Nedd4-2 isoforms ubiquitinate individual epithelial sodium channel subunits and reduce surface expression and function of the epithelial sodium channel

Abstract

We previously reported the existence of multiple isoforms of human Nedd4-2 (Am J Physiol Renal Physiol 285: F916-F929, 2003). When overexpressed in M-1 collecting duct epithelia, full-length Nedd4-2 (Nedd4-2), Nedd4-2 lacking the NH(2)-terminal C2 domain (Nedd4-2DeltaC2), and Nedd4-2 lacking WW domains 2 and 3 (Nedd4-2DeltaWW2,3) variably reduce benzamil-sensitive Na(+) transport. We investigated the effect of each of the Nedd4-2 isoforms on cell surface expression and ubiquitination of ENaC subunits. We find that alphaENaC when transfected alone or with beta and gammaENaC is expressed at the cell surface and this membrane expression is variably reduced by coexpression with each of the Nedd4-2 isoforms. Nedd4-2 reduces the half-life of ENaC subunits and enhances the ubiquitination of alpha, beta, and gammaENaC subunits when expressed alone or together suggesting that each subunit is a target for Nedd4-2-mediated ubiquitination. As has been reported recently, we confirm that the surface-expressed pool of ENaC is multi-ubiquitinated. Inhibitors of the proteasome increase ubiquitination of ENaC subunits and stimulate Na(+) transport in M-1 cells consistent with a role for the ubiquitin-proteasome pathway in regulating Na(+) transport in the collecting duct.

Figure 1. Nedd4-2 isoforms differentially regulate ENaC activity in M-1 cells

Panel A. M-1 cells transduced with adenovirus expressing Nedd4-2, Nedd4-2ΔC2, Nedd4-2ΔWW2,3 or empty virus and Isc measured in Ussing chambers under control conditions and after treatment with 100 nM dexamethasone (dex) for 24 hr. M-1 cells overexpressing all 3 isoforms had significantly lower currents as compared to empty virus (**P < 0.001; Tukey’s pairwise multiple comparison, SigmaStat; n = 15; mean ± SE). Following steroid treatment, currents were significantly different in between each of the Nedd4-2 isoforms (## P < 0.001 compared to Nedd4-2ΔWW2,3; $P < 0.05 compared to Nedd4-2ΔWW2,3; ¶P < 0.05 compared to Nedd4-2; Tukey’s pairwise multiple comparison, SigmaStat; n = 15; mean ± SE). Panel B. Representative western blot of transduced M-1 cells demonstrate relatively equal expression of each of the Nedd4-2 isoforms. Full length Nedd4-2 migrates at ~116 kDa; Nedd4-2ΔC2 (N4-2ΔC2) migrates at ~101 kDa and Nedd4-2ΔWW2,3 (N4-2ΔWW2,3) migrates at ~83 kDa.

Figure 2. Surface expression and the half-life of ENaC is greatly reduced by Nedd4-2

Panel A: Nedd4-2 isoforms or a control empty vector was transfected with ENaC subunits as indicated into HEK293 cells and biotinylated surface proteins (panel above) and total cellular lysates (panel below) were blotted with an anti-FLAG antibody, the epitope carried by the αENaC subunit. Each of the Nedd4-2 isoforms reduces surface expression of αENaC in a dose dependent manner with Nedd4-2ΔC2 being most potent. Representative of at least two separate experiments. Panel B: ENaC subunits were cotransfected with Nedd4-2 in HEK 293 cells. Cells were treated with 20 μg/ml cycloheximide to arrest translation and at indicated time points lysates prepared and immunoblotted with an anti-FLAG antibody and after stripping re-probed with anti-α tubulin antibody. Overexpression of Nedd4-2 decreases αENaC stability.

Figure 3. Inactivation of the catalytic domain of Nedd4-2 abolishes its effect on ENaC

Panel A. Active or inactive Nedd4-2 (Nedd4-2CS) or a control empty vector was transfected with ENaC subunits as indicated into HEK293 cells and biotinylated surface proteins and total cellular lysates were blotted with an anti-FLAG antibody, or an actin antibody. The data demonstrates that Nedd4-2 CS does not affect surface expression of ENaC. Actin was immunoblotted in total and surface fractions to confirm that intracellular proteins are excluded in affinity purified surface protein preparations. Panel B. Active or inactive Nedd4-2 (Nedd4-2CS) or a control empty vector were transfected with ENaC subunits and HA-tagged ubiquitin (HA-Ub) as indicated into HEK293 cells. ENaC subunits were immunoprecipitated with an anti-FLAG antibody and immunoblotted with anti-HA to detect ubiquitinated ENaC. Ubiquitinated proteins appear as high molecular mass smear. Compared to active Nedd4-2, Nedd4-2CS has no effect on ubiquitination of ENaC in HEK293 cells. Panel C: Adenoviral vectors expressing active or inactive Nedd4-2 (Nedd4-2CS) or a control empty virus were transduced into M-1 cells. Compared to active Nedd4-2, Nedd4-2CS has no effect on amiloride-sensitive Isc in M-1 cells. (*P < 0.05; Dunn’s pairwise multiple comparison, SigmaStat; n = 6; mean ± SD). Panel D. Representative western blot of transduced M-1 cells demonstrate relatively equal expression of the Nedd4-2 isoforms.

Figure 4. Each of the Nedd4-2 isoforms, enhances the ubiquitination of ENaC

Nedd4-2 isoforms or a control empty vector were transfected with ENaC subunits and HA-tagged ubiquitin (HA-Ub) as indicated into HEK293 cells. ENaC subunits were immunoprecipitated with an anti-FLAG antibody and immunoblotted with anti-HA to detect ubiquitinated ENaC. Total cellular lysates were immunoblotted with anti-FLAG antibody. Ubiquitinated proteins appear as high molecular mass smear. αENaC when expressed alone can be ubiquitinated and ubiquitination of the ENaC complex is increased in the presence of each Nedd4-2 isoform.

