title: The phosphorylation of endogenous Nedd4-2 In Na(+)-absorbing human airway epithelial cells.
abstract: Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalisation / degradation of epithelial Na(+) channel subunits (α-, β- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser(221), -Ser(327) and -Thr(246). This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na(+) absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na(+) transport. Effects on Nedd4-2 phosphorylation/abundance and the surface expression of ENaC were monitored by western analysis, whilst Na(+) absorption was quantified electrometrically. Acutely (20min) activating PKA in glucocorticoid-deprived (24h) cells increased the abundance of Ser(221)-phosphorylated, Ser(327)-phosphorylated and total Nedd4-2 without altering the abundance of Thr(246)-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, β- and γ-ENaC and had only a very small effect upon electrogenic Na(+) absorption. Activating PKA (20min) in glucocorticoid-treated (0.2µM dexamethasone, 24h) cells, on the other hand, increased the abundance of Ser(221)-, Ser(327)- and Thr(246)-phosphorylated and total Nedd4-2; increased the surface abundance of α-, β- and γ-ENaC and evoked a clear stimulation of Na(+) transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na(+) absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, UK; Biochemistry Department, Faculty of Medicine, University Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.
authors.forename: Noor A S
authors.initials: NA
authors.lastname: Ismail
authors.affiliation: Biomedical Sciences Research Centre, St Georges University of London, Cranmer Terrace, London, UK.
authors.forename: Deborah L
authors.initials: DL
authors.lastname: Baines
authors.affiliation: Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry & Nursing, University of Dundee, Dundee DD1 9SY, UK; Wolfson Research Institute, School of Medicine, Pharmacy and Health, Durham University Queen׳s Campus, Stockton on Tees, TS17 6BH, UK. Electronic address: Stuart.wilson@durham.ac.uk.
authors.forename: Stuart M
authors.initials: SM
authors.lastname: Wilson
mesh_terms: D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D002460:Cell Line; D000242:Cyclic AMP / Q000819:agonists; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology; D056827:Endosomal Sorting Complexes Required for Transport / Q000187:drug effects / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D053503:Epithelial Sodium Channels / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D000075702:Nedd4 Ubiquitin Protein Ligases; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020545:Respiratory Mucosa / Q000187:drug effects / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000187:drug effects / Q000378:metabolism*; D000099184:Serum-Glucocorticoid Regulated Kinases
chemical_list: D000893:Anti-Inflammatory Agents; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D003907:Dexamethasone; D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D017874:Immediate-Early Proteins; D000075702:Nedd4 Ubiquitin Protein Ligases; D017346:Protein Serine-Threonine Kinases; D012964:Sodium; D044767:Ubiquitin-Protein Ligases; D000099184:Serum-Glucocorticoid Regulated Kinases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human
keywords: Cellular signalling; Epithelial Na(+) channel; Protein kinase A; Pulmonary Na(+) absorption; Serum and glucocorticoid regulated kinase 1