CFTR ACTIVATION ADDITIVE EFFECTS OF STIMULATORY AND INHIBITORY PHOSPHORYLATION SITES IN THE R-DOMAIN

To investigate the functional significance of individual consensus pho sphorylation sites within the R domain of cystic fibrosis transmembran e conductance regulator (CFTR), serines were eliminated by substitutin g them with alanine. Included in this analysis were serine-660, -670, -686, -700, -712, -737, -768, -795, and -813, which lie within protein kinase A consensus sequences, and serine-641, which does not. Elimina tion of single potential phosphorylation sites altered the sensitivity of CFTR (expressed in Xenopus oocytes) to activating conditions in a manner that was highly site dependent. Substitution at serine-660, -67 0, -700, -795, or -813 significantly increased the half-maximal activa tion constant (K-A) for activation by 3-isobutyl-1-methylxanthine, whi ch is consistent with the hypothesis that phosphorylation at any of th ese sites promotes CFTR activation. The effect of substitution at seri ne-813 was significantly greater than at the other sites. In contrast, alanine substitution at serine-737 or -768 actually decreased the K-A for activation, suggesting that phosphorylation at either of these si tes is inhibitory. Substitution at serine-641, -686, and -712 had no s ignificant effect on activation sensitivity. The effects of multiple s erine to alanine substitutions were consistent with the notion that ph osphorylation at individual sites produced roughly additive effects, s uggesting that the effect produced by phosphorylation of any one serin e was not dependent on the phosphorylation state of other serines. The se results are consistent with the notion that, although none of the p hosphorylation sites studied here are absolutely necessary for activat ion of CFTR, individual sites contribute differently to the gating of the channel.