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Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/?-TrCP axis.

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 ?-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.

SUBMITTER: Rada P

PROVIDER: S-EPMC3422007 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/β-TrCP axis.

Rada Patricia P Rojo Ana I AI Evrard-Todeschi Nathalie N Innamorato Nadia G NG Cotte Axelle A Jaworski Tomasz T Tobón-Velasco Julio C JC Devijver Herman H García-Mayoral María Flor MF Van Leuven Fred F Hayes John D JD Bertho Gildas G Cuadrado Antonio A Cuadrado Antonio A

Molecular and cellular biology 20120702 17

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 phosph ...[more]

PMID: 22751928

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Project description:Evidence suggests that the glycogen synthase kinase 3 (GSK3)-dictated nuclear exclusion and degradation of Nrf2 is pivotal in switching off the self-protective antioxidant stress response after injury. Here, we examined the mechanisms underlying this regulation in glomerular disease. In primary podocytes, doxorubicin elicited cell death and actin cytoskeleton disorganization, concomitant with overactivation of GSK3β (the predominant GSK3 isoform expressed in glomerular podocytes) and minimal Nrf2 activation. SB216763, a highly selective small molecule inhibitor of GSK3, exerted a protective effect that depended on the potentiated Nrf2 antioxidant response, marked by increased Nrf2 expression and nuclear accumulation and augmented production of the Nrf2 target heme oxygenase-1. Ectopic expression of the kinase-dead mutant of GSK3β in cultured podocytes reinforced the doxorubicin-induced Nrf2 activation and prevented podocyte injury. Conversely, a constitutively active GSK3β mutant blunted the doxorubicin-induced Nrf2 response and exacerbated podocyte injury, which could be abolished by treatment with SB216763. In murine models of doxorubicin nephropathy or nephrotoxic serum nephritis, genetic targeting of GSK3β by doxycycline-inducible podocyte-specific knockout or pharmacologic targeting by SB216763 significantly attenuated albuminuria and ameliorated histologic signs of podocyte injury, including podocytopenia, loss of podocyte markers, podocyte de novo expression of desmin, and ultrastructural lesions of podocytopathy (such as foot process effacement). This beneficial outcome was likely attributable to an enhanced Nrf2 antioxidant response in glomerular podocytes because the selective Nrf2 antagonist trigonelline abolished the proteinuria-reducing and podocyte-protective effect. Collectively, our results suggest the GSK3β-regulated Nrf2 antioxidant response as a novel therapeutic target for protecting podocytes and treating proteinuric glomerulopathies.

Project description:Glycogen synthase kinase-3 (GSK3) is a constitutively active protein kinase that is involved in neuronal regulation and is a potential pharmacological target of neurological disorders. We found previously that GSK3? selectively interacts with 5-hydroxytryptamine-1B receptors (5-HT1BR) that have important functions in serotonin neurotransmission and behavior. In this study, we provide new information supporting the importance of GSK3? in 5-HT1BR-regulated signaling, physiological function, and behaviors. Using molecular, biochemical, pharmacological, and behavioral approaches, we tested 5-HT1BR's interaction with G(i)?(2) and ?-arrestin2 and 5-HT1BR-regulated signaling in cells, serotonin release in mouse cerebral cortical slices, and behaviors in wild-type and ?-arrestin2 knockout mice. Molecular ablation of GSK3? and GSK3 inhibitors abolished serotonin-induced change of 5-HT1BR coupling to G(i)?(2) and associated signaling but had no effect on serotonin-induced recruitment of ?-arrestin2 to 5-HT1BR. This effect is specific for 5-HT1BR because GSK3 inhibitors did not change the interaction between serotonin 1A receptors and G(i)?(2). Two GSK3 inhibitors, N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea (AR-A014418) and 3-(5-bromo-1-methyl-1H-indol-3-yl)-4-(benzofuran-3-yl)pyrrole-2,5-dione (BIP-135), efficiently abolished the inhibitory effect of the 5-HT1BR agonist anpirtoline on serotonin release in mouse cerebral cortical slices. GSK3 inhibitors also facilitated the 5-HT1BR agonist anpirtoline-induced behavioral effect in the tail suspension test but spared anpirtoline-induced locomotor activity. These results suggest that GSK3? is a functional selective modulator of 5-HT1BR-regulated signaling, and GSK3 inhibitors fine-tune the physiological and behavioral actions of 5-HT1BR. Future studies may elucidate the significant roles of GSK3 in serotonin neurotransmission and implications of GSK3 inhibitors as functional selective modulators of 5-HT1BR.

Project description:BACKGROUND: Plant Glycogen Synthase Kinase 3/ SHAGGY-like kinases (GSKs) have been implicated in numerous biological processes ranging from embryonic, flower, stomata development to stress and wound responses. They are key regulators of brassinosteroid signaling and are also involved in the cross-talk between auxin and brassinosteroid pathways. In contrast to the human genome that contains two genes, plant GSKs are encoded by a multigene family. Little is known about Liliopsida resp. Poaceae in comparison to Brassicaceae GSKs. Here, we report the identification and structural characterization of two GSK homologs named TaSK1 and TaSK2 in the hexaploid wheat genome as well as a widespread phylogenetic analysis of land plant GSKs. RESULTS: Genomic and cDNA sequence alignments as well as chromosome localization using nullisomic-tetrasomic lines provided strong evidence for three expressed gene copies located on homoeolog chromosomes for TaSK1 as well as for TaSK2. Predicted proteins displayed a clear GSK signature. In vitro kinase assays showed that TaSK1 and TaSK2 possessed kinase activity. A phylogenetic analysis of land plant GSKs indicated that TaSK1 and TaSK2 belong to clade II of plant GSKs, the Arabidopsis members of which are all involved in Brassinosteroid signaling. Based on a single ancestral gene in the last common ancestor of all land plants, paralogs were acquired and retained through paleopolyploidization events, resulting in six to eight genes in angiosperms. More recent duplication events have increased the number up to ten in some lineages. CONCLUSIONS: To account for plant diversity in terms of functionality, morphology and development, attention has to be devoted to Liliopsida resp Poaceae GSKs in addition to Arabidopsis GSKs. In this study, molecular characterization, chromosome localization, kinase activity test and phylogenetic analysis (1) clarified the homologous/paralogous versus homoeologous status of TaSK sequences, (2) pointed out their affiliation to the GSK multigene family, (3) showed a functional kinase activity, (4) allowed a classification in clade II, members of which are involved in BR signaling and (5) allowed to gain information on acquisition and retention of GSK paralogs in angiosperms in the context of whole genome duplication events. Our results provide a framework to explore Liliopsida resp Poaceae GSKs functions in development.

Dataset Information

Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/?-TrCP axis.

Publications

Structural and functional characterization of Nrf2 degradation by the glycogen synthase kinase 3/β-TrCP axis.

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