Diffusion dynamics of the Keap1-Cullin3 interaction in single live cells

Biochem Biophys Res Commun. 2013 Mar 29;433(1):58-65. doi: 10.1016/j.bbrc.2013.02.065. Epub 2013 Feb 26.


Transcription factor NF-E2 p45-related factor 2 (Nrf2) regulates the expression of a network of genes encoding drug-detoxification, anti-inflammatory, and metabolic enzymes, as well as proteins involved in the regulation of cellular redox homeostasis. Under basal conditions, Kelch-like ECH associated protein 1 (Keap1) targets Nrf2 for ubiquitination and proteasomal degradation via association with Cullin3 (Cul3)-based Rbx1 E3 ubiquitin ligase. Various small molecules (inducers) activate Nrf2 leading to upregulation of cytoprotective gene expression. Inducers chemically modify specific cysteine residues of Keap1 which ultimately loses its ability to target Nrf2 for degradation. Dissociation of the Keap1-Cul3 complex by inducers is one possible mechanism, but evidence in single live cells is lacking. To investigate the diffusion dynamics of the Keap1-Cul3 interaction and the effect of inducers, we developed a quantitative fluorescence recovery after photobleaching (FRAP)-based system using Keap1-EGFP and mCherry-Cul3 fusion proteins. We show that Keap1-EGFP and mCherry-Cul3 interact in single live cells. Exposure for 1h to small-molecule inducers of 4 different types, the oleanane triterpenoid CDDO, the isothiocyanate sulforaphane, the sulfoxythiocarbamate STCA, and the oxidant hydrogen peroxide which target distinct cysteine sensors within Keap1 with potencies which differ by nearly 4000-fold, does not dissociate the Keap1-Cul3 complex. As inducers cause conformational changes in Keap1, we conclude that changes in conformation rather than dissociation from Cul3 inactivate the repressor function of Keap1 leading to Nrf2 stabilization.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Carrier Proteins / metabolism
  • Cullin Proteins / chemistry
  • Cullin Proteins / genetics
  • Cullin Proteins / metabolism*
  • Facilitated Diffusion
  • Fluorescence Recovery After Photobleaching
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • HEK293 Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Kelch-Like ECH-Associated Protein 1
  • Kinetics
  • Luminescent Proteins / chemistry
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Multiprotein Complexes / chemistry
  • Multiprotein Complexes / metabolism
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism
  • Protein Conformation
  • Protein Stability
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Red Fluorescent Protein
  • Ubiquitination


  • CUL3 protein, human
  • Carrier Proteins
  • Cullin Proteins
  • Intracellular Signaling Peptides and Proteins
  • KEAP1 protein, human
  • Kelch-Like ECH-Associated Protein 1
  • Luminescent Proteins
  • Multiprotein Complexes
  • NF-E2-Related Factor 2
  • NFE2L2 protein, human
  • RBX1 protein, human
  • Recombinant Fusion Proteins
  • enhanced green fluorescent protein
  • Green Fluorescent Proteins