Author:

Keywords:

Science & Technology, Life Sciences & Biomedicine, Biochemistry & Molecular Biology, Cell Biology, TRANSCRIPTION FACTOR NRF2, UBIQUITIN-LIGASE COMPLEX, CUL3-BASED E3 LIGASE, BETA-TRCP PROTEIN, F-BOX PROTEINS, OXIDATIVE STRESS, SIGNALING PATHWAYS, LIPID-PEROXIDATION, DESTRUCTION MOTIF, OXIDANT DAMAGE, Amino Acid Sequence, Animals, Cells, Cultured, Glycogen Synthase Kinase 3, Glycogen Synthase Kinase 3 beta, HEK293 Cells, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Models, Molecular, Molecular Sequence Data, Mutagenesis, Site-Directed, NF-E2-Related Factor 2, Phosphorylation, Protein Structure, Tertiary, Serine, beta-Transducin Repeat-Containing Proteins, 06 Biological Sciences, 11 Medical and Health Sciences, Developmental Biology, 31 Biological sciences, 32 Biomedical and clinical sciences, 42 Health sciences

Abstract:

The 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 β-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 3β (GSK-3β) in the sequence DSGISL. Nuclear magnetic resonance studies defined key residues of this phosphosequence involved in docking to the WD40 propeller of β-TrCP, through electrostatic and hydrophobic interactions. We also identified three arginine residues of β-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-3β 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/β-TrCP axis and its functional relevance in the regulation of Nrf2 by the signaling pathways that impinge on GSK-3.