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. 2010 Aug 13;5(8):e12163.
doi: 10.1371/journal.pone.0012163.

Interaction of serum- and glucocorticoid regulated kinase 1 (SGK1) with the WW-domains of Nedd4-2 is required for epithelial sodium channel regulation

Dominik Wiemuth  1 , J Shaun Lott, Kevin Ly, Ying Ke, Paul Teesdale-Spittle, Peter M Snyder, Fiona J McDonald
Affiliations

Interaction of serum- and glucocorticoid regulated kinase 1 (SGK1) with the WW-domains of Nedd4-2 is required for epithelial sodium channel regulation

Dominik Wiemuth et al. PLoS One. .

Abstract

Background: The epithelial sodium channel (ENaC) is an integral component of the pathway for Na(+) absorption in epithelial cells. The ubiquitin ligases Nedd4 and Nedd4-2 bind to ENaC and decrease its activity. Conversely, Serum- and Glucocorticoid regulated Kinase-1 (SGK1), a downstream mediator of aldosterone, increases ENaC activity. This effect is at least partly mediated by direct interaction between SGK and Nedd4-2. SGK binds both Nedd4 and Nedd4-2, but it is only able to phosphorylate Nedd4-2. Phosphorylation of Nedd4-2 reduces its ability to bind to ENaC, due to the interaction of phosphorylated Nedd4-2 with 14-3-3 proteins, and hence increases ENaC activity. WW-domains in Nedd4-like proteins bind PY-motifs (PPXY) present in ENaC subunits, and SGK also has a PY-motif.

Principal finding: Here we show that single or tandem WW-domains of Nedd4 and Nedd4-2 mediate binding to SGK and that different WW-domains of Nedd4 and Nedd4-2 are involved. Our data also show that WW-domains 2 and 3 of Nedd4-2 mediate the interaction with SGK in a cooperative manner, that activated SGK has increased affinity for the WW-domains of Nedd4-2 in vitro, and a greater stimulatory effect on ENaC Na(+) transport compared to wildtype SGK. Further, SGK lacking a PY motif failed to stimulate ENaC activity in the presence of Nedd4-2.

Conclusions: Binding of Nedd4-2 WW-domains to SGK is necessary for SGK-induced ENaC activity.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Nedd4-2 and Nedd4 WW-domains interact with SGK in vitro.
(A) ΔN60SGK-HA and Nedd4-2C821A–FLAG were transiently co-expressed, or expressed alone as indicated, in COS7 cells. Complexes were immunoprecipitated with anti-HA antibody. Immunoprecipitates were analyzed by 10% SDS-PAGE and sequential western blotting (IB) with anti-FLAG antibody (Nedd4-2) and anti-HA antibody (ΔN60SGK). Expression of the proteins (5% input) is shown in the bottom panel, n = 3. Either ΔN60SGK-HA (B, E) or SGK-FLAG (C, D) was transiently expressed in COS7 cells. Cells were lysed 48 h after transfection with TBS +1% Triton-X-100. The lane labelled “lysate” shows expression of SGK in whole cell lysate. COS7 cell lysates were incubated with the indicated Nedd4-2 or Nedd4 WW-domain GST-fusion proteins, or GST alone, to test for interaction with SGK. Bound SGK was analyzed by immunostaining with anti-FLAG/HA antibody. The bands indicate that WW-domains 2 and 3 of Nedd4-2, and WW-domains 1 and 3 of Nedd4 interact with SGK, n = 7. The two SGK bands observed in some experiments are likely to be unphosphorylated and phosphorylated forms of SGK .
Figure 2
Figure 2. WW-domains 2 and 3 bind with low affinity to an SGK PY-motif peptide.
(A) Typical BIAcore response curves produced by injecting different concentrations of the isolated Nedd4-2 WW2 domain over immobilized SGK PY-motif peptide. Each concentration was measured in duplicate. (B) Estimation of K D from steady state equilibrium binding measurements. The equilibrium SPR response, R eq was measured by taking the average value in the equilibrium region of the response curve between 120 and 180 seconds. R eq, was measured in triplicate for each concentration of WW2, and the mean value of R eq was then plotted against the concentration of WW2. The solid line represents the best fit to the data using the equation described in Materials and Methods (χ2 = 0.34). Binding affinities of the other WW-domains are summarized in Table 1.
Figure 3
Figure 3. SGKS422D binding to Nedd4-2 and stimulation of ENaC is enhanced compared to wildtype SGK.
(A) SGK-FLAG or SGKS422D-FLAG were expressed in COS7 cells. After 48 hr cells were lysed, pre-cleared with GST bound to glutathione-Sepharose beads and then incubated with 50 µg of GST fusion proteins WW-domain 2, WW-domain 3, WW-domains 2 and 3 of Nedd4-2, or GST alone (left) as indicated. After washing the beads, the bound proteins were eluted and analyzed by SDS-PAGE and western blotting with anti-FLAG. The right panel shows interaction of SGK with the WW-domains, and also the interaction of SGKS422D with the WW-domains. (B) The resulting bands were quantified by densitometry and normalized to total expression of either SGK or SGKS422D. Bars represent mean +/− S.E. (n = 9 for WW2 and WW3, n = 6 for WW2–3). (C) Relative amiloride-sensitive short-circuit sodium current in FRT cells after transfection with α-, β- and γENaC together with SGK-HA or SGKS422D-HA under serum (FCS) or serum-free conditions, n = 4. ***indicates P<0.001, **indicates P<0.01 and *indicates P<0.05, analysis of variance.
Figure 4
Figure 4. The SGK PY motif is necessary for SGK to inhibit Nedd4-2.
Amiloride-sensitive short-circuit sodium current (relative to 0 µg of Nedd4-2) in FRT cells 48 hr after transfection with α-, β- and γENaC and SGK (closed circles) or SGKY298A (open circles) or GFP (closed squares), in the presence of increasing amounts of Nedd4-2, n = 18. Nedd4-2 is expressed as a fraction of the amount of ENaC cDNA. Expression of SGK and SGKY298A was reported in Figure 1B of Snyder et al. .
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