14-3-3 isoforms are induced by aldosterone and participate in its regulation of epithelial sodium channels.
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.
Liang
Xiubin
X
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, 3500 Terrace Street, Pittsburgh, PA 15261, USA.
Peters
Kathryn W
KW
Butterworth
Michael B
MB
Frizzell
Raymond A
RA
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
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