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

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 1 h 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. (c) 2013 Elsevier Inc. All rights reserved.