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Background/Aims: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a basic leucine-zipper transcription factor essential for cellular responses to oxidative stress. Degradation of Nrf2 in the cytoplasm, mediated by Keap1-Cullin3/RING box1 (Cul3-Rbx1) E3 ubiquitin ligase and the proteasome, is considered the primary pathway controlling the cellular abundance of Nrf2. Although the nucleus has been implicated in the degradation of Nrf2, little information is available on how this compartment participates in degrading Nrf2. Methods: Here, we fused the photoconvertible fluorescent protein Dendra2 to Nrf2 and capitalized on the irreversible change in color (green to red) that occurs when Dendra2 undergoes photoconversion to study degradation of Dendra2-Nrf2 in single live cells. Results: Using this approach, we show that the half-life (t1/2) of Dendra2-Nrf2 in the whole cell, under homeostatic conditions, is 35 min. Inhibition of the proteasome with MG-132 or induction of oxidative stress with tert-butylhydroquinone (tBHQ) extended the half-life of Dendra2-Nrf2 by 6- and 28-fold, respectively. By inhibiting nuclear export using Leptomycin B, we provide direct evidence that degradation of Nrf2 also occurs in the nucleus and involves PML-NBs (Promyelocytic Leukemia-nuclear bodies). We further demonstrate that co-expression of Dendra2-Nrf2 and Crimson-PML-I lacking two PML-I sumoylation sites (K65R and K490R) changed the decay rate of Dendra2-Nrf2 in the nucleus and stabilized the nuclear derived Nrf2 levels in whole cells. Conclusion: Altogether, our findings provide direct evidence for degradation of Nrf2 in the nucleus and suggest that modification of Nrf2 in PML nuclear bodies contributes to its degradation in intact cells.

Background/Aims: The transcription factor Nrf2 is a master regulator of the antioxidant defense system, protecting cells from oxidative damage. We previously reported that the SUMO-targeted E3 ubiquitin ligase (STUbL), RING finger protein 4 (RNF4) accelerated the degradation rate of Nrf2 in promyelocytic leukemia-nuclear body (PML-NB)-enriched fractions and decreased Nrf2-mediated gene transcription. The mechanisms that regulate Nrf2 nuclear levels are poorly understood. In this study, we aim to explore the role of the second mammalian STUbL, Arkadia/RNF111 on Nrf2. Methods: Arkadia mediated ubiquitination was detected using co-immunoprecipitation assays in which whole cell lysates were immunoprecipated with anti-Nrf2 antibody and Western blotted with anti-hemagglutinin (HA) antibody or anti-Lys-48 ubiquitin-specific antibody. The half-life of Nrf2 was detected in whole cell lysates and promyelocytic leukemia-nuclear body enriched fractions by cycloheximide-chase. Reporter gene assays were performed using the antioxidant response element (ARE)-containing promoter Heme oxygenase-1 (HO-1). Results: We show that Arkadia/RNF111 is able to ubiquitinate Nrf2 resulting in the stabilization of Nrf2. This stabilization was mediated through Lys-48 ubiquitin chains, contrary to traditionally degradative role of Lys-48 ubiquitination, suggesting that Lys-48 ubiquitination of Nrf2 protects Nrf2 from degradation thereby allowing Nrf2-dependent gene transcription. Conclusion: Collectively, these findings highlight a novel mechanism to positively regulate nuclear Nrf2 levels in response to oxidative stress through Arkadia-mediated K48-linked ubiquitination of Nrf2.