Hydrogen peroxide inhibition of nuclear protein import is mediated by the mitogen-activated protein kinase, ERK2

Abstract

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.