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Originally published In Press as doi:10.1074/jbc.M009322200 on October 20, 2000

J. Biol. Chem., Vol. 275, Issue 52, 41000-41003, December 29, 2000
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Phorbol Ester-induced Generation of Reactive Oxygen Species Is Protein Kinase Cbeta -dependent and Required for SAPK Activation*

Rakesh DattaDagger §, Kazufumi YoshinagaDagger , Masao Kaneki, Pramod Pandey, and Donald Kufe

From the Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115

Treatment of human U-937 myeloid leukemia cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) is associated with protein kinase C (PKC) beta II-mediated activation of the stress-activated protein kinase (SAPK) pathway. The present studies demonstrate that the TPA response of U-937 cells includes the generation of reactive oxygen species (ROS). By contrast, the TPA-resistant U-937 cell variant (TUR), which is deficient in PKCbeta II expression, failed to respond to TPA with the induction of ROS. Moreover, we show that TPA-induced ROS production is restored in TUR cells stably transfected to express PKCbeta II. The results also demonstrate that TPA-induced ROS production is required for activation of the MEK kinase-1 (MEKK-1)right-arrow SAPK pathway. In concert with this observation, treatment of U-937 with H2O2 as a source of ROS is associated with activation of the MEKK-1right-arrowSAPK cascade. These findings indicate that PKCbeta II is required for TPA-induced ROS production and that the MEKK-1right-arrowSAPK pathway is activated by a ROS-mediated mechanism.

* This work was supported by National Institutes of Health United States Public Health Services Grants GM58200 (to R. D.) and CA42802 (to D. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Both authors contributed equally to this work.

§ To whom correspondence should be addressed. Tel.: 617-632-2939; Fax: 617-632-2933; E-mail: rakesh_datta@dfci.harvard.edu.

Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
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