β₁ Pix exchange factor stabilizes the ubiquitin ligase Nedd4-2 and plays a critical role in ENaC regulation by AMPK in kidney epithelial cells
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2018-07-20
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Journal Article
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yes
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Abstract
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 β₁Pix 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 β₁Pix expression in CCD cells attenuated the inhibitory effect of AMPK activators on ENaC currents. Moreover, overexpression of a β₁Pix dimerization–deficient mutant unable to bind 14-3-3 proteins (Δ602–611) increased ENaC currents in CCD cells, whereas overexpression of WT β₁Pix had the opposite effect. Using additional immunoblotting and co-immunoprecipitation experiments, we found that treatment with AMPK activators promoted the binding of β₁Pix 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, β₁Pix knockdown, or overexpression of WT β₁Pix or the β₁Pix-Δ602–611 mutant. Moreover, we found that β1Pix 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 β₁Pix, 14-3-3 proteins, and Nedd4-2 into a complex that inhibits ENaC by enhancing Nedd4-2 binding to ENaC and its degradation.
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published
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Volume
293 (29)
Pages / Article No.
11612 - 11624
Publisher
American Society for Biochemistry and Molecular Biology
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Subject
AMP-activated kinase (AMPK); epithelial sodium channel (ENaC); epithelial sodium channel (ENaC); sodium transport; 14-3-3 protein; kidney; β-Pix
Organisational unit
02207 - Functional Genomics Center Zurich / Functional Genomics Center Zurich