An Obligatory Heterodimer of 14-3-3β and 14-3-3ϵ Is Required for Aldosterone Regulation of the Epithelial Sodium Channel

Xiubin Liang,Michael B Butterworth,Kathryn W Peters,William H Walker,Raymond A Frizzell

doi:10.1074/jbc.m803687200

Xiubin Liang, Michael B Butterworth + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m803687200

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Abstract

Increased distal nephron sodium absorption in response to aldosterone involves Nedd4-2 phosphorylation, which blocks its ability to ubiquitylate ENaC and increases apical membrane channel density by reducing its endocytosis. Our prior work (Liang, X., Peters, K. W., Butterworth, M. B., and Frizzell, R. A. (2006) J. Biol. Chem. 281, 16323-16332) showed that aldosterone selectively increased 14-3-3 protein isoform expression and that the association of 14-3-3beta with phospho-Nedd4-2 was required for sodium transport stimulation. The knockdown of 14-3-3beta alone nearly eliminated the response to aldosterone, despite the expression of other 14-3-3 isoforms in cortical collecting duct (CCD) cells. To further examine this marked effect of 14-3-3beta knockdown, we evaluated the hypothesis that phospho-Nedd4-2 binding prefers a heterodimer composed of two different 14-3-3 isoforms. We tested this concept in polarized CCD cells using RNA interference and assays of sodium transport and of the interaction of Nedd4-2 with 14-3-3epsilon, a second aldosterone-induced isoform. As observed previously for 14-3-3beta knockdown, small interfering RNA-induced reduction of 14-3-3epsilon markedly attenuated aldosterone-stimulated ENaC expression and sodium transport and increased the interaction of Nedd4-2 with ENaC toward prealdosterone levels. After aldosterone induction, 14-3-3beta and 14-3-3epsilon were quantitatively co-immunoprecipitated from CCD cell lysates, and the association of both isoforms with Nedd4-2 increased. Finally, the knockdown of either 14-3-3beta or 14-3-3epsilon reduced the association of Nedd4-2 with the other isoform. We conclude that the two aldosterone-induced 14-3-3 isoforms, beta and epsilon, interact with phospho-Nedd4-2 as an obligatory heterodimer, blocking its interaction with ENaC and thereby increasing apical ENaC density and sodium transport.

Highlights

Across renal cortical collecting duct (CCD)2 epithelia, where the epithelial sodium channel, ENaC, provides the limiting entry step in this process [1]
We detected the expression of five of the seven mammalian 14-3-3 isoforms in mouse cortical collecting duct epithelia by reverse transcription-PCR and immunoblot, and we showed that the expression of the 14-3-3␤ and ⑀ isoforms were elevated 3- and 12-fold, respectively, by physiological levels of aldosterone, whereas the expression of other expressed isoforms, 14-3-3␥, -␪, and -␴, were unaffected by the steroid
The physiological significance of the 14-3-3␤-phosphoNedd4-2 interaction was revealed by 14-3-3␤ knockdown, which increased ENaC-Nedd4-2 interactions to prealdosterone levels, blunted the steroid-induced increase in ␣-ENaC expression, and virtually eliminated the aldosterone-induced stimulation of sodium absorption across mouse cortical collecting duct (mCCD) epithelia

Summary

EXPERIMENTAL PROCEDURES

Antibodies—Antibodies specific for 14-3-3␤ (A-15), 14-3-3⑀ (T-16) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Thereafter, mCCD epithelia were either maintained without additives or treated with aldosterone (10 nM; Sigma). Immunoblot Analyses—Equal amounts of protein from either aldosterone-treated or nontreated, polarized mCCD cells or the immunoprecipitates described above were resolved by 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The filters were washed 24 h after transfection and maintained under control conditions or treated with aldosterone (10 nM) for 48 h. In addition to the siRNA control, the results were compared with data obtained from untreated CCD cells. A, preconfluent mCCD epithelia were transfected with siRNAs targeting 14-3-3⑀ expression or with control siRNAs, as described under “Experimental Procedures.”. Expression of the indicated proteins was determined by immunoblot using mCCD epithelia that had been maintained under control (base-line) conditions or stimulated with 10 nM aldosterone for 48 h before cell lysis.

RESULTS

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DISCUSSION