Cells respond to oxidants and electrophiles by activating receptor/transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) to coordinate the induction of cytoprotective genes critical for defense against oxidative and other stresses. Activation involves blocking the ubiquitination-proteasomal degradation of Nrf2. Modification of cysteine thiol groups by inducers in the linker region of Kelch-like ECH-associated protein-1 (Keap1), which congregates Nrf2 into the Keap1/Cul3 E3 complex for ubiquitination, is important but not sufficient for activation of Nrf2. Here we show that evolutionarily conserved cysteine residues of Nrf2 are critical for Nrf2 regulation. FlAsH (an arsenic-based fluorophore) and phenylarsine oxide (PAO) potently induce Nrf2 target genes and bind to Nrf2 in vitro and in vivo. Binding is inhibited by prototypical inducers arsenic and tert-butylhydroquinone. PAO affinity pull-down and mutation of individual cysteine to alanine reveal that Cys235, Cys311, Cys316, Cys414, and Cys506 are critical for binding, and binding is modulated by intramolecular interactions. To corroborate the functions of cysteine residues, Nrf2 wild-type or mutants are expressed in Nrf2 knockout cells to reconstitute Nrf2 regulation. Nrf2 mutants have reduced t(1/2) that inversely correlates with increased binding to Keap1 and polyubiquitination of mutant proteins. It is remarkable that the mutants fail to respond to arsenic for Nrf2 activation and gene induction. Furthermore, mutations at Cys119, Cys235, and Cys506 impede binding of Nrf2 to endogenous antioxidant response element and to coactivator cAMP response element-binding protein-binding protein/p300. The findings demonstrate that Nrf2 cysteine residues critically regulate oxidant/electrophile sensing, repress Keap1-dependent ubiquitination-proteasomal degradation, and promote recruitment of coactivators, such that chemical sensing, receptor activation, and transcription activation are integrated at the receptor molecule.
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