β1Pix Stabilizes Nedd4‐2 and Plays a Critical Role in ENaC Regulation by AMPK in Kidney Epithelial Cells

Abstract

The epithelial Na+ channel (ENaC) is expressed in many salt‐reabsorbing epithelia at the apical membrane, including in the aldosterone‐sensitive distal nephron of the kidney where it is the final regulator of Na+ balance, blood volume, and blood pressure. Our previous work has established that the metabolic sensor AMP‐activated protein kinase (AMPK) inhibits ENaC by promoting the binding of the ubiquitin ligase Nedd4‐2 to ENaC. Here, we have further demonstrated that functional β1Pix, a Rho‐GEF signaling protein, is required for AMPK‐dependent ENaC inhibition in CHO cells. Epithelial volt‐ohmmeter measurements were performed to examine the role of β1Pix in regulating ENaC‐dependent currents in polarized kidney cortical collecting duct (CCD) cells. Lentiviral shRNA‐mediated knockdown of β1Pix expression in mpkCCDcl1 cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β1Pix dimerization‐deficient mutant unable to bind 14‐3‐3 proteins (Δ602‐611) increased ENaC currents in mpkCCDc14 cells, whereas overexpression of wild‐type β1Pix had an opposite effect. Through additional immunoblotting and co‐immunoprecipitation studies, we explored the role of β1Pix in the regulation of ENaC by AMPK and the interplay of β1Pix, 14‐3‐3 proteins and Nedd4‐2. Treatment with AMPK activators promoted the binding of β1Pix to 14‐3‐3 proteins in CCD cells. However, the association between Nedd4‐2 and 14‐3‐3 proteins was not significantly altered by AMPK activation, β1Pix knockdown, or overexpression of wild‐type or β1Pix‐Δ602–611 mutant in cells. Moreover, β1Pix knockdown or overexpression of β1Pix‐Δ602–611 inhibited Nedd4‐2 protein expression in CCD cells. This inhibition was associated with a parallel reduction in Nedd4‐2 phosphorylation at Ser‐328, a site that is phosphorylated by several kinases including AMPK which is critical for cellular Nedd4‐2 protein stability. Decreased Nedd4‐2 protein stability in the setting of β1Pix knockdown was confirmed by cycloheximide chase studies. Together, these results suggest that functional β1Pix is important for Nedd4‐2 Ser‐328 phosphorylation and thus Nedd4‐2 stability. Overall, our findings suggest that AMPK activation promotes, potentially via AMPK‐dependent β1Pix phosphorylation, the association of β1Pix, 14‐3‐3 proteins, and Nedd4‐2 into a complex that inhibits ENaC by enhancing Nedd4‐2 binding to ENaC followed by ENaC ubiquitination and targeting for degradation. This regulation of ENaC and other epithelial transport proteins by AMPK may have a pro‐survival role by inhibiting energy‐consuming ion transport processes during conditions of ischemia and other cellular stresses that occur with acute kidney injury.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.