Arachidonic Acid Induces Direct Interaction of the p67phox-Rac Complex with the Phagocyte Oxidase Nox2, Leading to Superoxide Production [Enzymology]

July 23rd, 2014 by Matono, R., Miyano, K., Kiyohara, T., Sumimoto, H.

The phagocyte NADPH oxidase Nox2, heterodimerized with p22phox 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 simultaneously turned on: a conformational change of the cytosolic protein p47phox and GDP/GTP exchange on the small GTPase Rac. These proteins in an active form bind to their respective targets p22phox and p67phox, 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 p47phox 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 p47phox 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 p67phox, AA induces direct interaction of Rac-GTP-bound p67phox with the C-terminal cytosolic region of Nox2. p67phox-Rac-Nox2 assembly and superoxide production are both abrogated by alanine substitution for Tyr-198, Leu-199, and Val-204 in the p67phox activation domain that localizes C-terminal to the Rac-binding domain. Thus the "third" switch (AA-inducible interaction of p67phox-Rac-GTP with Nox2) is required to be turned on at the same time for Nox2 activation.
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