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J. Biol. Chem., Vol. 279, Issue 40, 41985-41990, October 1, 2004

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IB Kinase- (IKK) Modulation of Epithelial Sodium Channel Activity^*

Jonathan Lebowitz, Robert S. Edinger, Bing An, Clint J. Perry, Sergio Onate¶, Thomas R. Kleyman, and John P. Johnson

From the Renal-Electrolyte Division, Department of Medicine, and the ^¶Center for Research in Reproductive Physiology, Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

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 -ENaC and IB kinase- (IKK), the kinase that phosphorylates I and results in nuclear targeting of NF-B. A true two-hybrid reaction employing full-length IKK and the carboxyl termini of all three subunits confirmed a strong interaction with -ENaC, a weak interaction with -ENaC, and no interaction with -ENaC. Co-immunoprecipitation studies for IKK were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with -ENaC, but not -ENaC, resulted in co-immunoprecipitation of IKK. To examine the direct effects of IKK on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for -ENaC with or without cRNA for IKK. Co-injection of IKK 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 IKK (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCD_c14 cells with aldosterone increased whole cell amounts of IKK. Because this result suggested that aldosterone might activate NF-B, mpkCCD_c14 cells were transiently transfected with a luciferase reporter gene responsive to NF-B activation. Both aldosterone and tumor necrosis factor- (TNF) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKK interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKK is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-B transcription pathway and steroid regulatory pathways in epithelial cells.

Received for publication, April 8, 2004 , and in revised form, July 14, 2004.

^* This work was supported by National Institutes of Health Grants DK 067143 (to J. L.), DK 047874 (to J. P. J.), and DK 54354 (to T. R. K.). 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.

To whom correspondence should be addressed: Renal-Electrolyte Division, A915 Scaife Hall, 3550 Terrace St., Pittsburgh, PA 15261. Tel.: 412-647-7157; Fax: 412-647-6222; E-mail: Lebowitzj{at}msx.deptmed.pitt.edu.

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