HANRAHAN, J.W.; HINKSON, D.A.R.; EVAGELIDIS, A.; LUO, Y.; RIORDAN, J.R.; CHANG, X.-B. *more*; McGill Univ.; McGill Univ.; McGill Univ.; McGill Univ.; Mayo Clinic Scottsdale; Mayo Clinic Scottsdale *more*: PHOSPHORYLATION CONTROL OF CFTR CHANNEL ACTIVITY AND DOMAIN-DOMAIN INTERACTIONS

CFTR is controlled by its regulatory or �R� domain, which has many consensus sequences for phosphorylation by PKA and PKC that are highly conserved from lower vertebrates to humans. In vivo phosphorylation of some sites has been demonstrated, however the mechanism of activation by multi-site phosphorylation and its physiological significance remain obscure. PKA is the main stimulus of channel activity whereas PKC enhances responses to PKA. The actions of PKC require direct phosphorylation of CFTR, since they are abolished when all nine PKC sites in the R domain and adjacent nucleotide binding domain (NBD1) are removed. Point mutations suggest that the PKC dependence is mediated by only three sites, and the relative importance of each site parallels its phosphorylation. To investigate how phosphorylation might regulate the channel, pNUT-CFTR was converted into a bicistronic expression vector by replacing the region that encoding the R domain with an internal ribosomal entry sequence (IRES) so that N- and C-terminal halves lacking the R domain would be translated as separate polypeptides from a single mRNA. Stable expression of this �split delta R CFTR� construct in mammalian cells induced halide permeability that was not responsive to cAMP. The front and back halves of �split� channels associated with each other, and also with exogenously-added GST/R domain fusion protein. Surprisingly, interaction with GST/R was enhanced when it was pre-phosphorylated. The results suggest that PKC increases the response to PKA through direct phosphorylation of CFTR, and that phosphorylation alters domain-domain interactions. Support: CCFF, CIHR, NIDDK