cAMP and serum and glucocorticoid-inducible kinase (SGK) regulate the epithelial Na+ channel through convergent phosphorylation of Nedd4-2

Department of Internal Medicine, University of Iowa, Iowa City, Iowa, United States
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2004; 279(44):45753-8. DOI: 10.1074/jbc.M407858200
Source: PubMed


The epithelial Na(+) channel (ENaC) functions as a pathway for epithelial Na(+) transport, contributing to Na(+) homeostasis and blood pressure control. Vasopressin increases ENaC expression at the cell surface through a pathway that includes cAMP and cAMP-dependent protein kinase (PKA), but the mechanisms that link PKA to ENaC are unknown. Here we found that cAMP regulates Na(+) transport in part by inhibiting the function of Nedd4-2, an E3 ubiquitin-protein ligase that targets ENaC for degradation. Consistent with this model, we found that cAMP inhibited Nedd4-2 by decreasing its binding to ENaC. Moreover, decreased Nedd4-2 expression (RNA interference) or overexpression of a dominant negative Nedd4-2 construct disrupted ENaC regulation by cAMP. Nedd4-2 was a substrate for phosphorylation by PKA in vitro and in cells; three Nedd4-2 residues were phosphorylated by PKA and were required for cAMP to inhibit Nedd4-2 (relative functional importance Ser-327 > Ser-221 > Thr-246). Previous work found that these residues are also phosphorylated by serum and glucocorticoid-inducible kinase (SGK), a downstream mediator by which aldosterone regulates epithelial Na(+) transport. Consistent with a functional interaction between these pathways, overexpression of SGK blunted ENaC stimulation by cAMP, whereas inhibition of SGK increased stimulation. Conversely, cAMP agonists decreased ENaC stimulation by SGK. The data suggest that cAMP regulates ENaC in part by phosphorylation and inhibition of Nedd4-2. Moreover, Nedd4-2 is a central convergence point for kinase regulation of Na(+) transport.

