Journal
JOURNAL OF BIOLOGICAL CHEMISTRY
Volume 279, Issue 40, Pages 41985-41990Publisher
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DOI: 10.1074/jbc.M403923200
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Funding
- NIDDK NIH HHS [DK 54354, DK 067143, DK 047874] Funding Source: Medline
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Using the yeast two-hybrid system, we identified a number of proteins that interacted with the carboxyl termini of murine epithelial sodium channel ( ENaC) subunits. Initial screens indicated an interaction between the carboxyl terminus of beta-ENaC and IkappaB kinase-beta ( IKKbeta), the kinase that phosphorylates Ikappabeta and results in nuclear targeting of NF-kappaB. A true two-hybrid reaction employing full-length IKKbeta and the carboxyl termini of all three subunits confirmed a strong interaction with beta-ENaC, a weak interaction with gamma-ENaC, and no interaction with alpha-ENaC. Co-immunoprecipitation studies for IKKbeta were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with beta-ENaC, but not gamma-ENaC, resulted in co-immunoprecipitation of IKKbeta. To examine the direct effects of IKKbeta on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for alphabetagamma-ENaC with or without cRNA for IKKbeta. Co-injection of IKKbeta significantly increased the amiloride-sensitive current above controls. Using cell surface ENaC labeling, we determined that an increase of ENaC in the plasma membrane accounted for the increase in current. The injection of kinase-dead IKKbeta (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCD(c14) cells with aldosterone increased whole cell amounts of IKKbeta. Because this result suggested that aldosterone might activate NF-kappaB, mpkCCD(c14) cells were transiently transfected with a luciferase reporter gene responsive to NF-kappaB activation. Both aldosterone and tumor necrosis factor-alpha (TNFalpha) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKKbeta interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKKbeta is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-kappaB transcription pathway and steroid regulatory pathways in epithelial cells.
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