Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex

Donna D. Zhang, Shih Ching Lo, Janet V. Cross, Dennis J. Templeton, Mark Hannink

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Abstract

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of keap1-dependent ubiquitination of Nrf2 correlates with decreased association of keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between keap1 and Nrf2. Our results suggest that the ability of keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

Original languageEnglish (US)
Pages (from-to)10941-10953
Number of pages13
JournalMolecular and cellular biology
Volume24
Issue number24
DOIs
StatePublished - Dec 1 2004
Externally publishedYes

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ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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