Abstract

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 suggest that AMPK promotes the association of the PAK-interacting exchange factor ß1Pix, 14-3-3 proteins, and the ubiquitin ligase Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and ENaC degradation. Functional ß1Pix is required for ENaC inhibition by AMPK and promotes Nedd4-2 phosphorylation and stability in mouse kidney cortical collecting duct (CCD) cells. Here, we report that AMPK directly phosphorylates ß1Pix in vitro. Among several AMPK phosphorylation sites on ß1Pix detected by mass spectrometry (MS), Ser-71 was validated as functionally significant. Compared to wild-type ß1Pix, overexpression of a phosphorylation-deficient ß1Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both ß1Pix and Nedd4-2 to 14-3-3 proteins in CCD cells. Similarly, overexpression of a ß1Pix-?602-611 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 ß1Pix is critical for the formation of a ß1Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both ß1Pix and Nedd4-2 to 14-3-3 proteins were reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of ß1Pix at Ser-71, which promotes 14-3-3 interactions with ß1Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.

View details for DOI 10.1152/ajprenal.00592.2018

View details for PubMedID 31566435