Functional Regulation of the Epithelial Na+ Channel by I kappa B Kinase-beta Occurs via Phosphorylation of the Ubiquitin Ligase Nedd4-2

Edinger, Robert S.; Lebowitz, Jonathan; Li, Hui; Alzamora, Rodrigo; Wang, Huamin; Johnson, John P.; Hallows, Kenneth R.


We have previously shown that I kappa B kinase-beta(IKK beta) interacts with the epithelial Na+ channel (ENaC) beta-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKK beta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCD(c14)) cells, as RNA interference-mediated knockdown of endogenous IKK beta in these cells by similar to 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKK beta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKK beta in vitro. Because IKK beta stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKK beta-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. P-32 labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKK beta phosphorylation target. ENaC stimulation by IKK beta was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKK beta are required for IKK beta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs throughIKK beta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.

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Volumen: 284
Número: 1
Editorial: Elsevier
Fecha de publicación: 2009
Página de inicio: 150
Página final: 157
Idioma: English