Cytosolic guanine nucleotide-binding protein Rac2 operates in vivo as a component of the neutrophil respiratory burst oxidase. Transfer of Rac2 and the cytosolic oxidase components p47phox and p67phox to the submembranous actin cytoskeleton during oxidase activation.
The respiratory burst oxidase is responsible for O2- production in stimulated neutrophils and B lymphocytes. Components of this oxidase include cytochrome b558, a membrane-bound flavohemoprotein; the cytosolic polypeptides p47phox and p67phox; and one or more small G proteins including Rac1, Rac2, and/or Rap1A. We found that when normal neutrophils were activated, small percentages of each of the cytosolic proteins p47phox, p67phox, and Rac2 were transferred to the membrane cytoskeleton. However, Rac2 was not transferred to the membrane during activation of p47phox-deficient neutrophils. In normal cells, some p47phox also became associated with the non-cytoskeletal portion of the plasma membrane, but p67phox, Rac2, and O(2-)-forming activity were restricted to the cytoskeleton. Neutrophil activation also causes the phosphorylation of multiple serines in p47phox. The most heavily phosphorylated forms of p47phox were found solely in the membrane cytoskeleton. These results suggest that 1) the membrane cytoskeleton participates in respiratory burst oxidase activation, 2) the fully phosphorylated p47phox is located in the active oxidase, which resides in the membrane cytoskeleton, and 3) Rac2 acts like a dedicated component of the respiratory burst oxidase.
el Benna
J
J
Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, California 92037.
Ruedi
J M
JM
Babior
B M
BM
D000199:Actins / Q000097:blood; D000328:Adult; D002462:Cell Membrane / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D006105:Granulomatous Disease, Chronic / Q000097:blood / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D009252:NADPH Dehydrogenase / Q000302:isolation & purification / Q000378:metabolism*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D013481:Superoxides / Q000097:blood; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020744:rac GTP-Binding Proteins
D000199:Actins; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C022709:superoxide-forming enzyme; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate