| The Glutamate Cysteine Ligase (GCL) holoenzyme catalyzes the first and rate-limiting step in Glutathione synthesis and is composed of a catalytic (GCLC) and modulatory (GCLM) subunit. GCLM and GCLC are transcriptionally regulated through Electrophile/Antioxidant Response Elements (EpREs/AREs) located in the promoters of each gene. The transcription factor Nrf2, which is sequestered in the cytoplasm by the cytoskeletal binding protein Keap1, binds and transactivates through the ARE sequence. Exposure to an oxidative/electrophilic stress, releases Nrf2 from Keap1 cytoplasmic repression and results in Nrf2 nuclear translocation. Inhibition of Erk1&2 or p38 decreased Pyrrolidine Dithiocarbamate (PDTC) induction of GCL subunit genes by ∼50%, whereas simultaneous inhibition eliminated induction. Inhibition of Erk and/or p38 pathways also decreased Nrf2:ARE binding suggesting Erk or p38 phosphorylation event(s) were required for Nrf2 binding, perhaps by direct phosphorylation of Nrf2. Mutation of conserved MAPK consensus phosphorylation sites (S/T-P) in Nrf2 did not effect Nrf2 transactivation of a GCLM-ARE luciferase transgene or Nrf2:Keap1 interaction. Inhibition of Erk decreased PDTC induced Nrf2 nuclear localization. To determine if Erk phosphorylation of Keap1 was involved in Nrf2 release, we mutated the potential MAPK phosphorylation sites in Keap1. Keap1-S104A mutants did not sequester Nrf2 in the cytoplasm and were unable to repress Nrf2 transactivation. S104 is a highly conserved serine which lies in the BTB domain of Keap1, a domain involved in dimerization and multimerization. Wild-type Keap1 proteins were able to form homodimers and higher order complexes both in vitro and in vivo, however Keap1-S104A proteins were inefficient at complex formation. The correlation between inefficient dimerization and lack of Nrf2 cytoplasmic retention suggested Keap1 dimerization is involved in Nrf2 sequestration. Exposure to PDTC disrupted formation of Keap1-containing complexes independent of Erk and p38 activation. The functional changes associated with the Keap1-S104A mutant were not due to loss of an Erk/p38 phosphorylation site, but rather due to inefficient dimerization/complex formation resulting from BTB domain mutation. Collectively, our data are consistent with a model in which the BTB dimerization function of Keap1 is involved in Nrf2 sequestration, and disruption of dimerization after oxidant/electrophile exposure correlates with Nrf2 release and nuclear translocation. |
