Phosphatidic acid and diacylglycerol directly activate NADPH oxidase by interacting with enzyme components.
The enzyme NADPH oxidase is regulated by phospholipase D in intact neutrophils and is activated by phosphatidic acid (PA) plus diacylglycerol (DG) in cell-free systems. We showed previously that cell-free NADPH oxidase activation by these lipids involves both protein kinase-dependent and -independent pathways. Here we demonstrate that only the protein kinase-independent pathway is operative in a cell-free system of purified and recombinant NADPH oxidase components. Activation by PA + DG was ATP-independent and unaffected by the protein kinase inhibitor staurosporine, indicating the lack of protein kinase involvement. Both PA and DG were required for optimal activation to occur. The drug reduced activation of NADPH oxidase by either arachidonic acid or PA + DG, with IC(50) values of 46 and 25 microm, respectively. The optimal concentration of arachidonic acid or PA + DG for oxidase activation was shifted to the right with, indicating interference of the drug with the interaction of lipid activators and enzyme components. inhibited the lipid-induced aggregation/sedimentation of oxidase components p47(phox) and p67(phox), suggesting a disruption of the lipid-mediated assembly process. The direct effects of on NADPH oxidase activation complicate its use as a "specific" inhibitor of DG kinase. We conclude that the protein kinase-independent pathway of NADPH oxidase activation by PA and DG involves direct interaction with NADPH oxidase components. Thus, NADPH oxidase proteins are functional targets for these lipid messengers in the neutrophil.
Palicz
A
A
Department of Infectology and Pediatric Immunology, Medical and Health Science Center, University of Debrecen, H-4012, Debrecen, Nagyerdei krt.98, Hungary.
Foubert
T R
TR
Jesaitis
A J
AJ
Marodi
L
L
McPhail
L C
LC
D002474:Cell-Free System; D004075:Diglycerides / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D066298:In Vitro Techniques; D019255:NADPH Oxidases / Q000187:drug effects / Q000378:metabolism*; D009504:Neutrophils / Q000201:enzymology / Q000378:metabolism; D010712:Phosphatidic Acids / Q000494:pharmacology*; D010750:Phosphoproteins / Q000737:chemistry; D010880:Piperidines / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D052999:Quinazolinones; D011994:Recombinant Proteins / Q000737:chemistry
D004075:Diglycerides; D010712:Phosphatidic Acids; D010750:Phosphoproteins; D010880:Piperidines; D011799:Quinazolines; D052999:Quinazolinones; D011994:Recombinant Proteins; C079789:neutrophil cytosol factor 67K; C058544:R 59949; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1