AMPK-Mediated Phosphorylation of Nrf2 at S374/S408/S433 Favors Its beta TrCP2-Mediated Degradation in KEAP1-Deficient Cells

Nrf2 is a transcription factor facilitating cells' resilience against redox and various other forms of stress. In the absence of stressors, KEAP1 and/or beta TrCP mediate the ubiquitination of Nrf2 and prevent Nrf2-dependent gene expression and detoxification. AMPK regulates cellular energy homeostasis and redox balance. Previous studies indicated a potential Nrf2-AMPK cooperativity. In line with this, our lab had previously identified three AMPK-dependent phosphorylation sites (S374/408/433) in Nrf2. Given their localization in or near the Neh6 domain, known to regulate fiTrCP-mediated degradation, we examined whether they may influence the beta TrCP-driven degradation of Nrf2. By employing expression plasmids for WT and triple mutant (TM)-Nrf2 (Nrf2S(374/408/433 -> A)), (co)immunoprecipitation, proximity ligation, protein half-life, knockdown, ubiquitination experiments, and qPCR in Keap1-null mouse embryonic fibroblasts, we show that TM-Nrf2(S -> A374/408/433) had enhanced stability due to impeded interaction with beta TrCP2 and reduced ubiquitination in comparison to WT-Nrf2. In addition, TM-Nrf2 elicited higher expression of the Nrf2 target gene Gclc, potentiated in the presence of a pharmacological AMPK activator. Overall, we propose that AMPK-dependent phospho-sites of Nrf2 can favor its fiTrCP2-mediated degradation and dampen the extent of Nrf2 target gene expression. Therefore, targeting AMPK might be able to diminish Nrf2-mediated responses in cells with overactive Nrf2 due to KEAP1 deficiency.

Automated summary

Nrf2 is a transcription factor that enhances cells' resilience against stress. It has been found that AMPK, a kinase that regulates cellular energy balance, has a potential cooperativity with Nrf2. Our study shows that AMPK-dependent phosphorylation sites in Nrf2 can influence its degradation and gene expression. This research is important for understanding the regulation of Nrf2 by AMPK.