title: AS160 modulates aldosterone-stimulated epithelial sodium channel forward trafficking.
abstract: Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (I(sc)). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and I(sc) 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and I(sc) under basal conditions to approximately 80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, beta and epsilon. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3beta and epsilon and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.
authors.affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.
authors.forename: Xiubin
authors.initials: X
authors.lastname: Liang
authors.forename: Michael B
authors.initials: MB
authors.lastname: Butterworth
authors.forename: Kathryn W
authors.initials: KW
authors.lastname: Peters
authors.forename: Raymond A
authors.initials: RA
authors.lastname: Frizzell
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism; D000450:Aldosterone / Q000494:pharmacology*; D000818:Animals; D016764:Cell Polarity / Q000187:drug effects; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D055785:Gene Knockdown Techniques; D015640:Ion Channel Gating / Q000187:drug effects*; D007685:Kidney Tubules, Collecting / Q000166:cytology; D051379:Mice; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D012964:Sodium / Q000378:metabolism
chemical_list: D048948:14-3-3 Proteins; D053503:Epithelial Sodium Channels; D020690:GTPase-Activating Proteins; C505647:Tbc1d4 protein, mouse; D000450:Aldosterone; D012964:Sodium
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