PKC-mediated stimulation of amphibian CFTR depends on a single phosphorylation consensus site. Insertion of this site confers PKC sensitivity to human CFTR

Mutations of the CFTR, a phosphorylation-regulated Cl- channel, cause cysti c fibrosis. Activation of CFTR by PKA stimulation appears to be mediated by a complex interaction between several consensus phosphorylation sites in t he regulatory domain (R domain). None of these sites has a critical role in this process. Here, we show that although endogenous phosphorylation by PK C is required for the effect of PKA on CFTR, stimulation of PKC by itself h as only a minor effect on human CFTR. In contrast, CFTR from the amphibians Necturus maculosus and Xenopus laevis (XCFTR) can be activated to similar degrees by stimulation of either PKA or PKC. Furthermore, the activation of XCFTR by PKC is independent of the net charge of the R domain, and mutagen esis experiments indicate that a single site (Thr(665)) is required for the activation of XCFTR. Human CFTR lacks the PKC phosphorylation consensus si te that includes Thr(665), but insertion of an equivalent site results in a large activation upon PKC stimulation. These observations establish the pr esence of a novel mechanism of activation of CFTR by phosphorylation of the R domain, i.e., activation by PKC requires a single consensus phosphorylat ion site and is unrelated to the net charge of the R domain.