β1Pix stabilizes Nedd4-2 and plays a critical role in ENaC Regulation by AMPK in kidney epithelial cells
Ho PY, Li H, Pavlov TS, Staruschenko A, and Hallows KR. β1Pix stabilizes Nedd4-2 and plays a critical role in ENaC Regulation by AMPK in kidney epithelial cells. FASEB Journal 2018; 32(1 Suppl):747.9.
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.