Functional regulation of the epithelial Na+ channel by IkappaB kinase-beta occurs via phosphorylation of the ubiquitin ligase Nedd4-2.
We have previously shown that IkappaB kinase-beta (IKKbeta) 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 IKKbeta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKbeta in these cells by approximately 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKbeta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKbeta in vitro. Because IKKbeta 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. IKKbeta-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 IKKbeta phosphorylation target. ENaC stimulation by IKKbeta 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 IKKbeta are required for IKKbeta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKbeta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.
Edinger
Robert S
RS
Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Lebowitz
Jonathan
J
Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Li
Hui
H
Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Alzamora
Rodrigo
R
Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Wang
Huamin
H
Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Johnson
John P
JP
Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
Hallows
Kenneth R
KR
Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261; Renal-Electrolyte Division, Departments of Medicine and Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261. Electronic address: hallows@pitt.edu.
D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D016764:Cell Polarity / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000502:physiology*; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics / Q000378:metabolism*; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism; D007685:Kidney Tubules, Collecting / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D034622:RNA Interference; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*; D029867:Xenopus Proteins / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis; D000099184:Serum-Glucocorticoid Regulated Kinases
D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D051550:I-kappa B Kinase; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D044767:Ubiquitin-Protein Ligases; D029867:Xenopus Proteins; D000099184:Serum-Glucocorticoid Regulated Kinases; C507049:Scnn1b protein, mouse; C000618482:Nedd4 protein, Xenopus; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; C000618460:nedd4l protein, Xenopus; C496562:Ikbkb protein, mouse