Ubiquitination of Keap1, a BTB-Kelch Substrate Adaptor Protein for Cul3, Targets Keap1 for Degradation by a Proteasome-independent Pathway

Donna D Zhang,Shih-Ching Lo,Zheng Sun,Geetha M Habib,Michael W Lieberman,Mark Hannink

doi:10.1074/jbc.m501279200

Donna D Zhang, Shih-Ching Lo + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m501279200

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Abstract

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.

Highlights

Oxidative stress results from an imbalance between the production and removal of reactive oxygen species and has been implicated in numerous pathophysiological settings, including cancer, neurodegeneration, aging, and cardiovascular disease [1,2,3,4,5]
Ubiquitination of substrate adaptor proteins may play an important role in cellular physiology
To examine the possibility that Keap1 is ubiquitinated by a Cul3-Rbx1 complex in vitro, the Keap1-Cul3-Rbx1 complex was purified from COS-1 cells transfected with expression vectors for Keap1, Cul3, and Rbx1

Summary

Introduction

Oxidative stress results from an imbalance between the production and removal of reactive oxygen species and has been implicated in numerous pathophysiological settings, including cancer, neurodegeneration, aging, and cardiovascular disease [1,2,3,4,5]. Recent reports by several groups have demonstrated that Keap functions as a substrate adaptor protein for a Cul3-Rbx E31 ubiquitin ligase complex (19 – 22). Under conditions of homeostatic cell growth, Keap brings Nrf into the Cul3-Rbx complex and enables ubiquitin conjugation onto specific lysine residues located within the N-terminal Neh domain of Nrf2 [21]. Following exposure of cells to a wide variety of chemical inducers of Nrf2-dependent transcription, Keap1-dependent ubiquitination of Nrf is blocked, enabling Nrf to accumulate in the nucleus and activate expression of Nrf2-dependent genes [21, 24]. The molecular definition of Keap as a substrate adaptor protein for Cul provides a conceptual framework for understanding how Keap1-dependent ubiquitination of Nrf is regulated. Cullin-based E3 ubiquitin ligases typically catalyze the addition of a multiubiquitin chain onto the substrate protein and thereby target the substrate protein for proteasome-mediated degradation.

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