Molecular determinants of the interaction between E3 ubiquitin ligase Nedd4‐2 and ENaC (893.5)

Phenotypic characterization of Nedd4‐2 knockout mice demonstrates the critical importance of this E3 ubiquitin ligase to activity of the epithelial sodium channel (ENaC) in several tissues including lung and kidney. However, determinants of the interaction between Nedd4‐2 and ENaC are not well understood and have implications for the treatment of several diseases including cystic fibrosis and hypertension. We characterized several novel determinants of this interaction using cell‐based biochemical assays to analyze exogenously or endogenously expressed Nedd4‐2 and ENaC. Nedd4‐2 preferentially binds to proteolytically cleaved ENaC in transfected HEK293T cells (10.9:1 +/‐ 0.009, p < 0.05). The preference for Nedd4‐2 to interact with cleaved channels is independent of Nedd4‐2 species and Nedd4‐2 ligase activity. Additionally, we demonstrate that neither an ENaC channel inhibitor, amiloride, nor ENaC channel mutants with increased or decreased activity alter the preference of Nedd4‐2 for cleaved channels. However, the calcium‐binding C2 domain of Nedd4‐2 decreases the binding of Nedd4‐2 to cleaved and uncleaved ENaC channels by approximately 5‐fold (p < 0.01). Surprisingly, active serum‐and‐glucocorticoid‐induced protein kinase 1 (SGK1) increased interaction between Nedd4‐2 and ENaC. Ongoing studies of the subcellular localization of Nedd4‐2 and cleaved or uncleaved ENaC channels is critical to understanding the determinants of this interaction. Proteolytic cleavage, but not activity, of ENaC; regulatory domains on Nedd4‐2; and unexpectedly, SGK1 kinase activity enhance Nedd4‐2 and ENaC interaction and can inform our understanding of how to target these pathways for treating lung and kidney disease. Grant Funding Source : NIH