AMPK phosphorylation of the βPix exchange factor regulates the assembly and function of an ENaC inhibitory complex in kidney epithelial cells.

The metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na channel (ENaC), a key regulator of salt reabsorption by the kidney and thus total body volume and blood pressure. Recent studies have suggested that AMPK promotes the association of p21-activated kinase-interacting exchange factor-β βPix, 14-3-3 proteins, and the ubiquitin ligase neural precursor cell expressed developmentally downregulated protein (Nedd)4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional βPix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct cells. Here, we report that AMPK directly phosphorylates βPix in vitro. Among several AMPK phosphorylation sites on βPix detected by mass spectrometry, Ser was validated as functionally significant. Compared with wild-type βPix, overexpression of a phosphorylation-deficient βPix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both βPix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a βPix-Δ602-611 deletion tract mutant unable to bind 14-3-3 proteins decreased the interaction between Nedd4-2 and 14-3-3 proteins, suggesting that 14-3-3 binding to βPix is critical for the formation of a βPix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both βPix and Nedd4-2 to 14-3-3 proteins was reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of βPix at Ser, which promotes 14-3-3 interactions with βPix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.