Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

Details

Serval ID
serval:BIB_A490D71A413D
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.
Journal
Journal of Biological Chemistry
Author(s)
Ruffieux-Daidié D., Staub O.
ISSN
1083-351X (Electronic)
ISSN-L
0021-9258
Publication state
Published
Issued date
2011
Volume
286
Number
4
Pages
2416-2424
Language
english
Notes
Publication types: Journal Article ; Research Support, Non-U.S. Gov't
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.
Keywords
Amino Acid Motifs, Chymotrypsin/pharmacology, Endopeptidases/genetics, Endopeptidases/metabolism, Endosomal Sorting Complexes Required for Transport/genetics, Endosomal Sorting Complexes Required for Transport/metabolism, Epithelial Sodium Channel/genetics, Epithelial Sodium Channel/metabolism, Furin/genetics, Furin/metabolism, HEK293 Cells, Humans, Mutation, Signal Transduction/drug effects, Signal Transduction/physiology, Trypsin/pharmacology, Ubiquitin-Protein Ligases/genetics, Ubiquitin-Protein Ligases/metabolism, Ubiquitination/drug effects, Ubiquitination/physiology
Pubmed
Web of science
Open Access
Yes
Create date
20/03/2011 17:24
Last modification date
20/10/2020 10:12
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