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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    • "The cysteine residue in the M a n u s c r i p t 4 HECT domains (Gallagher et al., 2006) phosphorylation of T899 in the HECT domain of Nedd4-2 increases the activity by unknown mechanism (Hallows et al., 2010). Several kinases are involved in phosphorylation of Nedd4 family members in vivo, including PKA (Snyder et al., 2004). Rsp5 contains 59 possible sites of phosphorylation which match consensus sequences recognized by protein kinase A (PKA), protein kinase C, casein kinase I and several other kinases (Net.phos2.0 at Phosphorylation of several sites of Rsp5 has been found in studies of the yeast phosphoproteome (Beltrao et al., 2009; Bodenmiller et al., 2008; Breitkreutz et al., 2010; Gnad et al., 2009; Holt et al., 2009; Soufi et al., 2009; Yachie et al., 2011), but the significance of these phosphorylation is still unknown. "
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    ABSTRACT: Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28°C and did not grow at 37°C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.
    European journal of cell biology 10/2015; DOI:10.1016/j.ejcb.2015.10.005 · 3.83 Impact Factor
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    • "Selectively activating SGK1 by brief (3 h) exposure to dexamethasone increased the abundance of Ser221-, Ser327- and Thr246-phosphorylated Nedd4-2 indicating that these residues are SGK1 substrates (Debonneville et al., 2001; Snyder et al., 2004a; Snyder et al., 2002). However, these effects were accompanied by clear increases in overall Nedd4-2 abundance and, since the magnitudes of the two responses were similar, activating SGK1 did not change the relative phosphorylation status of Nedd4-2-Ser221, -Ser327 or -Thr246. "
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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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