title: Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.
abstract: The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.
authors.affiliation: Department of Pharmacology & Toxicology, University of Lausanne, 1005 Lausanne, Switzerland.
authors.forename: Dorothée
authors.initials: D
authors.lastname: Ruffieux-Daidié
authors.forename: Olivier
authors.initials: O
authors.lastname: Staub
mesh_terms: D020816:Amino Acid Motifs; D002918:Chymotrypsin / Q000494:pharmacology; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D056827:Endosomal Sorting Complexes Required for Transport / Q000235:genetics / Q000378:metabolism; D053503:Epithelial Sodium Channels / Q000235:genetics / Q000378:metabolism*; D045683:Furin / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D000075702:Nedd4 Ubiquitin Protein Ligases; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014357:Trypsin / Q000494:pharmacology; D043222:Ubiquitin Thiolesterase; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism; D054875:Ubiquitination / Q000187:drug effects / Q000502:physiology*
chemical_list: D056827:Endosomal Sorting Complexes Required for Transport; D053503:Epithelial Sodium Channels; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; D044767:Ubiquitin-Protein Ligases; D010450:Endopeptidases; C108665:USP2 protein, human; D043222:Ubiquitin Thiolesterase; D002918:Chymotrypsin; D014357:Trypsin; C468731:FURIN protein, human; D045683:Furin