Original Article

Hydrogen Peroxide Inhibition of Nuclear Protein Import Is Mediated by the Mitogen-activated Protein Kinase, ERK2

Correspondence to: Grant N. Pierce, Division of Stroke and Vascular Disease, St. Boniface General Hospital Research Centre, 351 Tache Avenue, Winnipeg, Manitoba, Canada R2H 2A6. Tel:(204) 235-3414 Fax:(204) 231-1151 E-mail:grant_pierce{at}sbrc.umanitoba.ca.

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.

Key Words: phosphorylation, confocal microscopy, permeablized cells, free radicals, signal transduction

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

• Faustino, R. S., Cheung, P., Richard, M. N., Dibrov, E., Kneesch, A. L., Deniset, J. F., Chahine, M. N., Lee, K., Blackwood, D., Pierce, G. N. (2008). Ceramide regulation of nuclear protein import. J. Lipid Res. 49: 654-662 [Abstract] [Full Text]  
• Kelley, J. B., Paschal, B. M. (2007). Hyperosmotic Stress Signaling to the Nucleus Disrupts the Ran Gradient and the Production of RanGTP. Mol. Biol. Cell 18: 4365-4376 [Abstract] [Full Text]  
• Richard, M. N., Deniset, J. F., Kneesh, A. L., Blackwood, D., Pierce, G. N. (2007). Mechanical Stretching Stimulates Smooth Muscle Cell Growth, Nuclear Protein Import, and Nuclear Pore Expression through Mitogen-activated Protein Kinase Activation. J. Biol. Chem. 282: 23081-23088 [Abstract] [Full Text]  
• Faustino, R. S., Stronger, L. N. W., Richard, M. N., Czubryt, M. P., Ford, D. A., Prociuk, M. A., Dibrov, E., Pierce, G. N. (2007). RanGAP-Mediated Nuclear Protein Import in Vascular Smooth Muscle Cells Is Augmented by Lysophosphatidylcholine. Mol. Pharmacol. 71: 438-445 [Abstract] [Full Text]  
• Miyamoto, Y., Saiwaki, T., Yamashita, J., Yasuda, Y., Kotera, I., Shibata, S., Shigeta, M., Hiraoka, Y., Haraguchi, T., Yoneda, Y. (2004). Cellular stresses induce the nuclear accumulation of importin {alpha} and cause a conventional nuclear import block. J. Cell Biol. 165: 617-623 [Abstract] [Full Text]  
• Furuta, M., Kose, S., Koike, M., Shimi, T., Hiraoka, Y., Yoneda, Y., Haraguchi, T., Imamoto, N. (2004). Heat-shock induced nuclear retention and recycling inhibition of importin {alpha}. GENES CELLS 9: 429-441 [Abstract] [Full Text]  
• Zettler, M. E., Prociuk, M. A., Austria, J. A., Zhong, G., Pierce, G. N. (2004). Oxidized Low-Density Lipoprotein Retards the Growth of Proliferating Cells by Inhibiting Nuclear Translocation of Cell Cycle Proteins. Arterioscler. Thromb. Vasc. Bio. 24: 727-732 [Abstract] [Full Text]  
• Gao, H., Sumanaweera, N., Bailer, S. M., Stochaj, U. (2003). Nuclear Accumulation of the Small GTPase Gsp1p Depends on Nucleoporins Nup133p, Rat2p/Nup120p, Nup85p, Nic96p, and the Acetyl-CoA Carboxylase Acc1p. J. Biol. Chem. 278: 25331-25340 [Abstract] [Full Text]  
• Zettler, M. E., Prociuk, M. A., Austria, J. A., Massaeli, H., Zhong, G., Pierce, G. N. (2003). OxLDL stimulates cell proliferation through a general induction of cell cycle proteins. Am. J. Physiol. Heart Circ. Physiol. 284: H644-H653 [Abstract] [Full Text]  
• Whitehurst, A. W., Wilsbacher, J. L., You, Y., Luby-Phelps, K., Moore, M. S., Cobb, M. H. (2002). ERK2 enters the nucleus by a carrier-independent mechanism. Proc. Natl. Acad. Sci. USA 99: 7496-7501 [Abstract] [Full Text]  
• Kehlenbach, R. H., Gerace, L. (2000). Phosphorylation of the Nuclear Transport Machinery Down-regulates Nuclear Protein Import in Vitro. J. Biol. Chem. 275: 17848-17856 [Abstract] [Full Text]  
• Lam, M. H. C., Michell, B. J., Fodero-Tavoletti, M. T., Kemp, B. E., Tonks, N. K., Tiganis, T. (2001). Cellular Stress Regulates the Nucleocytoplasmic Distribution of the Protein-tyrosine Phosphatase TCPTP. J. Biol. Chem. 276: 37700-37707 [Abstract] [Full Text]  

Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents