Interactions of β and γENaC with Nedd4 can be facilitated by an ERK-mediated phosphorylation

Phosphorylation of the epithelial Na ⁺ channel (ENaC) has been suggested to play a role in its regulation. Here we demonstrate that phosphorylating the carboxyl termini of the β and γ subunits facilitates their interactions with the ubiquitin ligase Nedd4 and inhibits channel activity. Three protein kinases, which phosphorylate the carboxyl termini of β and γENaC, have been identified by an in vitro assay. One of these phosphorylates βThr-613 and γThr-623, well-conserved C-tail threonines in the immediate vicinity of the PY motifs. Phosphorylation of γThr-623 has also been demonstrated in vivo in channels expressed in Xenopus oocytes, and mutating βThr-613 and γThr-623 into alanine increased the channel activity by 3.5-fold. Effects of the above phosphorylations on interactions between ENaC and Nedd4 have been studied using surface plasmon resonance. Peptides having phospho-threonine at positions β613 or γ623 bind the WW domains of Nedd4 two to three times better than the non-phosphorylated analogues, due to higher association rate constants. Using a number of different approaches it was demonstrated that the protein kinase acting on βThr-613 and γThr-623 is the extracellular regulated kinase (ERK). It is suggested that an ERK-mediated phosphorylation of βThr-613 and γThr-623 down-regulates the channel by facilitating its interaction with Nedd4.