title: 14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels.
abstract: Aldosterone increases sodium absorption across renal collecting duct cells primarily by increasing the apical membrane expression of ENaC, the sodium entry channel. Nedd4-2, a ubiquitin-protein isopeptide ligase, tags ENaC with ubiquitin for internalization and degradation, but when it is phosphorylated by the aldosterone-induced kinase, SGK1, Nedd4-2 is inhibited and apical ENaC density and sodium absorption increase. We evaluated the hypothesis that 14-3-3 proteins participate in the aldosterone-mediated regulation of ENaC by associating with phosphorylated Nedd4-2. Mouse cortical collecting duct (mCCD) epithelia cultured on filters expressed several 14-3-3 isoforms; this study focused on an isoform whose expression was induced 3-fold by aldosterone, 14-3-3beta. In polarized mCCD epithelia, aldosterone elicited significant, time-dependent increases in the expression of alpha-ENaC, SGK1, phospho-Nedd4-2, and 14-3-3beta without altering total Nedd4-2. Aldosterone decreased the interaction of alpha-ENaC with Nedd4-2, and with similar kinetics increased the association of 14-3-3beta with phospho-Nedd4-2. Short interfering RNA-induced knockdown of 14-3-3beta blunted the aldosterone-induced increase in alpha-ENaC expression, returned alpha-ENaC-Nedd4-2 binding toward prealdosterone levels, and blocked the aldosterone-stimulated increase in transepithelial sodium transport. Incubation of cell extracts with a selective phospho-Nedd4-2 antibody blocked the aldosterone-induced association of 14-3-3beta with Nedd4-2, implicating SGK1 phosphorylation at Ser-328 as the primary site of 14-3-3beta binding. Our studies show that aldosterone increases the expression of 14-3-3beta, which interacts with phospho-Nedd4-2 to block its interaction with ENaC, thus enhancing sodium absorption by increasing apical membrane ENaC density.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, Pittsburgh, PA 15261, USA.
authors.forename: Xiubin
authors.initials: X
authors.lastname: Liang
authors.forename: Kathryn W
authors.initials: KW
authors.lastname: Peters
authors.forename: Michael B
authors.initials: MB
authors.lastname: Butterworth
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
mesh_terms: D048948:14-3-3 Proteins / Q000737:chemistry*; D000450:Aldosterone / Q000378:metabolism; D000818:Animals; D001692:Biological Transport; D002478:Cells, Cultured; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D007700:Kinetics; D051379:Mice; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D011485:Protein Binding; D020033:Protein Isoforms; D015222:Sodium Channels / Q000737:chemistry* / Q000378:metabolism; D025801:Ubiquitin / Q000737:chemistry; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D020033:Protein Isoforms; D015222:Sodium Channels; D025801:Ubiquitin; D000450:Aldosterone; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases