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Abstract: FR-OR063

Phosphorylation of β1Pix by AMPK and Its Role in ENaC Inhibition in Kidney Epithelial Cells

Session Information

Category: Fluid and Electrolytes

  • 901 Fluid and Electrolytes: Basic


  • Ho, Pei-Yin, USC Keck School of Medicine, Los Angeles, California, United States
  • Li, Hui, USC Keck School of Medicine, Los Angeles, California, United States
  • Cheng, Lei, Aarhus university, Aarhus, Denmark
  • Fenton, Robert A., Aarhus university, Aarhus, Denmark
  • Hallows, Kenneth R., USC Keck School of Medicine, Los Angeles, California, United States

AMP-activated kinase (AMPK) inhibits the epithelial Na+ channel (ENaC) by increasing the binding of the ubiquitin ligase Nedd4-2 to ENaC. We recently showed that AMPK inhibits ENaC by direct phosphorylation of Nedd4-2 at a site critical for Nedd4-2 stability. We also demonstrated that the Pak-interacting exchange factor β1Pix is required for ENaC inhibition by AMPK and for Nedd4-2 stability in mouse kidney cortical collecting duct (CCD) cells. AMPK activators increase the binding of β1Pix to 14-3-3. Thus, AMPK may enhance Nedd4-2-dependent ENaC degradation by promoting the formation of a β1Pix/Nedd4-2/14-3-3 complex. Here, we further investigate the mechanisms of how AMPK promotes a β1Pix-14-3-3 interaction and whether 14-3-3 binding is important for ENaC inhibition by AMPK.


Protein mass spectrometry (MS), in vitro translation and phosphorylation assays were used to detect AMPK phosphorylation sites in β1Pix. Various constructs were transiently expressed in polarized mpkCCDc14 cells. An epithelial volt-ohmmeter was used to measure amiloride-sensitive equivalent short-circuit currents in mpkCCDc14 cells ± combined treatment with the AMPK activators AICAR and A-769662 for 24 h. Coimmunoprecipitation assays were used to examine modulation of β1Pix/14-3-3/Nedd4-2 interactions by AMPK.


AMPK directly phosphorylates β1Pix in vitro, and six potential AMPK phosphorylation sites were detected by MS. Among these sites, the Ser-71 phosphorylation site on β1Pix was found to be functionally significant. Compared to wild-type β1Pix, overexpression of the β1Pix-S71A mutant significantly reduced ENaC inhibition and the binding of both β1Pix and Nedd4-2 to 14-3-3 caused by AMPK activation in mpkCCDc14 cells. Moreover, overexpression of a β1Pix-Δ602-611 mutant unable to bind 14-3-3 decreased the interaction between Nedd4-2 and 14-3-3. Finally, in preliminary studies, overexpression of the R18 peptide, which inhibits 14-3-3-target interactions, reduced the binding of both β1Pix and Nedd4-2 to 14-3-3 and attenuated ENaC inhibition by AMPK in CCD cells.


Our results suggest that phosphorylation of β1Pix at Ser-71 by AMPK is involved in the formation of a β1Pix/Nedd4-2/14-3-3 complex, which promotes ENaC degradation caused by AMPK activation.


  • NIDDK Support