Abstract
Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosolic factors including p47phox, p67phox and p21rac2. We compared NADPH oxidase activity with the membrane translocation of p47phox, p67phox, and p21rac2. In a cell-free system, GTP gamma S stimulated translocation of p47phox and p67phox to the plasma membrane only in the presence of arachidonate, and this translocation correlated with NADPH oxidase activity of the reisolated plasma membranes (R = 0.94 and 0.97, respectively). In contrast, GTP gamma S-stimulated p21rac2 translocation with or without arachidonate, and the extent of translocation did not correlate with oxidase activity (R = 0.17). Neutrophil cytoplasts were used to relate membrane translocation of p47phox, p67phox and p21rac2 to membrane oxidase activity in response to the inflammatory agonists. Whereas N-formyl-methionyl-leucyl-phenylalanine stimulated equimolar, transient membrane translocation of p47phox and p67phox which kinetically paralleled NADPH oxidase activity, relatively little p21rac2 translocated (moles of p47phox/p21rac2 = 16.6). Moreover, although phorbol 12-myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac2 translocation. From these data we conclude that membrane translocation of p21rac2 does not regulate NADPH oxidase activity stoichiometrically.
Highlights
:j: To whom correspondence and reprint requests should be addressed. 1 The abbreviations used are: PM, plasma membrane; CS, cytosol; Over the last decade, the development of cell-free systems for studying NADPH oxidase activation [3,4,5,6] has permitted the identification of other proteins associated with the oxidase [7]
Studies using neutrophils from patients with chronic granulomatous disease lacking either cytochrome b558, p47phox, or p67phox suggest that cytochrome b558 is required for translocation of both p47Phox and p67phox and that p47phox can translocate to the PM in the absence of p67phox but p67phox translocation requires p47phox [21]. p47phox and p67phox have been shown to translocate to the PM in cell-free systems in response to anionic amphiphiles and GTPI'S [22,23,24,25], cofactors required for NADPH oxidase activation
We have extended our initial observation of GTPyS-stimulated translocation of p21rac2 to membranes in a cell-free system [32] by demonstrating that this process is rapid «1 min), stable (> 1 h), dependent on GTP and its analogs, and dependent on membranes from neutrophils but not erythrocytes
Summary
11514-11521, 1995 Printed in U.S.A. Translocation of p21rac from Cytosol to Plasma Membrane Is Neither Necessary nor Sufficient for Neutrophil NADPH Oxidase Activity*. Activation of the membrane-associated NADPH oxidase of neutrophils requires several cytosoIic factors including p47phox, p67phox and p21rac. Phorbol 12myristate 13-acetate stimulated both the stable translocation of p47phox and p67phox and sustained NADPH oxidase activity, it did not stimulate p21rac translocation. GTPyS, guanosine 5'-O-(3-thiotriphosphate); GDPj3S, guanyl-5'-yl thiophosphate; GMP-PNP, guanyl-5'-yl imidodiphosphate; ATPyS, adenosine 5'-O-(3-thiotriphosphate); PMA, phorbol 12-myristate 13-acetate; GDI, GDP dissociation inhibitors; AFC, N-acetyl-S-trans,trans-farnesyl-L-cysteine; AGGC, N-acetyl-S-all trans-geranylgeranyl-L-cysteine Using a cell-free system and enucleate neutrophil cytoplasts, we have tested the hypothesis that translocation of p21rac from CS to PM regulates NADPH oxidase activity by promoting stoichiometric assembly of the oxidase complex. Preliminary reports of these results have been presented [33,34]
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