| First Author | Ho PY | Year | 2018 |
| Journal | J Biol Chem | Volume | 293 |
| Issue | 29 | Pages | 11612-11624 |
| PubMed ID | 29858246 | Mgi Jnum | J:267094 |
| Mgi Id | MGI:6197329 | Doi | 10.1074/jbc.RA118.003082 |
| Citation | Ho PY, et al. (2018) beta1Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells. J Biol Chem 293(29):11612-11624 |
| abstractText | Our previous work has established that the metabolic sensor AMP-activated protein kinase (AMPK) inhibits the epithelial Na(+) channel (ENaC) by promoting its binding to neural precursor cell-expressed, developmentally down-regulated 4-2, E3 ubiquitin protein ligase (Nedd4-2). Here, using MS analysis and in vitro phosphorylation, we show that AMPK phosphorylates Nedd4-2 at the Ser-444 (Xenopus Nedd4-2) site critical for Nedd4-2 stability. We further demonstrate that the Pak-interacting exchange factor beta1Pix is required for AMPK-mediated inhibition of ENaC-dependent currents in both CHO and murine kidney cortical collecting duct (CCD) cells. Short hairpin RNA-mediated knockdown of beta1Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a beta1Pix dimerization-deficient mutant unable to bind 14-3-3 proteins (Delta602-611) increased ENaC currents in CCD cells, whereas overexpression of WT beta1Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of beta1Pix to 14-3-3 proteins in CCD cells. However, the association between Nedd4-2 and 14-3-3 proteins was not consistently affected by AMPK activation, beta1Pix knockdown, or overexpression of WT beta1Pix or the beta1Pix-Delta602-611 mutant. Moreover, we found that beta1Pix is important for phosphorylation of the aforementioned Nedd4-2 site critical for its stability. Overall, these findings elucidate novel molecular mechanisms by which AMPK regulates ENaC. Specifically, they indicate that AMPK promotes the assembly of beta1Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation. |
