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Activation of the transcription factor Nrf2 regulates expression of phase II enzymes and other adaptive responses to electrophile and oxidant stress. Nrf2 concentrations are regulated by the thiol-rich sensor protein Keap1, which is an adaptor protein for Cul3-dependent ubiquitination and degradation of Nrf2. However, the links between site specificity of Keap1 modification by electrophiles and mechanisms of Nrf2 activation are poorly understood. We studied the actions of the prototypical Nrf2 inducer tert-butylhydroquinone (tBHQ) and two biotin-tagged, thiol-reactive electrophiles, N-iodoacetyl-N-biotinylhexylenediamine (IAB) and 1-biotinamido-4-(4'-[maleimidoethyl-cyclohexane]-carboxamido)butane (BMCC). Both IAB and tBHQ induced antioxidant response element (ARE)-directed green fluorescent protein (GFP) expression in ARE/thymidine kinase GFP HepG2 cells, and both initiated nuclear Nrf2 accumulation and induction of heme oxygenase 1 in HEK293 cells. In contrast, BMCC produced none of these effects. Liquid chromatography tandem mass spectrometry (MS-MS) analysis of human Keap1 modified by IAB or BMCC in vitro indicated that IAB adduction occurred primarily in the central linker domain, whereas BMCC modified other Keap1 domains. Treatment of FLAG-Keap1-transfected HEK293 with the Nrf2-activating compounds IAB and tBHQ generated high molecular weight Keap1 forms, which were identified as K-48-linked polyubiquitin conjugates by immunoblotting and liquid chromatography MS-MS. Keap1 polyubiquitination coincided with Nrf2 stabilization and nuclear accumulation. In contrast, BMCC did not induce Keap1 polyubiquitination. Our results suggest that Nrf2 activation is regulated through the polyubiquitination of Keap1, which in turn is triggered by specific patterns of electrophile modification of the Keap1 central linker domain. These results suggest that Keap1 adduction triggers a switching of Cul3-dependent ubiquitination from Nrf2 to Keap1, leading to Nrf2 activation.