Hydrogen Peroxide Inhibition of Nuclear Protein Import Is Mediated by the Mitogen-Activated Protein Kinase, Erk2

H₂O₂ alters gene expression in many cell types. Alterations in nuclear import of transcription factors or similar key proteins may be responsible for these changes. To investigate this possibility, a cytosolic nuclear import cocktail was treated with varying [H₂O₂] and used in import assays. H₂O₂ caused a dose- and time-dependent inhibition of import at concentrations as low as 100 μM. Catalase reversed this effect. H₂O₂ treatment of permeablized cells did not affect import, suggesting that H₂O₂ was acting on a cytosolic factor. Treatment of import cocktail with two different free radical generating systems had no effect, but treatment of permeablized cells inhibited import, suggesting H₂O₂ works via a distinct process from hydroxyl or superoxide radicals. Pretreatment of import cocktail with genistein reversed the effect of H₂O₂ on import. Western blotting revealed that H₂O₂ activated ERK2. The specific MEK1/2 inhibitor, PD98059, completely blocked the effects of H₂O₂ on import. Activated ERK2 mimicked H₂O₂'s effect on import. Immunocytochemistry revealed that H₂O₂ treatment of whole cells increased cytosolic Ran/TC4 levels, an effect reversible by catalase or PD98059. These data demonstrate that H₂O₂ inhibits nuclear protein import and that this effect is mediated by mitogen-activated protein (MAP) kinase activation, possibly by altering Ran/TC4 function.