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Originally published In Press as doi:10.1074/jbc.M008326200 on December 18, 2000

J. Biol. Chem., Vol. 276, Issue 12, 8910-8917, March 23, 2001
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Roles for beta II-Protein Kinase C and RACK1 in Positive and Negative Signaling for Superoxide Anion Generation in Differentiated HL60 Cells*

Helen M. KorchakDagger and Laurie E. Kilpatrick

From the Departments of Pediatrics and Biochemistry/Biophysics, University of Pennsylvania School of Medicine, The Joseph Stokes, Jr. Research Institute of the Children's Hospital of Philadelphia, Philadelphia, Pennsylvania 19104

beta -Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O&cjs1138;2). Neutrophils and neutrophilic HL60 cells contain both beta I and beta II-PKC, isotypes that are derived by alternate splicing. beta I-PKC-positive and beta I-PKC null HL60 cells generated equivalent amounts of O&cjs1138;2 in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of beta II-PKC from beta I-PKC null cells inhibited ligand-initiated O&cjs1138;2 generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47phox, with beta II-PKC but not with RACK1, a binding protein for beta II-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of beta -PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O&cjs1138;2 generation. Therefore, beta II-PKC but not beta I-PKC is essential for activation of O&cjs1138;2 generation and plays a positive role in signaling for NADPH oxidase activation in association with p47phox. In contrast, RACK1 is involved in negative signaling for O&cjs1138;2 generation. RACK1 binds to beta II-PKC but not with the p47phox·beta II-PKC complex. RACK1 may divert beta II-PKC to other signaling pathways requiring beta -PKC for signal transduction. Alternatively, RACK1 may sequester beta II-PKC to down-regulate O&cjs1138;2 generation.

* This work was supported by National Institutes of Health Grant AI 24840.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 To whom correspondence should be addressed: Immunology Section, Room 1208C Abramson Bldg., Children's Hospital of Philadelphia, 34th and Civic Center Blvd., Philadelphia, PA 19104. Tel.: 215-590-2136; Fax: 610-525-1190; E-mail: korchak@email.chop.edu.

Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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