A novel assay system implicates PtdIns(3,4)P(2), PtdIns(3)P, and PKC delta in intracellular production of reactive oxygen species by the NADPH oxidase

Activated neutrophils assemble an NADPH oxidase enzyme complex to produce superoxide for microbial killing. Much of the initial oxidase assembly occurs on intracellular granules, followed by movement of the oxidase to phagolysosomes and the plasma membrane. We have developed a novel assay system usi...

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Bibliographic Details
Published in:Molecular cell 2003-01, Vol.11 (1), p.35
Main Authors: Brown, Glenn E, Stewart, Mary Q, Liu, Hui, Ha, Vi-Luan, Yaffe, Michael B
Format: Article
Language:English
Subjects:
Adenosine Triphosphate - metabolism
Bacterial Proteins
Cell Fractionation
Cytoplasm - metabolism
Enzyme Inhibitors - pharmacology
Guanosine 5'-O-(3-Thiotriphosphate) - metabolism
Humans
NADPH Oxidases - metabolism
Neutrophil Activation - physiology
Neutrophils - drug effects
Neutrophils - metabolism
Neutrophils - ultrastructure
Phosphatidylinositol 3-Kinases - metabolism
Phosphatidylinositol Phosphates - metabolism
Phosphoproteins - metabolism
Protein Kinase C - metabolism
Protein Kinase C-delta
Protein Transport - physiology
Reactive Oxygen Species - metabolism
Signal Transduction - physiology
Streptolysins - metabolism
Tetradecanoylphorbol Acetate - pharmacology
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Summary:Activated neutrophils assemble an NADPH oxidase enzyme complex to produce superoxide for microbial killing. Much of the initial oxidase assembly occurs on intracellular granules, followed by movement of the oxidase to phagolysosomes and the plasma membrane. We have developed a novel assay system using Streptolysin-O permeabilized neutrophils that recapitulates the initial intracellular activation process while maintaining the ultrastructural features of this granulocytic cell type. Using this system, we biochemically dissect molecular events and signaling pathways involved in NADPH oxidase assembly and demonstrate specific roles for PKC delta, PI(3,4)P(2)/PI(3,4,5)P(3), and PI(3)P in the PMA-dependent intracellular activation process. This system should be of great utility for the study of cell signaling events that regulate the intracellular production of reactive oxygen species by neutrophils.
ISSN:1097-2765