Olivier Staub

Ubiquitylation—that is, the covalent attachment of ubiquitin polypeptides to target proteins—involves the sequential action of the E1 ubiquitin-activating enzyme, E2 ubiquitin-conjugating enzymes, and E3 ubiquitin-protein ligases. Similarly, the conjugation of ubiquitin-like proteins is thought to occur through parallel, but nonidentical, cascades. This concept of strict separation of these modification pathways is now being challenged by the evidence, showing that there are enzymes that play a role both in ubiquitylation and isgylation.

The epithelial Na⁺ channel (ENaC) is a heteromeric protein complex playing a fundamental role in Na⁺ homeostasis and blood pressure regulation. Specific mutations inactivating PY motifs in ENaC C termini cause Liddle's syndrome, an inherited form of hypertension. Previously we showed that these PY motifs serve as binding sites for the E3 enzyme Nedd4-2, implying ubiquitination as a regulatory mechanism of ENaC. Ubiquitination involves the sequential action of E1, E2, and E3 enzymes. Here we identify the E2 enzyme UBE2E3, which acts in concert with Nedd4-2, and show by coimmunoprecipitation that UBE2E3 and Nedd4-2 interact together. In Xenopus laevis oocytes, UBE2E3 reduces ENaC activity marginally, consistent with Nedd4-2 being the rate-limiting factor in this process, whereas a catalytically inactive mutant of UBE2E3 (UBE2E3-CS) causes elevated ENaC activity by increasing cell surface expression. No additive effect is observed when UBE2E3-CS is coexpressed with an inactive Nedd4-2 mutant, and the stimulatory role of UBE2E3-CS depends on the integrity of the PY motifs (Nedd4-2 binding sites) and the ubiquitination sites on ENaC. In renal mpkCCD_cl4 cells, displaying ENaC-dependent transepithelial Na⁺ transport, Nedd4-2 and UBE2E3 can be coimmunoprecipitated and overexpression of UBE2E3 affects Na⁺ transport, corroborating the concept of a concerted action of UBE2E3 and Nedd4-2 in ENaC regulation.

Ubiquitylation has emerged as an important mechanism for controlling surface expression of membrane proteins. This post-translational modification involves the sequential action of several enzymes including a ubiquitin-activating enzyme E1, a ubiquitin-conjugating enzyme E2 and a ubiquitin-protein ligase E3. E3s are responsible for substrate recognition. Here we describe the role of the Nedd4/Nedd4-like family of ubiquitin-protein ligases in the regulation of proteins involved in epithelial transport. The Nedd4/Nedd4-like proteins are composed of a N-terminal C2 domain, several WW domains and a catalytic HECT domain. The epithelial Na⁺ channel ENaC is the best studied example of a Nedd4/Nedd4-like substrate. Its cell surface expression is regulated by the ubiquitin-protein ligase Nedd4–2 via direct PY motif/WW domain interaction. This regulatory mechanism is impaired in Liddle's disease, an inherited form of human hypertension, and is controlled by Sgk1, an aldosterone-inducible kinase which phosphorylates Nedd4–2. The regulation of ENaC by Nedd4–2 is a paradigm for the control of epithelial membrane proteins, as evidenced by the regulation of the ClC-5 chloride channel by the ubiquitin-protein ligase WWP2 or the tight junction protein Occludin by Itch.

The gap junction protein connexin43 is known to have a rapid turnover,involving degradation by both the proteasomal and lysosomal systems, but the structural features of connexin43 that govern these actions are not known. The connexin43 C-terminal sequence contains a proline-rich region corresponding to the consensus of a protein-protein interaction PY-motif (xPPxY), and an overlapping putative tyrosine-based sorting signal (Yxxϕ;ϕ=hydrophobic), known to play a role in the intracellular trafficking of many membrane proteins. As both motifs may control turnover of connexin43, we used a combination of metabolic radiolabelling, immuno-precipitation and functional assays to determine the possible role of these motifs in controlling degradation of human connexin43 expressed in SKHep1 cells. Mutation V289D in the tyrosine-based sorting motif increased the steady-state pool of connexin43 by approximately 3.5-fold, while mutation P283L in the PY-motif produced a comparatively modest augmentation (1.7-fold). No additive effect was observed when the overlapping tyrosine was mutated. In pulse-chase experiments, the Y286A substitution increased the half-life of connexin43 from 2 to 6 hours, indicating that the increased steady-state levels reflected reduced protein degradation. Moreover, expression at the junctional membrane,as well as gap junction-mediated intercellular communication (GJC), were nearly abolished by lysosomal inhibitors and Brefeldin A in cells expressing wild-type connexin43, but were unaffected in the tyrosine mutant. These results provide strong evidence that the tyrosine-based motif of human connexin43 is a prime determinant controlling connexin43 stability, and consequently GJC, by targeting connexin43 for degradation in the endocytic/lysosomal compartment.

