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

Area of Science:

• Cellular biology
• Molecular signaling
• Biochemistry

Background:

• Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and AMP-activated protein kinase (AMPK) are key regulators of cellular stress responses.
• While cooperative interactions are known, direct post-translational modification of Nrf2 by AMPK in vivo remains poorly understood.

Purpose of the Study:

• To investigate the direct phosphorylation of Nrf2 by AMPK in living cells.
• To identify specific phosphorylation sites on Nrf2 modified by AMPK.
• To determine the functional consequences of Nrf2 phosphorylation by AMPK on target gene expression.

Main Methods:

• Mass spectrometry-based analysis of immunoprecipitated Nrf2.
• In vitro kinase assays using recombinant proteins.
• Co-immunoprecipitation to assess protein interactions.
• Site-directed mutagenesis of Nrf2 phosphorylation sites.
• Analysis of reporter gene and endogenous gene expression.

Main Results:

• Mass spectrometry identified serine 374, 408, and 433 on human Nrf2 as hyperphosphorylated upon AMPK activation.
• In vitro assays confirmed direct phosphorylation of these sites by AMPK.
• Mutating these phosphosites did not significantly alter Nrf2 half-life or nuclear accumulation.
• While some Nrf2 target genes showed reduced induction upon phosphosite mutation, others were unaffected.
• Genes sensitive to phosphosite mutation exhibited decreased induction in AMPKα1 knockout cells.

Conclusions:

• AMPK directly phosphorylates Nrf2 at serine 374, 408, and 433.
• This AMPK-mediated phosphorylation regulates the transactivation extent of specific Nrf2 target genes.
• The findings elucidate a novel regulatory mechanism in cellular adaptive responses to stress.