The Role of Nedd8 in the Regulation of the Human Epithelial Sodium Channel ENaC

Abstract

The amiloride-sensitive epithelial sodium channel ENaC is a facilitator of sodium and water homeostasis expressed in the kidney, colon, lung and other epithelial cells. ENaC is a heteromeric, membrane-spanning complex formed from a combination of α, β and γENaC subunits. The number of functional sodium channels at the apical epithelial cell surface is diversely regulated, for example by aldosterone and vasopressin, and by post-translational modifications such as glycosylation, phosphorylation and ubiquitination. Ubiquitination is a conserved and widespread process, performed by a cascade of Ubiquitin activating, conjugating and ligase enzymes to covalently bind the small protein ubiquitin as a monomer or in polyubiquitin chains to a substrate protein. Ubiquitin ligase enzymes are a diverse group providing specificity to substrate ubiquitination. The Nedd4 family of ubiquitin ligases facilitates ubiquitination of α- and γENaC, resulting in a decreased number of ENaC channels at the cell surface. PY-motifs (PPPXY, where P = proline, Y = tyrosine, X = any amino acid) of ENaC C-terminal regions bind to Nedd4 WW domains, and are necessary for ENaC ubiquitination. Loss-of-function mutations in the PY-motif result in decreased ubiquitination, an increase in the number of functional epithelial sodium channels at the apical cell membrane and the inherited hypertension of Liddle's syndrome. A study of Type 2 diabetes in the Goto-Kakizaki rat model showed that as the disease progressed, expression of αENaC mRNA decreased whereas expression of mRNA for Nedd8 increased. Nedd8 is a ubiquitin-like protein known to bind covalently (in a process known as neddylation) to the cullin components of SCF (Skp1-Cullin1-F-box) ubiquitin ligases, upregulating the ubiquitinating activity of these complexes. Functional investigations in Xenopus oocytes showed that Nedd8 downregulates ENaC in a dose-responsive manner. This study aimed therefore to explore the mechanism of regulation of human ENaC by Nedd8. Colocalisation studies were used to investigate the location of Nedd8, ENaC subunits and Nedd4 proteins in Cos7 cells. Nedd8, when expressed alone, was predominantly found in the nucleus, but Nedd8 localisation included both nuclear and perinuclear cytoplasmic regions when cotransfected with ENaC subunits, or with the ubiquitin ligases Nedd4, Nedd4-2 and WWP2. Binding studies, however, showed that Nedd8 did not conjugate to either ENaC subunits or Nedd4 proteins. Mutation of the ubiquitination sites of αENaC altered the pattern of Nedd8 and αENaC colocalisation. The role of the Nedd8 pathway in regulating ubiquitination of αENaC was investigated by disrupting neddylation using the Nedd8 conjugating enzyme Dominant-negative mutant Ubc12(C111S). When increased amounts of either mutant or wild type Ubc12 were transfected, ubiquitination of αENaC was observed to decrease. In contrast, overexpressed Nedd8 upregulated ubiquitination of αENaC. A role for cycles of neddylation and deneddylation in modulating ENaC ubiquitination is supported by these findings. Potential mediators for the Nedd8-regulated ubiquitination of ENaC include SCF ligase components Skp1A(2) and Cull, both of which were shown to colocalise with ENaC subunits in the cytoplasm. In addition, Skp1A(2) was found to bind to αENaC. Taken together, these results support a hypothesis that αENaC is ubiquitinated at least in part by an SCF ligase activity that is upregulated by Nedd8, and introduce Nedd8 as a new regulator of the epithelial sodium channel.