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    • "Moreover, whilst the direct effects of dexamethasone upon Na+ transport were abolished by GSK650394, cAMP agonists could still activate the PKA – Nedd4-2 – ENaC pathway in GSK650394-treated cells. Whilst these data accord with earlier observations which suggest that cAMP agonists control Na+-transport via a mechanism that is independent of SGK1 (Inglis et al., 2009; Mansley and Wilson, 2010; Snyder et al., 2004a), they also show that SGK1 cannot mediate the permissive effects of dexamethasone which we now report. "
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    ABSTRACT: Neural precursor cell expressed, developmentally down-regulated protein 4-2 (Nedd4-2) mediates the internalization / degradation of epithelial Na+ channel subunits (α-, β- and γ-ENaC). Serum / glucocorticoid inducible kinase 1 (SGK1) and protein kinase A (PKA) both appear to inhibit this process by phosphorylating Nedd4-2-Ser221, -Ser327 and -Thr246. This Nedd4-2 inactivation process is thought to be central to the hormonal control of Na+ absorption. The present study of H441 human airway epithelial cells therefore explores the effects of SGK1 and / or PKA upon the phosphorylation / abundance of endogenous Nedd4-2; the surface expression of ENaC subunits, and electrogenic Na+ transport. Effects on Nedd4-2 phosphorylation / abundance and the surface expression of ENaC were monitored by western analysis, whilst Na+ absorption was quantified electrometrically. Acutely (20 min) activating PKA in glucocorticoid-deprived (24 h) cells increased the abundance of Ser221-phosphorylated, Ser327-phosphorylated and total Nedd4-2 without altering the abundance of Thr246-phosphorylated Nedd4-2. Activating PKA under these conditions did not cause a co-ordinated increase in the surface abundance of α-, β- and γ-ENaC and had only a very small effect upon electrogenic Na+ absorption. Activating PKA (20 min) in glucocorticoid-treated (0.2 µM dexamethasone, 24 h) cells, on the other hand, increased the abundance of Ser221-, Ser327- and Thr246-phosphorylated and total Nedd4-2; increased the surface abundance of α-, β- and γ-ENaC and evoked a clear stimulation of Na+ transport. Chronic glucocorticoid stimulation therefore appears to allow cAMP-dependent control of Na+ absorption by facilitating the effects of PKA upon the Nedd4-2 and ENaC subunits.
    European journal of pharmacology 06/2014; 732(100). DOI:10.1016/j.ejphar.2014.03.005 · 2.53 Impact Factor
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    • "Because the cAMP-PKA pathway is a major intracellular signaling pathway that regulates hepatocyte responses to fasting, we sought E3 ubiquitin ligases expressed in liver that are known to be regulated by cAMP signaling. The HECT family E3 ubiquitin ligase NEDD4L (also called NEDD4-2) is expressed in liver [12] and is acutely inhibited by direct PKA phosphorylation in epithelial cells treated with vasopressin [13]. In vivo roles of NEDD4L in liver have not been examined, but NEDD4L is best known for inhibition of the epithelial sodium channel (ENaC) in the kidney [14]. "
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    ABSTRACT: During cycles of fasting and feeding, liver function is regulated by both transcriptional and post-translational events. Regulated protein degradation has recently emerged as a key mechanism to control abundance of specific hepatic proteins under different nutritional conditions. As glucagon signaling through cAMP and PKA is central to glucose output during fasting, we hypothesized that this signaling pathway may also regulate ubiquitin ligases in the fasted state. Here we show that fasting stimuli promote expression of the short isoform of the E3 ubiquitin ligase Nedd4l in primary mouse hepatocytes. Nedd4l-short mRNA and NEDD4L (short isoform) protein accumulate in glucagon-treated primary mouse hepatocytes and in liver tissues during fasting. We identified a functional cAMP response element in the alternate Nedd4l-short promoter; mutation of this element blunts cAMP-induced expression of a Nedd4l reporter construct. CREB occupies the endogenous Nedd4l locus near this element. CREB and its co-activator CRTC2, both activated by fasting stimuli, contribute to glucagon-stimulated Nedd4l-short expression in primary hepatocytes. siRNA-mediated Nedd4l depletion in primary hepatocytes did not affect gluconeogenic gene expression, glucose output or glycogen synthesis. Our findings reveal a new mechanism of Nedd4l transcriptional regulation in liver cells.
    PLoS ONE 10/2013; 8(10):e78522. DOI:10.1371/journal.pone.0078522 · 3.23 Impact Factor
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    • "Much like Sgk1, PKA can also phosphorylate Nedd4-2 and prevent it from inhibiting ENaC (Snyder et al., 2004a). Since PKA is activated by cAMP, which is generated by vasopressin release and binding to its V2 receptor, it is likely that vasopressin, like aldosterone, can increase ENaC cell surface abundance by inhibiting Nedd4-2. "
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    ABSTRACT: Nedd4-2 is a ubiquitin ligase previously demonstrated to regulate endocytosis and lysosomal degradation of the epithelial Na(+) channel (ENaC) and other ion channels and transporters. Recent studies using Nedd4-2 knockout mice specifically in kidney or lung epithelia has revealed a critical role for this E3 ubiquitin ligase in regulating salt and fluid transport in these tissues/organs and in maintaining homeostasis of body blood pressure. Interestingly, the primary targets for Nedd4-2 may differ in these two organs: in the lung Nedd4-2 targets ENaC, and loss of Nedd4-2 leads to excessive ENaC function and to cystic fibrosis - like lung disease, whereas in the kidney, Nedd4-2 targets the Na(+)/Cl(-) cotransporter (NCC) in addition to targeting ENaC. In accord, loss of Nedd4-2 in the distal nephron leads to increased NCC abundance and function. The aldosterone-responsive kinase, Sgk1, appears to be involved in the regulation of NCC by Nedd4-2 in the kidney, similar to its regulation of ENaC. Collectively, these new findings underscore the physiological importance of Nedd4-2 in regulating epithelial salt and fluid transport and balance.
    Frontiers in Physiology 06/2012; 3:212. DOI:10.3389/fphys.2012.00212 · 3.53 Impact Factor
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