Changes in the R‐region interactions depend on phosphorylation and contribute to PKA and PKC regulation of the cystic fibrosis transmembrane conductance regulator chloride channel

Abstract

The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R-region) through a mechanism, which is still not completely understood. We used a split-CFTR construct expressing the N-term-TMD1-NBD1 (Front Half; FH), TMD2-NBD2-C-Term (Back Half; BH), and the R-region as separate polypeptides (Split-R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R-region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R-region and each half of the Split-CFTR. We found that at basal conditions, the density of complexes formed between the R-region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R-region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH-R complexes by 40% from PKA level. In cells expressing the Split-R with the two inhibitory PKC sites on the R-region inactivated (SR-S641A/T682A), density of FH-R complexes was much higher than in Split-R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH-R complexes measured at all phosphorylation conditions. Since full-length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH-R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH-R interaction and prevented the PKC enhancing effect on CFTR function and FH-R interaction. The phospho-mimetic mutation (S686D) restored basal BH-R interaction and the PKC enhancing effect on CFTR function with enhanced FH-R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R-region, and this response is known to be enhanced 34 |

Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation

“…Here, we demonstrate how the requirement for S903 phosphorylation is also linked to transport activation. Nonetheless, CFTR studies have shown that combined PKA and PKC stimulation can drastically increase R-domain complex formation and activation (23, 46, 47). The phosphorylated S903 site phosphate engages a distinct triple ionic interaction with the basic groove in the N-terminal region (K617, K655, and K805), which stands out when compared to the other phospho-sites (S908, T911 and S914).…”

“…In purified samples, mutagenesis on CFTR and Ycf1 can diminish ATPase activity and can impact cellular activity (20, 21). Indeed, R-domain phospho-regulation seems to involve a complex kinase recruitment mechanism in which a myriad of phospho-sites that suggest PKA, PKC, CKII kinases can potentiate or suppress each other in the context of ABCC R-domain phosphorylation (22, 23). Recent work points to a specific PKA site (24), S813, as the predominant rate-limiting site, similar to the Ycf1 site S908 (20, 25).…”

Mutations of the cystic fibrosis transmembrane conductance regulator gene in males with congenital bilateral absence of the vas deferens: Reproductive implications and genetic counseling (Review)

“…1). Each MSD is composed of six transmembrane helices, of whichTM1-6 constitute MSD-1 and TM7-12 constitute MSd-2, followed by a cytoplasmic NBD (13). The MSD-2 and NBD-1 modules are connected by the r domain, a regulatory region with disordered structure that is unique to the CFTR gene and not found in other transporters (14).…”

“…In [12][13][14][15][16][17][18] week-old abortive fetuses with CFTR gene mutations, the vas deferens is obstructed and denatured by mucus, and the vas deferens may be further aggravated in embryonic development (26). Furthermore, Gaillard et al (26) demonstrated that the expression of the CFTR in epididymal epithelium in human fetuses carrying CFTR mutations at 10-33 weeks of gestation was low, resulting in the production of highly viscous mucus by epithelial cells of the male genital tract.…”

Cystic fibrosis transmembrane conductance regulator gene mutations in males with CBAVD versus nonobstructive azoospermia: A review

“…CBAVD as a genital type of cystic fibrosis is responsible for 2-6% of male infertility (Radpour et al, 2008). The clinical signs and symptoms of CBAVD, include slightly smaller or normal testicle size, atrophy in these testicles, or even lack of either the seminal vesicle or cauda epididymis or both.…”