CYTOSOLIC GUANINE-NUCLEOTIDE-BINDING PROTEIN RAC2 OPERATES IN-VIVO ASA COMPONENT OF THE NEUTROPHIL RESPIRATORY BURST OXIDASE - TRANSFER OFRAC2 AND THE CYTOSOLIC OXIDASE COMPONENTS P47(PHOX) AND P67(PHOX) TO THE SUBMEMBRANOUS ACTIN CYTOSKELETON DURING OXIDASE ACTIVATION

The respiratory burst oxidase is responsible for O-2(-) production in stimulated neutrophils and B lymphocytes. Components of this oxidase i nclude cytochrome b(558), a membrane-bound flavohemoprotein; the cytos olic polypeptides p47(phox) and p67(phox); and one or more small G pro teins including Rad1 Rac2, and/or Rap1A. We found that when normal neu trophils were activated, small percentages of each of the cytosolic pr oteins p47(phox) p67(phox), and Rac2 were transferred to the membrane cytoskeleton. However, Rac2 was not transferred to the membrane during activation of p47(phox)-deficient neutrophils. In normal cells, some p47(phox) also became associated with the non-cytoskeletal portion of the plasma membrane, but p67(Phox), Rac2, and O-2(-)-forming activity were restricted to the cytoskeleton. Neutrophil activation also causes the phosphorylation of multiple serines in p47(phox), The most heavil y phosphorylated forms of p47(phox) were found solely in the membrane cytoskeleton. These results suggest that 1) the membrane cytoskeleton participates in respiratory burst oxidase activation, 2) the fully pho sphorylated p47(phox) is located in the active oxidase, which resides in the membrane cytoskeleton, and 3) Rac2 acts like a dedicated compon ent of the respiratory burst oxidase.