Kidney Week

Abstract: FR-OR063

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

Session Information

• Fluids and Electrolytes: New Insights on Balance
  October 26, 2018 | Location: 8, San Diego Convention Center
  Abstract Time: 05:30 PM - 05:42 PM

Category: Fluid and Electrolytes

• 901 Fluid and Electrolytes: Basic

Authors

• Ho, Pei-Yin, USC Keck School of Medicine, Los Angeles, California, United States

Pei-Yin Ho, PhD

• Li, Hui, USC Keck School of Medicine, Los Angeles, California, United States

Hui Li,

• Cheng, Lei, Aarhus university, Aarhus, Denmark

Lei Cheng,

• Fenton, Robert A., Aarhus university, Aarhus, Denmark

Robert A. Fenton,

• Hallows, Kenneth R., USC Keck School of Medicine, Los Angeles, California, United States

Kenneth R. Hallows,

Background

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.

Methods

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 mpkCCD_c14 cells. An epithelial volt-ohmmeter was used to measure amiloride-sensitive equivalent short-circuit currents in mpkCCD_c14 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.

Results

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 mpkCCD_c14 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.

Conclusion

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.

Funding

• NIDDK Support