Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components

Anita Palicz, Thomas R. Foubert, Algirdas J. Jesaitis, L. Máródi, Linda C. McPhail

Research output: Contribution to journalArticle

90 Citations (Scopus)

Abstract

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 R59949 reduced activation of NADPH oxidase by either arachidonic acid or PA + DG, with IC50 values of 46 and 25 μM, respectively. The optimal concentration of arachidonic acid or PA + DG for oxidase activation was shifted to the right with R59949, indicating interference of the drug with the interaction of lipid activators and enzyme components. R59949 inhibited the lipid-induced aggregation/sedimentation of oxidase components p47phox and p67phox, suggesting a disruption of the lipid-mediated assembly process. The direct effects of R59949 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.

Original languageEnglish
Pages (from-to)3090-3097
Number of pages8
JournalJournal of Biological Chemistry
Volume276
Issue number5
DOIs
Publication statusPublished - Feb 2 2001

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Phosphatidic Acids
NADPH Oxidase
Diglycerides
R 59949
Chemical activation
Enzymes
Protein Kinases
Lipids
Cell-Free System
Arachidonic Acid
Oxidoreductases
Neutrophils
Enzyme Activators
Diacylglycerol Kinase
Phospholipase D
Staurosporine
Protein Kinase Inhibitors
Drug Interactions
Sedimentation
Pharmaceutical Preparations

ASJC Scopus subject areas

  • Biochemistry

Cite this

Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components. / Palicz, Anita; Foubert, Thomas R.; Jesaitis, Algirdas J.; Máródi, L.; McPhail, Linda C.

In: Journal of Biological Chemistry, Vol. 276, No. 5, 02.02.2001, p. 3090-3097.

Research output: Contribution to journalArticle

Palicz, Anita ; Foubert, Thomas R. ; Jesaitis, Algirdas J. ; Máródi, L. ; McPhail, Linda C. / Phosphatidic Acid and Diacylglycerol Directly Activate NADPH Oxidase by Interacting with Enzyme Components. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 5. pp. 3090-3097.
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