CFTR channel activation by PKA.
Activation of the cystic fibrosis transmembrane conductance regulator (CFTR) channel by protein kinase A (PKA) is enhanced by protein kinase C (PKC). However, the mechanism of modulation is not known and it remains uncertain whether PKC acts directly on CFTR or through phosphorylation of an ancillary protein. Using excised patches that had been pre-treated with phosphatases, we found that PKC exposure results in much larger PKA-activated currents and shifts the PKA concentration dependence. To examine if these effects are mediated by direct PKC phosphorylation of CFTR, a mutant was constructed in which serines or threonines at nine PKC consensus sequences on CFTR were replaced by alanines (i.e. the '9CA' mutant T582A/T604A/S641A/T682A/S686A/S707A/S790A/T791A/S809A). In excised patches, 9CA channels had greatly reduced responses to PKA (i.e. 5-10 % that of wild-type), which were not enhanced by PKC pre-treatment, although the mutant channels were still functional according to iodide efflux assays. Stimulation of iodide efflux by chlorophenylthio-cAMP (cpt-cAMP) was delayed in cells expressing 9CA channels, and a similar delay was observed when cells expressing wild-type CFTR were treated with the PKC inhibitor chelerythrine. This suggests that weak activation by PKA in excised patches and slow stimulation of iodide efflux from intact cells are specifically due to the loss of PKC phosphorylation. Finally, PKC caused a slight activation of wild-type channels when added to excised patches after phosphatase pre-treatment but had no effect on the mutant. We conclude that direct phosphorylation of CFTR at one or more of the nine sites mutated in 9CA is required for both the partial activation by PKC and for its modulation of CFTR responses to PKA.
Chappe
V
V
Department of Physiology, McGill University, Montréal, Québec, Canada. valerie.chappe1@mcgill.edu
Hinkson
D A
DA
Zhu
T
T
Chang
X-B
XB
Riordan
J R
JR
Hanrahan
J W
JW
D000470:Alkaloids; D000595:Amino Acid Sequence; D000818:Animals; D053119:Benzophenanthridines; D001665:Binding Sites; D002417:Cattle; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D006224:Cricetinae; D000242:Cyclic AMP / Q000031:analogs & derivatives* / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000378:metabolism*; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015151:Immunoblotting; D007454:Iodides / Q000378:metabolism; D008564:Membrane Potentials / Q000502:physiology; D008969:Molecular Sequence Data; D009154:Mutation / Q000502:physiology; D018408:Patch-Clamp Techniques; D010617:Phenanthridines / Q000494:pharmacology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats; D013873:Thionucleotides / Q000494:pharmacology
D000470:Alkaloids; D053119:Benzophenanthridines; D004791:Enzyme Inhibitors; D007454:Iodides; D010617:Phenanthridines; D013873:Thionucleotides; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C015929:8-((4-chlorophenyl)thio)cyclic-3',5'-AMP; D000242:Cyclic AMP; C016299:chelerythrine; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C