Abstract

Cells respond to oxidants and electrophiles by activating receptor/transcription factor Nrf2 to coordinate 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 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 evolutionally 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 tBHQ. PAO affinity pulldown and mutation of individual cysteine to alanine reveal that C235, C311, C316, C414, and C506 are critical for binding and binding is modulated by intra-molecular 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. Remarkably, the mutants fail to respond to arsenic for Nrf2 activation and gene induction. Furthermore, mutations at C119, C235, and C506 impede binding of Nrf2 to endogenous ARE and to coactivator CBP/p300. The findings demonstrate that Nrf2 cysteine residues critically regulate oxidant/electrophile sensing, repress Keap1-dependent ubiquitination-proteasomal degradation, and promote recruitment of co-activators, such that chemical sensing, receptor activation, and transcription activation are integrated at the receptor molecule.