Phosphorylation-dependent Changes in the R-region Interactions Contribute to Regulation of the CFTR Chloride Channel

Abstract

Cystic Fibrosis Transmembrane Conductance Regulator (CFTR), the defective protein in cystic fibrosis, is an ion channel regulated by PKA and PKC phosphorylation of its regulatory region (R-region) through a mechanism still not completely understood, preventing a full comprehension of the process involved in CFTR activation. To provide novel insights into the CFTR activation mechanism, I analyzed in situ and in vitro interactions of the R-region with other parts of CFTR at different phosphorylation conditions (PKA, PKC, PKA+PKC). For in situ analysis, I used a split-CFTR construct expressing the front half (FH;N-tail/TMD1/NBD1), back half (BH;TMD2/NBD2/C-tail) and the R-region as three separate polypeptides expressed in BHK cells. I found that PKA stimulation increased both FH-R and BH-R interactions and PKC+PKA further increased only FH-R interactions whereas PKC stimulation alone had no effect. Inactivation of PKC consensus site S686 (S686A) significantly reduced basal BH-R interaction and prevented the further enhancing of FH-R interactions by PKC+PKA phosphorylation that was found with the wild-type R-region. The opposite phosphomimetic mutation S686D restored basal BH-R and rescued 80% of FH-R interactions after PKC+PKA stimulation compared to wild-type levels. As the channel function is mainly stimulated by PKA phosphorylation of the R region, and this response is doubled by PKC+PKA phosphorylation, results from this thesis suggest that PKC enhances PKA effect by increasing R-region interactions with the FH. Also, PKC site S686 was found to be crucial for the PKC enhancing effect which appeared to be mediated by a permissive interaction of the R-region with the BH, allowing FH-R interactions to be enhanced by PKC. Analysis of in vitro interactions of the R-region with six polypeptides corresponding to the N- and C-terminal tails and the four cytoplasmic loops (CLs) of CFTR showed that phosphorylation strongly increased R-region interactions with both N- and C-tails of CFTR. Moreover, PKA and PKC+PKA phosphorylation reduced R-region interactions with CLs 4 and 3, respectively, a mechanism that permits the channel activation by PKA and PKC enhancing effect. All together, these results demonstrate that phosphorylation induces an R-region shift from the cytoplasmic loops to both amino and carboxyl ends of the protein, to activate the channel.