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Sequential Binding of Cytosolic Phox Complex to Phagosomes through Regulated Adaptor Proteins: Evaluation Using the Novel Monomeric Kusabira-Green System and Live Imaging of Phagocytosis¹

Takehiko Ueyama^*, Tomoko Kusakabe^*, Satoshi Karasawa^,,, Takumi Kawasaki^*, Aya Shimizu^*, Jeonghyun Son^*, Thomas L. Leto^¶, Atsushi Miyawaki and Naoaki Saito^2,*

^* Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, Kobe, Japan; Laboratory of Cell Function and Dynamics, Brain Science Institute, RIKEN, Saitama, Japan; Amalgaam, Tokyo, Japan; Medical and Biological Laboratories, Nagoya, Japan; and ^¶ Molecular Defenses Section, Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892

We engineered a method for detecting intramolecular and intermolecular phox protein interactions in cells by fluorescence microscopy using fusion proteins of complementary fragments of a coral fluorescent reporter protein (monomeric Kusabira-Green). We confirmed the efficacy of the monomeric Kusabira-Green system by showing that the PX and PB1 domains of p40^phox interact in intact cells, which we suggested maintains this protein in an inactive closed conformation. Using this system, we also explored intramolecular interactions within p47^phox and showed that the PX domain interacts with the autoinhibited tandem Src homology 3 domains maintained in contact with the autoinhibitory region, along with residues 341–360. Furthermore, we demonstrated sequential interactions of p67^phox with phagosomes involving adaptor proteins, p47^phox and p40^phox, during FcR-mediated phagocytosis. Although p67^phox is not targeted to phagosomes by itself, p47^phox functions as an adaptor for the ternary complex (p47^phox-p67^phox-p40^phox) in early stages of phagocytosis before phagosome closure, while p40^phox functions in later stages after phagosomal closure. Interestingly, a mutated "open" form of p40^phox linked p47^phox to closed phagosomes and prolonged p47^phox and p67^phox retention on phagosomes. These results indicate that binding of the ternary complex to phagosomes can be temporally regulated by switching between adaptor proteins that have PX domains with distinct lipid-binding specificities.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported in part by a Grant-in-Aid for Scientific Research from the Global Center of Excellence Program of the Ministry of Education, Culture, Sports, Science, and Technology of Japan; a Grant-in-Aid for Scientific Research on Priority Areas and (C) of Education, Culture, Sports, Science, and Technology in Japan; the SHISEIDO Grants for Scientific Research; a grant from Hyogo Science and Technology Association; and a grant from The Naito Foundation.

² Address correspondence and reprint requests to Dr. Naoaki Saito, Laboratory of Molecular Pharmacology, Biosignal Research Center, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan. E-mail address: naosaito{at}kobe-u.ac.jp

³ Abbreviations used in this paper: ROS, reactive oxygen species; CGD, chronic granulomatous disease; PA, phosphatidic acid; SH3, Src homology 3; AA, arachidonic acid; mKG, monomeric Kusabira Green; AIR, autoinhibitory region; EEA1, early endosome Ag-1; RT, room temperature; LZA, leucine zipper acidic protein; LZB, leucine zipper basic protein; AD, activation domain; IRES, internal ribosomal entry site; PR, proline rich.

⁴ The online version of this article contains supplemental material.

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