Roles for βII-protein kinase C and RACK1 in positive and negative signaling for superoxide anion generation in differentiated HL60 cells

beta -Protein kinase (PKC)is essential for ligand-initiate. assembly of the NADPH oxidase far generation of superoxide anion (O-2(radical anion). Neutrophils and neutrophilic HL60 cells contain both betaI and beta II-PKC, isotypes that are derived by alternate splicing. betaI-PKC-positive and betaI-PKC null HL60 cells generated equivalent amounts of O-2(radical anion) in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of beta II-PKC from betaI-PKC null cells inhibited ligand-initiated O-2(radical anion) generation, fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47(phox), 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-2(radical anion) generation. Therefore, beta II-PKC but not betaI-PKC is essential for activation of O-2(radical anion) generation and plays a positive role in signaling for NADPH oxidase activation in association with p47(phox). In contrast; RACK1 is involved in negative signaling for O-2(radical anion) generation. RACK1 binds to beta II-PKC but not with the p47(phox). 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-2(radical anion) generation.