Structural and Functional Characterization of Nrf2 Degradation by the Glycogen Synthase Kinase 3/β-TrCP Axis

Abstract

fThe transcription factor NF-E2-related factor 2 (Nrf2) is a master regulator of a genetic program, termed the phase 2 response, that controls redox homeostasis and participates in multiple aspects of physiology and pathology. Nrf2 protein stability is regulated by two E3 ubiquitin ligase adaptors, Keap1 and ␤-TrCP, the latter of which was only recently reported. Here, two-dimensional (2D) gel electrophoresis and site-directed mutagenesis allowed us to identify two serines of Nrf2 that are phosphorylated by glycogen synthase kinase 3␤ (GSK-3␤) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of ␤-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of ␤-TrCP that participate in Nrf2 docking. Intraperitoneal injection of the GSK-3 inhibitor SB216763 led to increased Nrf2 and heme oxygenase-1 levels in liver and hippocampus. Moreover, mice with hippocampal absence of GSK-3␤ exhibited increased levels of Nrf2 and phase 2 gene products, reduced glutathione, and decreased levels of carbonylated proteins and malondialdehyde. This study establishes the structural parameters of the interaction of Nrf2 with the GSK-3/␤-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.

Hrd1 suppresses Nrf2-mediated cellular protection during liver cirrhosis

“…Presently, all small chemical Nrf2 activators or endogenous proteins identified disrupt the DLG -Kelch interaction to suppress Nrf2 ubiquitylation, resulting in stabilization of Nrf2 and activation of the pathway (Magesh et al 2012;Jaramillo and Zhang 2013). Recently, b-TrCP-Skp1-Cul1-Rbx1 was also identified as an E3 ubiquitin ligase for Nrf2 (Rada et al 2011(Rada et al , 2012Chowdhry et al 2013). Two motifs, DSGIS and DSAPGS, in the Neh6 domains of Nrf2 were determined to interact with b-TrCP.…”

“…Two motifs, DSGIS and DSAPGS, in the Neh6 domains of Nrf2 were determined to interact with b-TrCP. Phosphorylation of the serine residues in DSGIS resulted in recruitment of Nrf2 by b-TrCP into the E3 ligase complex, with subsequent ubiquitylation of Nrf2 (Rada et al 2011(Rada et al , 2012Chowdhry et al 2013). However, phosphorylation of DSAPGS is not required (Chowdhry et al 2013).…”

“…To demonstrate that Hrd1 functions independently of Keap1 or b-TrCP, previously defined E3 ligases that control Nrf2 protein stability (Kobayashi et al 2004;Zhang et al 2004;Rada et al 2011Rada et al , 2012Chowdhry et al 2013), we performed several additional experiments. Overexpression of XBP1s or Hrd1 reduced Nrf2 levels in Keap1 À/À cells (Fig.…”

Transcription factor NFE2L2/NRF2 modulates chaperone-mediated autophagy through the regulation of LAMP2A

“…pTK-Renilla was used as an internal control vector. Luciferase assays were performed with the Dual-Luciferase Reporter Assay System (Promega, E1910) as described previously [53]. …”

Therapeutic Potential of the Activators of the Nuclear Factor Erythroid 2-Related Factor 2–Antioxidant Response Element Pathway in Brain Disorders

“…1,2) Glycogen synthase kinase (GSK)-3β phosphorylates the Neh6 domain of Nrf2 in these models. 3) Phosphorylated Neh6 on Nrf2 is recognized by an E3 ubiquitin ligase. Thus, GSK-3β-mediated phosphorylation of Neh6 causes ubiquitination and degradation of Nrf2 in place of Keap1.…”