AMP-activated Kinase Inhibits the Epithelial Na+ Channel through Functional Regulation of the Ubiquitin Ligase Nedd4-2

Vivek Bhalla,Nicholas M Oyster,Adam C Fitch,Marjolein A Wijngaarden,Dietbert Neumann,Uwe Schlattner,David Pearce,Kenneth R Hallows

doi:10.1074/jbc.m606045200

Abstract

We recently found that the metabolic sensor AMP-activated kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) through decreased plasma membrane ENaC expression, an effect requiring the presence of a binding motif in the cytoplasmic tail of the beta-ENaC subunit for the ubiquitin ligase Nedd4-2. To further examine the role of Nedd4-2 in the regulation of ENaC by AMPK, we studied the effects of AMPK activation on ENaC currents in Xenopus oocytes co-expressing ENaC and wild-type (WT) or mutant forms of Nedd4-2. ENaC inhibition by AMPK was preserved in oocytes expressing WT Nedd4-2 but blocked in oocytes expressing either a dominant-negative (DN) or constitutively active (CA) Nedd4-2 mutant, suggesting that AMPK-dependent modulation of Nedd4-2 function is involved. Similar experiments utilizing WT or mutant forms of the serum- and glucocorticoid-regulated kinase (SGK1), modulators of protein kinase A (PKA), or extracellular-regulated kinase (ERK) did not affect ENaC inhibition by AMPK, suggesting that these pathways known to modulate the Nedd4-2-ENaC interaction are not responsible. AMPK-dependent phosphorylation of Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for modulation of Nedd4-2 and thus cellular ENaC activity. Moreover, cellular AMPK activation significantly enhanced the interaction of the beta-ENaC subunit with Nedd4-2, as measured by co-immunoprecipitation assays in HEK-293 cells. In summary, these results suggest a novel mechanism for ENaC regulation in which AMPK promotes ENaC-Nedd4-2 interaction, thereby inhibiting ENaC by increasing Nedd4-2-dependent ENaC retrieval from the plasma membrane. AMPK-dependent ENaC inhibition may limit cellular Na+ loading under conditions of metabolic stress when AMPK becomes activated.

Highlights

epithelial Na؉ channel (ENaC) is regulated by the actions of several hormones, including aldosterone, vasopressin, and insulin, as well as various non-hormonal mechanisms
These findings suggest that AMPK may regulate ENaC via Nedd4-2 activation, enhancing Nedd4-2-dependent retrieval of ENaC from the plasma membrane
We have previously shown that injection of the AMPK activator ZMP into oocytes inhibits mouse ENaC (mENaC) currents in an AMPK-dependent fashion [37]

Summary

Introduction

ENaC is regulated by the actions of several hormones, including aldosterone, vasopressin, and insulin, as well as various non-hormonal mechanisms. The interaction between the PY motifs on the C termini of ␤and ␥-ENaC and the WW domains of Nedd, an E3 ubiquitin-protein ligase, has emerged as a critical locus for ENaC activity regulation by these signaling pathways [13, 15,16,17]. SGK1 phosphorylates Nedd at two key residues, Ser-338 and Ser-444 in Xenopus Nedd (xNedd4-2) [18] This phosphorylation disrupts the ENaC-Nedd interaction, apparently by enhancing the binding affinity of Nedd to 14-3-3 proteins, which sequester Nedd from ENaC, as we and others [19, 20] have recently shown. PKA phosphorylates Nedd at the same residues to inhibit ENaC internalization and may promote increased delivery and insertion of ENaC into the plasma membrane [14]. AMPK may modulate several important kinase-dependent cellular signaling pathways, including the MAPK/ERK and PKC signaling pathways known to regulate ENaC (34 –36)

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