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.

Regulation of the CFTR channel by phosphorylation.

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are regulated tightly by protein kinases and phosphatases. The regulatory domain of CFTR has about 20 potential sites for phosphorylation by protein kinases A (PKA) and C (PKC). The reason for this large number of sites is not known, however their conservation from fish to humans implies that they play important roles in vivo. PKA is an important activator, and its stimulation of CFTR is enhanced by PKC via mechanisms which are not fully understood. The physiological stimuli of CFTR are not known for some epithelia, and it appears likely that other serine/threonine and even tyrosine kinases also regulate CFTR in particular tissues. Phosphatases that deactivate CFTR have yet to be identified definitively at the molecular level, however CFTR is regulated by a membrane-bound form of protein phosphatase-2C (PP2C) in several cell types. Patch-clamp studies of channel rundown, co-immunoprecipitation, chemical cross-linking studies, and pull-down assays all indicate that CFTR and PP2C are closely associated within a stable regulatory complex. Understanding the regulation of CFTR by PP2C is a priority due to its potential as a target for pharmacotherapies in the treatment of cystic fibrosis.