CAMP-DEPENDENT PROTEIN KINASE-MEDIATED PHOSPHORYLATION OF CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR RESIDUE SER-753 AND ITS ROLE IN CHANNEL ACTIVATION

Hormonal regulation of the cystic fibrosis transmembrane conductance r egulator (CFTR) Cl- channel is largely mediated via cAMP-dependent pro tein kinase (PKA), CFTR contains 10 dibasic consensus sites for potent ial PKA phosphorylation ((R/K)(R/K)X(S/T*)). Previous studies (Chang, X-B., Tabcharani, J. A., Hou, Y.-X., Jensen, T. J., Kartner, N., Alon , N., Hanrahan, J.W., and Riordan, J.R (1993) J. Biol. Chem. 268, 1130 4-11311) showed that approximately 25% of the CFTR wild-type response to PKA activation remained upon inhibition of most detectable phosphor ylation by in vitro mutagenesis of all 10 dibasic consensus sites (10S A CFTR), To identify potential additional sites responsible for the re sidual activity, large amounts of this mutant CFTR were phosphorylated with PKA using high specific activity [gamma-P-32]ATP. Cyanogen bromi de cleavage indicated that a large portion of the observed PKA phospho rylation occurred within a 5.8-kDa fragment of the R domain between re sidues 722-773. Removal of serines at potential PKA sites in this frag ment showed that Ser-753 accounted for all of the gamma-P-32 labeling of the 5.8-kDa peptide. Replacement of Ser-753 with alanine reduced th e level of residual CFTR activity by a further 40%, indicating that ph osphorylation at this previously unidentified site contributes to the activation of 10SA CFTR.