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 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.
Brown
Glenn E
GE
Center for Cancer Research, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
Stewart
Mary Q
MQ
Liu
Hui
H
Ha
Vi-Luan
VL
Yaffe
Michael B
MB
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins; D002458:Cell Fractionation; D003593:Cytoplasm / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism*; D018375:Neutrophil Activation / Q000502:physiology; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D021381:Protein Transport / Q000502:physiology; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D013301:Streptolysins / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology
D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D017382:Reactive Oxygen Species; D013301:Streptolysins; C079789:neutrophil cytosol factor 67K; C060980:phosphatidylinositol 3,4-diphosphate; C025964:streptolysin O; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D000255:Adenosine Triphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D019869:Phosphatidylinositol 3-Kinases; C497258:PRKCD protein, human; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D013755:Tetradecanoylphorbol Acetate