8.A.30 The Nedd4-Family Interacting Protein-2 (Nedd4) Family

Epithelial Na⁺ absorption is regulated by Nedd4-2, an E3 ubiquitin-protein ligase that reduces expression of the epithelial Na⁺ channel ENaC at the cell surface. Nedd4-2 catalyzes ubiquitination of α-, β-, and γEnaC. Ubiquitination is disrupted by Liddle syndrome-associated mutations in ENaC or mutation of the catalytic HECT domain in Nedd4-2. The interaction between Nedd4-2 and ENaC is localized to the cell surface. First, Nedd4-2 bound to a population of ENaC at the cell surface. Second, Nedd4-2 catalyzed ubiquitination of cell surface ENaC. Third, Nedd4-2 selectively reduced ENaC expression at the cell surface but did not alter the quantity of immature ENaC in the biosynthetic pathway. Finally, Nedd4-2 induced degradation of the cell surface pool of ENaC (Zhou et al., 2007). Thus, Nedd4-2 binds to and ubiquitinates ENaC at the cell surface, which targets surface ENaC for degradation, and thus, reduces epithelial Na⁺ transport (Zhou, et al., 2007).

The carboxyl terminus of each ENaC subunit contains a PPXY motif in the C-terminus of either the α, β or γ-subunit of ENac, that is believed to be important for interaction with the WW domains of the ubiquitin-protein ligases, Nedd4 and Nedd4-2. Disruption of this interaction, as in Liddle's syndrome where mutations delete or alter the PPXY motif in the C-terminus of either the α, β or γ-subunit of ENac, has been shown to result in increased ENaC activity and arterial hypertension. N4WBP5A, a novel Nedd4/Nedd4-2-binding protein, is a potential regulator of ENaC. In Xenopus laevis oocytes N4WBP5A increased surface expression of ENaC by reducing the rate of ENaC retrieval. N4WBP5A prevents sodium feedback inhibition of ENaC, possibly by interfering with the Nedd4-2-mediated regulation of ENaC. As N4WBP5A binds Nedd4/Nedd4-2 via PPXY motif/WW domain interactions and appears to be associated with specific intracellular vesicles, N4WBP5A may function by regulating Nedd4/Nedd4-2 availability and trafficking. Because N4WBP5A is highly expressed in native renal collecting duct and other tissues that express ENaC, it may modulate ENaC function in vivo (Konstas et al., 2002).

Voltage-gated KCNQ2/3 and KCNQ3/5 K⁺ channels regulate neuronal excitability, and KCNQ2/3 and 3/5 channels are regulated by the ubiquitin ligase, Nedd4-2. The serum and glucocorticoid-induced kinase-1 (SGK-1) plays an important role in the regulation of epithelial ion transport. SGK-1 phosphorylation of Nedd4-2 decreases the ability of Nedd4-2 to ubiquitinate the epithelial sodium channel (ENaC) which increases the abundance of channel protein in the cell membrane. KCNQ channels expressed in Xenopus oocytes are up regulated by SGK-1 (Schuetz et al., 2008) whereas the kinase dead SGK-1 mutant had no effect. The cell surface levels of KCNQ2-HA/3 were also increased by SGK-1. Deletion of the KCNQ3 C terminal in the presence of SGK-1 did not affect the current amplitude of KCNQ2/3/5-mediated currents. Co-expression of Nedd4-2 and SGK-1 with either KCNQ2/3 or 3/5 did not significantly alter the current amplitudes. Only the Nedd4-2 mutant [S448A] exhibited a significant down-regulation of the KCNQ2/3/5 K(+) current amplitudes. Thus, SGK-1 regulates the activity of the ubiquitin ligase Nedd4-2 which regulates KCNQ channels (Schuetz et al., 2008).

Ubiquitination of proteins by the Nedd4 family of ubiquitin ligases is a significant mechanism in protein trafficking and degradation and provides for tight spatiotemporal regulation. Ubiquitination is gaining increasing recognition as a central mechanism underpinning the regulation of neuronal development and homeostasis in the brain (Donovan and Poronnik 2013). Ubiquitination signals not only orchestrate protein degradation events but also play roles in protein trafficking. The description of several novel Nedd4/4-2 targets in neurons is changing the way we conceptualize how neurons maintain normal function and how this is altered in disease.

The metal homeostasis protein (MHP) of Saccharomyces cerevisiae is probably a ubiquitin-protein ligase (by sequence similarity). Its defect can be complemented by the NRAMP1 heavy metal efflux pump (TC# 2.A.55.2.3) (Techau et al. 2007). Bsd2 may indirectly regulate protein trafficking between the endoplasmic reticulum, the plasma membrane and the endosomes. It may also mediate sensitivity to oxygen in intact yeast cells. Bsd2 has 321 amino acyl residues with three putative transmembrane α-helical spanners.