A regulatory role for ADP-ribosylation factor 6 (ARF6) in activation of the phagocyte NADPH oxidase.
In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2, 3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.
Laboratory of Host Defenses and the Flow Cytometry Section, NIAID, National Institutes of Health, Bethesda, Maryland 20892, USA.
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D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors; D003994:Bucladesine / Q000494:pharmacology; D002454:Cell Differentiation; D002460:Cell Line; D004789:Enzyme Activation; D005434:Flow Cytometry; D006098:Granulocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D006801:Humans; D008163:Luminescent Measurements; D008562:Membrane Glycoproteins / Q000032:analysis / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016897:Respiratory Burst; D014162:Transfection
D000089982:ADP-Ribosylation Factor 6; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D003994:Bucladesine; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human