title: Arachidonic acid induces direct interaction of the p67(phox)-Rac complex with the phagocyte oxidase Nox2, leading to superoxide production.
abstract: The phagocyte NADPH oxidase Nox2, heterodimerized with p22(phox) in the membrane, is dormant in resting cells but becomes activated upon cell stimulation to produce superoxide, a precursor of microbicidal oxidants. Nox2 activation requires two switches to be turned on simultaneously: a conformational change of the cytosolic protein p47(phox) and GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22(phox) and p67(phox), leading to productive oxidase assembly at the membrane. Although arachidonic acid (AA) efficiently activates Nox2 both in vivo and in vitro, the mechanism has not been fully understood, except that AA induces p47(phox) conformational change. Here we show that AA elicits GDP-to-GTP exchange on Rac at the cellular level, consistent with its role as a potent Nox2 activator. However, even when constitutively active forms of p47(phox) and Rac1 are both expressed in HeLa cells, superoxide production by Nox2 is scarcely induced in the absence of AA. These active proteins also fail to effectively activate Nox2 in a cell-free reconstituted system without AA. Without affecting Rac-GTP binding to p67(phox), AA induces the direct interaction of Rac-GTP-bound p67(phox) with the C-terminal cytosolic region of Nox2. p67(phox)-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67(phox) activation domain that localizes the C-terminal to the Rac-binding domain. Thus the "third" switch (AA-inducible interaction of p67(phox)·Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.
authors: From the Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors: Rumi
authors: R
authors: Matono
authors: From the Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors: Kei
authors: K
authors: Miyano
authors: From the Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
authors: Takuya
authors: T
authors: Kiyohara
authors: From the Department of Biochemistry, Kyushu University Graduate School of Medical Sciences, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan hsumi@med.kyushu-u.ac.jp.
authors: Hideki
authors: H
authors: Sumimoto
mesh_terms: D000818:Animals; D016718:Arachidonic Acid / Q000494:pharmacology*; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010586:Phagocytes / Q000201:enzymology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013481:Superoxides / Q000378:metabolism*; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008562:Membrane Glycoproteins; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D006153:Guanosine Diphosphate; D016718:Arachidonic Acid; D006160:Guanosine Triphosphate; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C086986:CYBA protein, human; C060505:neutrophil cytosolic factor 1; D020744:rac GTP-Binding Proteins
keywords: Arachidonic Acid (AA) (ARA); NADPH Oxidase; Nox2; Rac (Rac GTPase); Reactive Oxygen Species (ROS); Signal Transduction; Superoxide Ion; p67phox