The epithelial Na+ channel (ENaC) plays an essential role in the regulation of whole body Na+ balance and blood pressure. The cell surface expression of this channel, a complex of three subunits (α, β and γENaC), has been shown to be regulated by hormones such as aldosterone and vasopressin and by intracellular signaling, including ubiquitylation and/or phosphorylation. However, the molecular mechanisms involving phosphorylation in the regulation of ENaC are unclear. Here we show by expression studies in Xenopus laevis oocytes that the aldosterone‐induced Sgk1 kinase interacts with the ubiquitin protein ligase Nedd4‐2 in a PY motif‐dependent manner and phosphorylates Nedd4‐2 on Ser444 and, to a lesser extent, Ser338. Such phosphorylation reduces the interaction between Nedd4‐2 and ENaC, leading to elevated ENaC cell surface expression. These data show that phosphorylation of an enzyme involved in the ubiquitylation cascade (Nedd4‐2) controls cell surface density of ENaC and propose a paradigm for the control of ion channels. Moreover, they suggest a novel and complete signaling cascade for aldosterone‐dependent regulation of ENaC.

Liddle's syndrome is a form of inherited hypertension linked to mutations in the genes encoding the epithelial Na ⁺ channel (ENaC). These mutations alter or delete PY motifs involved in protein–protein interactions with a ubiquitin‐protein ligase, Nedd4. Here we show that Na ⁺ transporting cells, derived from mouse cortical collecting duct, express two Nedd4 proteins with different structural organization and characteristics of ENaC regulation: 1 ) the classical Nedd4 (herein referred to as Nedd4–1) containing one ammo‐terminal C2, three WW, and one HECT‐ubiquitin protein ligase domain and 2 ) a novel Nedd4 protein (Nedd4–2), homologous to Xenopus Nedd4 and comprising four WW, one HECT, yet lacking a C2 domain. Nedd4–2, but not Nedd4–1, inhibits ENaC activity when coexpressed in Xenopus oocytes and this property correlates with the ability to bind to ENaC, as only Nedd4–2 coimmunoprecipitates with ENaC. Furthermore, this interaction depends on the presence of at least one PY motif in the ENaC complex and on WW domains 3 and 4 in Nedd4–2. Thus, these results suggest that the novel suppressor protein Nedd4–2 is the regulator of ENaC and hence a potential susceptibility gene for arterial hypertension.—Kamynina, E., Debonneville, C., Bens, M., Vandewalle, A., Staub, O. A novel mouse Nedd4 protein suppresses the activity of the epithelial Na ⁺ channel. . FASEB J. 15, 204–214 (2001)

Liddle's syndrome is an inherited form of hypertension linked to mutations in the epithelial Na⁺ channel (ENaC). ENaC is composed of three subunits (α, β, γ), each containing a COOH-terminal PY motif (xPPxY). Mutations causing Liddle's syndrome alter or delete the PY motifs of β- or γ-ENaC. We recently demonstrated that the ubiquitin–protein ligase Nedd4 binds these PY motifs and that ENaC is regulated by ubiquitination. Here, we investigate, using the Xenopus oocyte system, whether Nedd4 affects ENaC function. Overexpression of wild-type Nedd4, together with ENaC, inhibited channel activity, whereas a catalytically inactive Nedd4 stimulated it, likely by acting as a competitive antagonist to endogenous Nedd4. These effects were dependant on the PY motifs, because no Nedd4-mediated changes in channel activity were observed in ENaC lacking them. The effect of Nedd4 on ENaC missing only one PY motif (of β-ENaC), as originally described in patients with Liddle's syndrome, was intermediate. Changes were due entirely to alterations in ENaC numbers at the plasma membrane, as determined by surface binding and immunofluorescence. Our results demonstrate that Nedd4 is a negative regulator of ENaC and suggest that the loss of Nedd4 binding sites in ENaC observed in Liddle's syndrome may explain the increase in channel number at the cell surface, increased Na⁺ reabsorption by the distal nephron, and hence the hypertension. J. Clin. Invest. 103:667–673 (1999)