Arachidonic acid induces direct interaction of the p67phox-Rac complex with the phagocyte oxidase Nox2, leading to superoxide production

Rumi Matono, Kei Miyano, Takuya Kiyohara, Hideki Sumimoto

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

The phagocyte NADPH oxidase Nox2, heterodimerized with p22phoxin 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 p47phoxand GDP/GTP exchange on the small GTPase Rac. These proteins, in an active form, bind to their respective targets, p22phoxand 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 p47phoxconformational 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 p47phoxand 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 the direct interaction of Rac-GTP-bound p67phoxwith 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 p67phoxactivation domain that localizes the 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.

Original languageEnglish
Pages (from-to)24874-24884
Number of pages11
JournalJournal of Biological Chemistry
Volume289
Issue number36
DOIs
Publication statusPublished - 2014

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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