EGF receptor-independent action of TGF-alpha protects Naked2 from AO7-mediated ubiquitylation and proteasomal degradation.

Naked family members (Drosophila Naked Cuticle and mammalian Naked1 and Naked2) have been identified as inducible antagonists of canonical Wnt signaling. We recently reported that Naked2, but not Naked1, interacts with the cytoplasmic tail of TGF-alpha, thereby coating TGF-alpha-containing exocytic vesicles and directing these vesicles to the basolateral corner of polarized epithelial cells. Here, we show that Naked2 is a short-lived protein with a half-life of 60 min caused by its rapid ubiquitin-mediated proteasomal degradation. Overexpression of TGF-alpha stabilizes Naked2 protein in an EGF receptor (EGFR)-independent manner; a physical interaction between the cytoplasmic tail of TGF-alpha and Naked2 is necessary and sufficient for this protection. We have identified a RING finger protein, AO7/RNF25, as a ubiquitin ligase for Naked2, and we have shown that overexpression of TGF-alpha reduces binding of AO7 to Naked2. These results identify an EGFR-independent action of TGF-alpha, in which it protects Naked2 from proteasomal degradation, thus ensuring its delivery to the basolateral surface of polarized epithelial cells.

The kinase Grk2 regulates Nedd4/Nedd4-2-dependent control of epithelial Na+ channels.

Epithelial Na(+) channels mediate the transport of Na across epithelia in the kidney, gut, and lungs and are required for blood pressure regulation. They are inhibited by ubiquitin protein ligases, such as Nedd4 and Nedd4-2, with loss of this inhibition leading to hypertension. Here, we report that these channels are maintained in the active state by the G protein-coupled receptor kinase, Grk2, which has been previously implicated in the development of essential hypertension. We also show that Grk2 phosphorylates the C terminus of the channel beta subunit and renders the channels insensitive to inhibition by Nedd4-2. This mechanism has not been previously reported to regulate epithelial Na(+) channels and provides a potential explanation for the observed association of Grk2 overactivity with hypertension. Here, we report a G protein-coupled receptor kinase regulating a membrane protein other than a receptor and provide a paradigm for understanding how the interaction between membrane proteins and ubiquitin protein ligases is controlled.

Regulation of the epithelial sodium channel by N4WBP5A, a novel Nedd4/Nedd4-2-interacting protein.

The amiloride-sensitive epithelial sodium channel (ENaC) plays a critical role in fluid and electrolyte homeostasis and consists of alpha, beta, and gamma subunits. The carboxyl terminus of each ENaC subunit contains a PPXY motif 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 of either the beta or gamma subunits, has been shown to result in increased ENaC activity and arterial hypertension. Here we present evidence that N4WBP5A, a novel Nedd4/Nedd4-2-binding protein, is a potential regulator of ENaC. In Xenopus laevis oocytes N4WBP5A increases surface expression of ENaC by reducing the rate of ENaC retrieval. We further demonstrate that N4WBP5A prevents sodium feedback inhibition of ENaC possibly by interfering with the xNedd4-2-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, we propose that N4WBP5A functions 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 is a likely candidate to modulate ENaC function in vivo.