Arkadia Mediates Sumoylation‐dependent Stabilization of Nrf2

Abstract

We have previously demonstrated that ubiquitylation of polysumoylated Nrf2 by RING finger protein 4 (RNF4), a SUMO‐targeted ubiquitin ligase (STUbL), targets Nrf2 for degradation in promyelocytic leukemia‐nuclear bodies (PML‐NBs) in HepG2 cells. Here, we show that Arkadia (also called RNF111), another SUMO‐targeted ubiquitin ligase, also ubiquitylated polysumoylated Nrf2 in these cells. The action of Arkadia stabilized Nrf2,measured in whole‐cell lysates or in PML‐NB‐enriched fractions. Using linkage (Lys 48‐ or Lys 63‐) specific ubiquitin antibodies, we show in co‐immunoprecipitation assays that Lys‐63 but not Lys‐48 is involved in Arkadia‐mediated ubiquitylation of polysumoylated Nrf2. Thus, Arkadia‐induced stabilization of Nrf2 is consistent with the notion that polyubiquitin chains linked through Lys‐63 residues do not target proteins for proteolytic destruction. Given that Arkadia has also been reported by others to ubiquitylate polysumoylated PML, thereby targeting it for proteolytic degradation, our study suggests that Arkadia functions as a dual specificity E3 ubiquitin ligase. Arkadia was able to enhance Nrf2‐mediated gene transcription from the heme‐oxygenase (HO‐1) luciferase gene construct whereas RNF4 decreased Nrf2‐mediated transcription from this reporter. RNF4 and Arkadia did not act synergistically suggesting that these two STUbLs affect Nrf2‐mediated gene transcription by two different mehanisms. Arkadia‐mediated ubiquitylation of polysumoylated Nrf2, resulting in stabilization of Nrf2, may explain the Arkadia‐induced increase in Nrf2‐dependent gene transcription.Supported by NIH grants # SC1CA143985, 5T32HL007737, 2 SD1MD000104, and ULITR000445