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J. Biol. Chem., Vol. 284, Issue 1, 150-157, January 2, 2009

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Functional Regulation of the Epithelial Na⁺ Channel by IB Kinase-β Occurs via Phosphorylation of the Ubiquitin Ligase Nedd4-2^*

Robert S. Edinger, Jonathan Lebowitz, Hui Li, Rodrigo Alzamora, Huamin Wang, John P. Johnson, and Kenneth R. Hallows¹

From the Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261

We have previously shown that IB kinase-β (IKKβ) interacts with the epithelial Na⁺ channel (ENaC) β-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKβ-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCD_c14) cells, as RNA interference-mediated knockdown of endogenous IKKβ in these cells by 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKβ binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKβ in vitro. Because IKKβ 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. IKKβ-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. ³²P 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 IKKβ phosphorylation target. ENaC stimulation by IKKβ 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 IKKβ are required for IKKβ regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKβ-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.

Received for publication, September 23, 2008 , and in revised form, October 28, 2008.

^* This work was supported, in whole or in part, by National Institutes of Health Grants K08 DK067143 (to J. L.), R01 DK057718 and DK047874 (to J. P. J.), and R01 DK075048 (to K. R. H.). This work was also supported by American Heart Association Postdoctoral Fellowship Award AHA 0825540D (to R. A.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Renal-Electrolyte Div., S976 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-648-9580; Fax: 412-383-8956; E-mail: hallows{at}pitt.edu.

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