title: An obligatory heterodimer of 14-3-3beta and 14-3-3epsilon is required for aldosterone regulation of the epithelial sodium channel.
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.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Xiubin
authors.initials: X
authors.lastname: Liang
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Butterworth
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Kathryn W
authors.initials: KW
authors.lastname: Peters
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: William H
authors.initials: WH
authors.lastname: Walker
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261.
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity / Q000187:drug effects / Q000235:genetics; D019281:Dimerization; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D007685:Kidney Tubules, Collecting / Q000378:metabolism*; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D012964:Sodium / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
chemical_list: D048948:14-3-3 Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D000450:Aldosterone; D012964:Sodium; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases