Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway

Keap1 is a BTB-Kelch protein that functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 targets its substrate, the Nrf2 transcription factor, for ubiquitination and subsequent degradation by the 26 S proteasome. Inhibition of Keap1-dependent ubiquitination of Nrf2 increases steady-state levels of Nrf2 and enables activation of cytoprotective Nrf2-dependent genes. In this report, we demonstrate that Keap1 and three other BTB-Kelch proteins, including GAN1, ENC1, and Sarcosin, are ubiquitinated by a Cul3-dependent complex. Ubiquitination of Keap1 is markedly increased in cells exposed to quinone-induced oxidative stress, occurs in parallel with inhibition of Keap1-dependent ubiquitination of Nrf2, and results in decreased steady-state levels of Keap1, particularly in cells that are unable to synthesize glutathione. Degradation of Keap1 is independent of the 26 S proteasome, because inhibitors of the 26 S proteasome do not prevent loss of Keap1 following exposure of cells to quinone-induced oxidative stress. Our results suggest that a switch from substrate to substrate adaptor ubiquitination is a critical regulatory step that controls steady-state levels of both BTB-Kelch substrate adaptor proteins and their cognate substrates.