Figure 5. The accessory ENaC subunits β and γ are also targets for ubiquitination

Tagged ENaC subunits were transfected alone or together with or without Nedd4-2 and HA-tagged ubiquitin (HA-Ub) as indicated into HEK293 cells. ENaC subunits were immunoprecipitated with an anti-V5 (β) or anti-myc (γ) antibody and immunoblotted with anti-HA to detect ubiquitinated ENaC subunits. Panel A. ENaC subunit β is ubiquitinated when expressed alone or together and when βENaC is expressed alone this ubiquitination is enhanced by co-expression of Nedd4-2. Panel B. ENaC subunit γ is ubiquitinated when expressed alone or together and as with βENaC this is enhanced by co-expression of Nedd4-2. Similar results were observed from at least three independent experiments for each subunit.

Figure 6. ENaC subunits at the cell surface contain monoubiquitin but not polyubiquitin chains

Panel A. Nedd4-2 or a control empty vector was transfected with ENaC subunits and HA-tagged ubiquitin as indicated into HEK293. Biotinylated surface proteins (panel above) were affinity purified and then immunoprecipitated with an anti-FLAG antibody and blotted with an anti-HA antibody (upper panel). Total cellular lysates were also immunoblotted with an anti-FLAG antibody (middle panel) and anti-Xpress antibody (bottom panel), the epitope tag attached to Nedd4-2. Panel B. and C. HEK 293 cells were co-transfected with increasing amounts of ENaC subunits (0, 5, 10, 15 μg of each subunit). The cells were treated with 1μM MG132 for 4 hours and biotinylated surface proteins were affinity purified and then immunoprecipitated with an anti-FLAG antibody and blotted with an anti-FK1 antibody (B) or anti-P4D1 (C). Total cellular lysates were also immunoblotted with these antibodies. The anti-polyubiquitin antibody FK1 detects ubiquitin chains only in cellular lysate and not in surface fractions whereas anti-P4D1 which identifies both monoubiquitin and polyubiquitin chains identifies ubiquitin in surface fractions.

Figure 7

Effect of Nedd4-2 isoforms on ubiquitination of individual ENaC subunits: Panel A: Each of the Nedd4-2 isoforms were transfected with individual ENaC subunits and HA-tagged ubiquitin (HA-Ub) as indicated into HEK293 cells. ENaC subunits were immunoprecipitated with an anti-FLAG/V5/Myc antibody and immunoblotted with anti-HA to detect ubiquitinated α, β and γENaC respectively. Ubiquitinated proteins appear as high molecular mass smear. Each of the ENaC subunits when expressed alone can be ubiquitinated in the presence of each Nedd4-2 isoform. Panel B: αENaC was transfected with each of Nedd4-2 isoforms into HEK293 cells. Then biotinylated surface proteins were affinity purified and then immunoprecipitated with an anti-FLAG antibody and blotted with an anti-HA antibody. Each Nedd4-2 isoform ubiquitinates a surface expressed fraction of αENaC. Panel C: αENaC was transfected with each of Nedd4-2 isoforms into HEK293 cells and biotinylated surface proteins were blotted with an anti-FLAG antibody. Each of the Nedd4-2 isoforms reduces surface expression of αENaC correlating with ubiquitination seen in panel A and B.

Figure 8

The effect of Nedd4-2 isoforms and proteasomal inhibitors on ENaC function: Panel A. ENaC subunits were expressed with and without HA-tagged Ubiquitin (HA-Ub) in HEK293 cells and cells were treated with or with-out 1μM MG132 or 100 μM chloroquine for 4 hours as indicated. Ubiquitinated ENaC subunits were detected by immunoprecipitation with anti-Flag followed by immunoblotting with anti-HA antibody. There appeared to be more ubiquitinated ENaC detectable in the presence of MG132. Panel B. M-1 cells grown in filters were treated with or without 1μM MG132 and Isc measured in Ussing chambers. Isc was expressed as fold change compared to the control value corresponding to the same time point. Currents increase significantly within an hr of MG132 and reaches a peak between 2-3 hr (**P < 0.001; *P < 0.05 Tukey’s pairwise multiple comparison, SigmaStat; n = 8 from three different experiment; means ± SE). Panel C. M-1 cells were transduced with adenovirus expressing Nedd4-2 or empty virus and Isc measured in Ussing chambers under control conditions and after treatment with MG132 or chloroquine for 2 hr. M-1 cells overexpressing Nedd4-2 had significantly lower currents as compared to empty virus. MG132 but not chloroquine partially reverses the effect of Nedd4-2 on Na⁺ transport (*P < 0.05 between vehicle and MG132 ; ¶P < 0.05 between vehicle and chloroquine; Tukey’s pairwise multiple comparison, SigmaStat; n = 12 from 3 experiments; mean ± SE). Panel D. M-1 cells were transduced with adenovirus expressing each of the Nedd4-2 isoforms and Isc measured in Ussing chambers under control conditions and after treatment with MG132 or chloroquine for 2 hr. M-1 cells overexpressing Nedd4-2ΔC2 had lower currents compared to Nedd4-2 and Nedd4-2ΔWW2,3. MG132 partially reverses the effect of Nedd4-2 and Nedd4-2ΔWW2,3, but not Nedd4-2ΔC2, on Na⁺ transport (*P < 0.05 between vehicle and MG132 ; ¶P < 0.05 between vehicle and chloroquine; Tukey’s pairwise multiple comparison, SigmaStat; n = 16 from 3 experiments; mean ± SE).