AMPK phosphorylation of the β₁Pix exchange factor regulates the assembly and function of an ENaC inhibitory complex...

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-β₁ β₁Pix, 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 β₁Pix 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 β₁Pix in vitro. Among several AMPK phosphorylation sites on β₁Pix detected by mass spectrometry, Ser⁷¹ was validated as functionally significant. Compared with wild-type β₁Pix, overexpression of a phosphorylation-deficient β₁Pix-S71A mutant attenuated ENaC inhibition and the AMPK-activated interaction of both β₁Pix and Nedd4-2 to 14-3-3 proteins in cortical collecting duct cells. Similarly, overexpression of a β₁Pix-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 β₁Pix is critical for the formation of a β₁Pix/Nedd4-2/14-3-3 complex. With expression of a general peptide inhibitor of 14-3-3-target protein interactions (R18), binding of both β₁Pix 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 β₁Pix at Ser⁷¹, which promotes 14-3-3 interactions with β₁Pix and Nedd4-2 to form a tripartite ENaC inhibitory complex, in the mechanism of ENaC regulation by AMPK.