Abstract
The bZIP transcription factor Nrf2 has emerged as a pivotal regulator of intracellular redox homeostasis by controlling the expression of many endogenous antioxidants and phase II detoxification enzymes. Upon oxidative stress, Nrf2 is induced at protein levels through redox-sensitive modifications on cysteine residues of Keap1, a component of the E3 ubiquitin ligase that targets Nrf2 for ubiquitin-dependent degradation. The mitogen activated protein kinases (MAPKs) have previously been proposed to regulate Nrf2 in response to oxidative stress. However, the exact role of MAPKs and the underlying molecular mechanism remain poorly defined. Here we report the first evidence that Nrf2 is phosphorylated in vivo by MAPKs. We have identified multiple serine/threonine residues as major targets of MAPK-mediated phosphorylation. Combined alanine substitution on those residues leads to a moderate decrease in the transcriptional activity of Nrf2, most likely due to a slight reduction in its nuclear accumulation. More importantly, Nrf2 protein stability, primarily controlled by Keap1, is not altered by Nrf2 phosphorylation in vivo. These data indicate that direct phosphorylation of Nrf2 by MAPKs has limited contribution in modulating Nrf2 activity. We suggest that MAPKs regulate the Nrf2 signaling pathway mainly through indirect mechanisms.
Highlights
Disruption of redox homeostasis is associated with the toxic effects of many environmental insults and pathogenesis of agingrelated diseases such as cancer and neurodegenerative disorders [1,2,3,4,5]
The nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant response element (ARE) system is responsible for both basal and inducible expression of many genes involved in antioxidant responses, such as NAD(P)H quinone oxidoreductase 1 (NQO1), heme oxygenase-1 (HO-1), glutathione S-transferase A1 (GSTA1, known as GST-Ya in mouse), glutamate-cysteine ligase modifier subunit (GCLM) and catalytic subunit (GCLC) [9,10,11,12]
The immunoprecipitates were analyzed by immunoblot using antibodies that recognize phosphorylated serine or threonine residue adjacent to a proline, the consensus motif phosphorylated by mitogen activated protein kinases (MAPKs)
Summary
Disruption of redox homeostasis is associated with the toxic effects of many environmental insults and pathogenesis of agingrelated diseases such as cancer and neurodegenerative disorders [1,2,3,4,5]. Mammalian intracellular redox homeostasis is maintained mainly through transcriptional control of an array of antioxidative genes upon exposure to environmental insults that generate oxidative stress. As a key component of such a control system, the antioxidant response element (ARE) is a conservative cis-acting element found in the promoter regions of many genes encoding antioxidants and detoxification enzymes. The corresponding trans-acting factor for the ARE is the nuclear factor erythroid 2-related factor 2 (Nrf2), a protein belonging to the bZIP (basic-leucine zipper) transcription factor family [6,7,8]. The importance of the Nrf2-ARE system is demonstrated by findings that Nrf[2] knockout mice display significantly increased sensitivity to chemical toxicants and carcinogens [13,14]
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