AMPK leads to phosphorylation of the transcription factor Nrf2, tuning transactivation of selected target genes

Abstract

The transcription factor Nrf2 (nuclear factor (erythroid-derived 2)-like 2) and the kinase AMPK (AMP-activated protein kinase) participate in the cellular adaptive response to redox or energy stress. Despite accumulating evidence for positive cooperativity between both proteins, information about direct post-translational modification of Nrf2 by AMPK in living cells is scarce. Here, MS-based analysis of immunoprecipitated Nrf2 revealed serine 374, 408 and 433 in human Nrf2 to be hyperphosphorylated as a function of activated AMPK. A direct phosphate-transfer by AMPK to those sites was indicated by in vitro kinase assays with recombinant proteins as well as interaction of AMPK and Nrf2 in cells, evident by co-immunoprecipitation. Mutation of serine 374, 408 and 433 to alanine did not markedly affect half-life, nuclear accumulation or induction of reporter gene expression upon Nrf2 activation with sulforaphane. However, some selected endogenous Nrf2 target genes responded with decreased induction when the identified phosphosites were mutated, whereas others remained unaffected. Notably, the genes susceptible to the mutation of the phosphorylation sites in Nrf2 consistently showed reduced induction in AMPKα1 −/−cells. Overall, our data reveal AMPK-triggered phosphorylation of Nrf2 at three serine residues, apparently determining the extent of transactivation of selected target genes.

BML‐111 treatment prevents cardiac apoptosis and oxidative stress in a mouse model of autoimmune myocarditis

“…36 This protein is capable of activating AMPKα, which, in turn, directly phosphorylates Nrf2 facilitating its nuclear accumulation. 22 When HL-1 cells were treated with BML-111 for short periods of time, we detected an early induction of CaMKK2 at 5 minutes followed by an increased phosphorylation of AMPKα from 30 minutes to 2 hours, which agrees with CaMKK2-AMPKα-Nrf2 signaling pathway ( Figure 7A). To further confirm the implication of AMPKα in BML-111-mediated Nrf2 activation in this model, we treated HL-1 cells with compound C, a potent, selective and reversible inhibitor of AMPK activity.…”

“…Nrf2 stability and activation is mainly mediated by Keap1 (Kelch‐like ECH associated protein‐1), which interacts with Nrf2 and inhibits its nuclear translocation 21 . In addition, Nrf2 activation can also be modulated by posttranslational modifications, particularly through phosphorylation by different kinases like AMPKα (AMP‐activated protein kinase alpha) 22 . AMPKα induces nuclear sequestration of Nrf2 by phosphorylating its nuclear exportation sequence (NES) 23 .…”

AMPK Enhances Transcription of Selected Nrf2 Target Genes via Negative Regulation of Bach1

“…The underlying ChIP-qPCR experiments may call for some caution, as they directly compare enrichment efficiencies obtained with two different antibodies leaving the risk of distinct performance in the immunoprecipitation step, which, however, is likely to affect wt and AMPK−/− cells to the same extent. Levels of nuclear Nrf2 upon activation (here by Sfn) were not different in wt and AMPK−/− cells, which is consistent with our previous observations (Zimmermann et al, 2015;Matzinger et al, 2020), and could therefore not directly account for the varying Nrf2 to Bach1 ratio. In contrast, bach1 mRNA and Bach1 protein levels showed a clear inverse correlation with AMPK presence in DMSO and Sfn-treated cells.…”

“…The degree of AMPK-(in)dependence was further confirmed by employment of the AMPK inhibitor SBI0206965 (SBI) ( Dite et al, 2018 ), as exemplified by impaired induction of hmox1 but not that of gclc upon SBI treatment ( Figure 2B ). Nrf2-dependent induction of all investigated genes was double-checked by qPCR analysis in Sfn-treated wt and Nrf2−/− MEFs ( Supplementary Figure 2 ; Matzinger et al, 2020 ). Overall, AMPK seems to regulate expression of only a subset of the Nrf2-dependent transcriptome.…”

“…Very likely, the number and exact sequence of ARE sites in the regulatory regions of a gene play a role (Reichard et al, 2007;Raghunath et al, 2018;Otsuki and Yamamoto, 2019). Moreover, we recently have uncovered AMPK-dependent phosphorylation of Nrf2 at three serine residues in living cells (Matzinger et al, 2020). Interestingly, this post-translational modification conferred Nrf2 with a boosted transactivation potential for almost the same target genes that are also enhanced in wt versus AMPK−/− cells.…”