Dual targeting of Keap1 and Gsk-3 by hexaraphane in the regulation of transcription factor Nrf2

6-(Methylsulfinyl)hexyl isothiocyanate, here referred to as hexaraphane, is a bioactive compound found in wasabi, which exhibits cytoprotective, anti-inflammatory, and chemopreventive properties. The beneficial effects of hexaraphane are largely mediated through derepression of transcription factor Nrf2, which is typically repressed via proteasomal degradation mediated by its two-site interaction with the ubiquitin E3 ligase adapter Keap1. Like other isothiocyanates, hexaraphane increases Nrf2 activity by perturbing the Nrf2-Keap1 interaction and stalling Keap1-directed ubiquitination. However, we found that hexaraphane modestly increases Nrf2 levels in Keap1-deficient cells, suggesting an additional Keap1-independent mechanism. Unlike other electrophilic Nrf2 activators, hexaraphane did not significantly impact Erk, p38^Mapk, Jnk, or Pten/Pi3k/Akt signaling. Instead, our data reveal that hexaraphane inhibits Gsk-3β, a kinase that targets Nrf2 for proteasomal degradation by phosphorylating a DSGIS degron in the transcription factor through which it interacts with the β-TrCP E3 ligase adaptor. Hexaraphane reduced Nrf2 ubiquitination and its binding to Gsk-3β, mimicking the effects of the Gsk-3β inhibitor SB216763. In vitro kinase assays confirmed that hexaraphane suppresses Nrf2 phosphorylation. Furthermore, simulations of molecular docking and dynamics predict a specific interaction between hexaraphane and the catalytic groove of Gsk-3β. These findings identify hexaraphane as a previously unrecognized dual-acting Nrf2 activator that stabilizes Nrf2 both by disturbing Keap1 binding and by inhibiting Gsk-3β that forms the DSGIpS³³⁸ phosphodegron.