Erk activation is required for Nrf2 nuclear localization during pyrrolidine dithiocarbamate induction of glutamate cysteine ligase modulatory gene expression in HepG2 cells

Pyrrolidine dithiocarbamate (PDTC) induction of the human glutamate cysteine ligase modulatory (GCLM) gene is dependent on activation of the mitogen-activated protein kinases (MAPKs) extracellular regulated kinase (Erk) and p38, and is not affected by protein kinase C (PKC) or PI3K inhibitors. Nrf2 binding to the electrophile response element (EpRE) located within the GCLM promoter is decreased after MAPK inhibition, suggesting that Nrf2 could be a downstream target of activated MAPK. To evaluate this hypothesis, a series of Nrf2 proteins harboring mutations in conserved consensus MAPK phosphorylation sites were developed and used in multiple functional assays. All mutated Nrf2 proteins tested interacted with the cytoplasmic repressor Keap1 in a manner indistinguishable from wild-type Nrf2. Furthermore, the mutant and wild-type Nrf2 proteins were similarly capable of transactivating an EpRE-containing GCLM/luciferase reporter transgene. Collectively these functional assays suggest that Nrf2 is not likely to be a direct downstream target of activated MAPK in vivo. However, treatment of HepG2 cells with MAPK inhibitors PD98059 and/or SB202190 prior to exposure to PDTC, reduced Nrf2 translocation to the nucleus, suggesting that MAPK-directed phosphorylation is a requirement for nuclear localization during PDTC induction of GCLM gene expression.