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J. Biol. Chem., Vol. 283, Issue 4, 2108-2119, January 25, 2008

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Phosphatidylinositol 3-Phosphate-dependent and -independent Functions of p40^phox in Activation of the Neutrophil NADPH Oxidase^*

Sarah A. Bissonnette, Christina M. Glazier, Mary Q. Stewart, Glenn E. Brown, Chris D. Ellson¹, and Michael B. Yaffe^¶2

From the Department of Biology and ^¶Division of Biological Engineering, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 and the Department of Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02130

In response to bacterial infection, the neutrophil NADPH oxidase assembles on phagolysosomes to catalyze the transfer of electrons from NADPH to oxygen, forming superoxide and downstream reactive oxygen species (ROS). The active oxidase is composed of a membrane-bound cytochrome together with three cytosolic phox proteins, p40^phox, p47^phox, and p67^phox, and the small GTPase Rac2, and is regulated through a process involving protein kinase C, MAPK, and phosphatidylinositol 3-kinase. The role of p40^phox remains less well defined than those of p47^phox and p67^phox. We investigated the biological role of p40^phox in differentiated PLB-985 neutrophils, and we show that depletion of endogenous p40^phox using lentiviral short hairpin RNA reduces ROS production and impairs bacterial killing under conditions where p67^phox levels remain constant. Biochemical studies using a cytosol-reconstituted permeabilized human neutrophil cores system that recapitulates intracellular oxidase activation revealed that depletion of p40^phox reduces both the maximal rate and total amount of ROS produced without altering the K_M value of the oxidase for NADPH. Using a series of mutants, p47PX-p40^phox chimeras, and deletion constructs, we found that the p40^phox PX domain has phosphatidylinositol 3-phosphate (PtdIns(3)P)-dependent and -independent functions. Translocation of p67^phox requires the PX domain but not 3-phosphoinositide binding. Activation of the oxidase by p40^phox, however, requires both PtdIns(3)P binding and an Src homology 3 (SH3) domain competent to bind to poly-Pro ligands. Mutations that disrupt the closed auto-inhibited form of full-length p40^phox can increase oxidase activity 2.5-fold above that of wild-type p40^phox but maintain the requirement for PX and SH3 domain function. We present a model where p40^phox translocates p67^phox to the region of the cytochrome and subsequently switches the oxidase to an activated state dependent upon PtdIns(3)P and SH3 domain engagement.

Received for publication, August 9, 2007 , and in revised form, November 5, 2007.

^* This work was supported in part by National Institutes of Health Grant GM59281 (to M. B. Y.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. 1.

¹ Supported by a fellowship from the Charles A. King Trust, Bank of America.

² To whom correspondence should be addressed: Massachusetts Institute of Technology, Center for Cancer Research, 77 Massachusetts Ave. E18-580, Cambridge, MA 02139. Tel.: 617-452-2103; Fax: 617-452-4978; E-mail: myaffe{at}mit.edu.

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