Roles for βII-Protein Kinase C and RACK1 in Positive and Negative Signaling for Superoxide Anion Generation in Differentiated HL60 Cells

• Published in: The Journal of biological chemistry 2001-03, Vol.276 (12), p.8910-8917
• Main Authors: Korchak, Helen M., Kilpatrick, Laurie E.
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Base Sequence; DNA Primers; Down-Regulation; Electroporation; HL-60 Cells; Humans; Isoenzymes - metabolism; Ligands; Molecular Sequence Data; NADPH Oxidases; Oligonucleotides, Antisense - pharmacology; Peptides - antagonists & inhibitors; Peptides - metabolism; Phosphoproteins - metabolism; Precipitin Tests; Protein Kinase C - metabolism; Protein Kinase C beta; Receptors for Activated C Kinase; Recombinant Proteins - metabolism; Signal Transduction; Superoxides - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M008326200

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