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Am J Physiol Renal Physiol. 2019 Sep 30. doi: 10.1152/ajprenal.00592.2018. [Epub ahead of print]

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

Author information

1
Medicine, Division of Nephrology and Hypertension, University of Southern California.
2
Biomedicine, Aarhus University.
3
Medicine / Nephrology, Stanford University, United States.
4
Biomedicine, Aarhus University, Denmark.
5
Medicine, Division of Nephrology and Hypertension, University of Southern California, United States.

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 β1Pix, 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 β1Pix 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 β1Pix in vitro. Among several AMPK phosphorylation sites on β1Pix detected by mass spectrometry (MS), Ser-71 was validated as functionally significant. Compared to 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 CCD 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 were reduced, and AMPK-dependent ENaC inhibition was also attenuated. Altogether, our results demonstrate the importance of AMPK-mediated phosphorylation of β1Pix at Ser-71, 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.

KEYWORDS:

14-3-3 protein; AMPK; ENaC; Nedd4-2; beta-Pix

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