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Vol. 18, Issue 2, 441-454, February 2007

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A Regulated Adaptor Function of p40^phox: Distinct p67^phox Membrane Targeting by p40^phox and by p47^phox

Takehiko Ueyama^*, Toshihiko Tatsuno^*, Takumi Kawasaki^*, Satoshi Tsujibe^*, Yasuhito Shirai^*, Hideki Sumimoto, Thomas L. Leto, and Naoaki Saito^*

*Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe 657-8501, Japan; Molecular Defenses Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; and Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan

Submitted August 21, 2006; Revised November 13, 2006; Accepted November 14, 2006
Monitoring Editor: Ralph Isberg

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47^phox, p67^phox, p40^phox, and Rac) translocate and associate with the membrane-spanning flavocytochrome b₅₅₈, leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40^phox that regulate its translocation to membranes upon stimulation. GFP-p40^phox translocates to early endosomes, whereas GFP-p47^phox translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67^phox does not translocate to membranes when expressed alone, but it is dependent on p40^phox and p47^phox for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40^phox or GFP-p47^phox to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67^phox translocation to either membrane is abolished by mutations that disrupt its interaction with p40^phox or p47^phox. Finally, we detected a head-to-tail (PX–Phox and Bem1 [PB1] domain) intramolecular interaction within p40^phox in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40^phox and p47^phox function as diverse p67^phox "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Naoaki Saito (naosaito{at}kobe-u.ac.jp)

Abbreviations used: AA, arachidonic acid; FcR, Fc receptor; PA, phosphatidic acid; ROS, reactive oxygen species.

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