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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 AMPK?1 -/-cells. Overall, our data reveal AMPK-triggered phosphorylation of Nrf2 at three serine residues, apparently determining the extent of transactivation of selected target genes.

SUBMITTER: Matzinger M

PROVIDER: S-EPMC6909106 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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

Matzinger Manuel M Fischhuber Katrin K Pölöske Daniel D Mechtler Karl K Heiss Elke H EH

Redox biology 20191127

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 hyperphosphoryla ...[more]

PMID: 31805502

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Project description:Eukaryotic cells adopt highly tuned stress response physiology under threats of exogenous stressors including viruses to maintain cellular homeostasis. Not surprisingly, avoidance of cellular stress response pathways is an essential facet of virus-induced obligatory host reprogramming to invoke a cellular environment conducive to viral perpetuation. Adaptive cellular responses to oxidative and electrophilic stress are usually taken care of by an antioxidant defense system, core to which lies the redox-responsive transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) and Nrf2-driven transcriptional cascade. Deregulation of host redox balance and redox stress-sensitive Nrf2 antioxidant defense have been reported for many viruses. In the current study, we aimed to study the modulation of the Nrf2-based host cellular redox defense system in response to Rotavirus (RV) infection in vitro. Interestingly, we found that Nrf2 protein levels decline sharply with progression of RV infection beyond an initial upsurge. Moreover, Nrf2 decrease as a whole was found to be accompanied by active nuclear vacuity of Nrf2, resulting in lowered expression of stress-responsive Nrf2 target genes heme oxygenase-1 (HO-1), NAD(P)H quinone dehydrogenase 1, and superoxide dismutase 1 both in the presence and absence of Nrf2-driven transcriptional inducers. Initial induction of Nrf2 concurred with RV-induced early burst of oxidative stress and therefore was sensitive to treatments with antioxidants. Reduction of Nrf2 levels beyond initial hours, however, was found to be independent of the cellular redox status. Furthermore, increasing the half-life of Nrf2 through inhibition of the Kelch-like erythroid cell-derived protein with CNC homology- (ECH-) associated protein 1/Cullin3-RING Box1-based canonical Nrf2 turnover pathway could not restore Nrf2 levels post RV-SA11 infection. Depletion of the Nrf2/HO-1 axis was subsequently found to be sensitive to proteasome inhibition with concurrent observation of increased K48-linked ubiquitination associated with Nrf2. Together, the present study describes robust downregulation of Nrf2-dependent cellular redox defense beyond initial hours of RV infection, justifying our previous observation of potent antirotaviral implications of Nrf2 agonists.

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

Publications

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

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