SCF/β-TrCP Promotes Glycogen Synthase Kinase 3-Dependent Degradation of the Nrf2 Transcription Factor in a Keap1-Independent Manner

Abstract

Regulation of transcription factor Nrf2 (NF-E2-related factor 2) involves redox-sensitive proteasomal degradation via the E3 ubiquitin ligase Keap1/Cul3. However, Nrf2 is controlled by other mechanisms that have not yet been elucidated. We now show that glycogen synthase kinase 3 (GSK-3) phosphorylates a group of Ser residues in the Neh6 domain of mouse Nrf2 that overlap with an SCF/β-TrCP destruction motif (DSGIS, residues 334 to 338) and promotes its degradation in a Keap1-independent manner. Nrf2 was stabilized by GSK-3 inhibitors in Keap1-null mouse embryo fibroblasts. Similarly, an Nrf2^ΔETGE mutant, which cannot be degraded via Keap1, accumulated when GSK-3 activity was blocked. Phosphorylation of a Ser cluster in the Neh6 domain of Nrf2 stimulated its degradation because a mutant Nrf2^{ΔETGE 6S/6A} protein, lacking these Ser residues, exhibited a longer half-life than Nrf2^ΔETGE. Moreover, Nrf2^{ΔETGE 6S/6A} was insensitive to β-TrCP regulation and exhibited lower levels of ubiquitination than Nrf2^ΔETGE. GSK-3β enhanced ubiquitination of Nrf2^ΔETGE but not that of Nrf2^{ΔETGE 6S/6A}. The Nrf2^ΔETGE protein but not Nrf2^{ΔETGE 6S/6A} coimmunoprecipitated with β-TrCP, and this association was enhanced by GSK-3β. Our results show for the first time that Nrf2 is targeted by GSK-3 for SCF/β-TrCP-dependent degradation. We propose a “dual degradation” model to describe the regulation of Nrf2 under different pathophysiological conditions.

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ACKNOWLEDGMENTS

This work was supported by grants SAF2007-62646 and SAF2010-17822 from the Spanish Ministry of Science and Innovation (awarded to A.C.), by grants C4909/A5942 and C4909/A9990 from Cancer Research UK (awarded to J.D.H.), and by a grant from Tenovus Scotland (awarded to M.M. and J.D.H.). P.R. is a recipient of a Formación de Profesorado Universitario fellowship from the Spanish Ministry of Science and Innovation.

We thank Ronald T. Hay, Calum Sutherland, and Lindsay A. Allan for valuable discussions.