Phosphorylation of p47phox directs phox homology domain from SH3 domain toward phosphoinositides, leading to phagocyte NADPH oxidase activation Tetsuro Ago*†, Futoshi Kuribayashi*†, Hidekazu Hiroaki‡, Ryu Takeya*†, Takashi Ito§, Daisuke Kohda‡, and Hideki Sumimoto*†¶ *Medical Institute of Bioregulation, Kyushu University, Fukuoka 812-8582, Japan; †Department of Molecular and Structural Biology, Kyushu University Graduate School of Medical Science, Fukuoka 812-8582, Japan; ‡Department of Structural Biology, Biomolecular Engineering Research Institute, Osaka 565-0874, Japan; and §Division of Genome Biology, Cancer Research Institute, Kanazawa University, Kanazawa 920-0934, Japan Edited by Bernard M. Babior, The Scripps Research Institute, La Jolla, CA, and approved February 7, 2003 (received for review September 18, 2002) Protein–phosphoinositide interaction participates in targeting pro- p47phox PX structure reveals a positively charged deep pocket (8) teins to membranes where they function correctly and is often that is supposed to interact with a negatively charged small modulated by phosphorylation of lipids. Here we show that molecule. Indeed it has been uncovered recently that PX do- protein phosphorylation of p47phox, a cytoplasmic activator of the mains function as a phosphoinositide-binding module (9–14). microbicidal phagocyte oxidase (phox), elicits interaction of However, it is presently unknown how the lipid-binding activity p47phox with phosphoinositides. Although the isolated phox ho- of PX domains is regulated. mology (PX) domain of p47phox can interact directly with phos- The phagocyte oxidase, dormant in resting cells, becomes phoinositides, the lipid-binding activity of this protein is normally activated during phagocytosis to produce superoxide, a precur- suppressed by intramolecular interaction of the PX domain with sor of microbicidal oxidants, in consumption with NADPH (6, the C-terminal Src homology 3 (SH3) domain, and hence the 15–18). The significance of the NADPH oxidase in host defense wild-type full-length p47phox is incapable of binding to the lipids. is exemplified by recurrent and life-threatening infections that The W263R substitution in this SH3 domain, abrogating the inter- occur in patients with chronic granulomatous disease, the phago- action with the PX domain, leads to a binding of p47phox to cytes of which lack the superoxide-producing system (15–18). phosphoinositides. The findings indicate that disruption of the The catalytic core of the enzyme is a membrane-integrated intramolecular interaction renders the PX domain accessible to the flavocytochrome, namely cytochrome b558, comprised of the two lipids. This conformational change is likely induced by phosphor- subunits gp91phox and p22phox. The activation of the oxidase ylation of p47phox, because protein kinase C treatment of the requires the proteins p47phox and p67phox and the small GTPase wild-type p47phox but not of a mutant protein with the S303͞ Rac, which exist in the cytoplasm of resting phagocytes and 304͞328A substitution culminates in an interaction with phos- translocate after cell stimulation to membranes to interact with phoinositides. Furthermore, although the wild-type p47phox the cytochrome, leading to superoxide production (6, 15–19). translocates upon cell stimulation to membranes to activate the It is well established that p47phox plays a central role in oxidase, neither the kinase-insensitive p47phox nor lipid-binding- membrane translocation of cytosolic factors, an event that is defective proteins, one lacking the PX domain and the other essential for activation of the NADPH oxidase: In chronic carrying the R90K substitution in this domain, migrates. Thus the granulomatous disease patients with p47phox deficiency, p67phox protein phosphorylation-driven conformational change of p47phox fails to migrate, whereas p47phox becomes targeted to membranes enables its PX domain to bind to phosphoinositides, the interaction in stimulated phagocytes from p67phox-defective patients (20). phox of which plays a crucial role in recruitment of p47 from the The resting form of p47phox is likely in a closed inactive confor- cytoplasm to membranes and subsequent activation of the phago- mation where its two SH3 domains are masked via an intramo- cyte oxidase. lecular interaction with the C-terminal region of this protein (7, 21). Upon cell stimulation, p47phox becomes phosphorylated at ne of the most dominant themes in current cell biology is the C-terminal quarter, which causes a conformational change Oacute and sophisticated targeting of proteins to new cellular that leads to exposure of the SH3 domains (7, 21, 22). The locations, e.g., to membranes, the nucleus, and so forth. Re- unmasked SH3 domains directly bind to p22phox, an interaction cruitment of proteins to cell membranes is often triggered by that is required for membrane translocation of p47phox and phosphorylation of the lipid phosphatidylinositol (PtdIns), which resultant activation of the oxidase (21, 23). Thus the SH3 can create targeting sites for proteins (1, 2). The phosphorylation domains of p47phox participate in the interaction with p22phox, or hydrolysis of inositol-containing lipids in cell membranes is whereas the C-terminal region negatively regulates the translo- currently known to orchestrate numerous complex cellular cation of p47phox via the intramolecular interaction with the SH3 events (3, 4). A variety of protein modules such as pleckstrin domains. However, the role of the N-terminal PX domain has homology and FYVE domains recognize specific phospho- remained to be elucidated. inositides (phosphorylated forms of PtdIns) to recruit proteins Here we show that the PX domain of p47phox exhibits a to appropriate cell membranes (1, 2). phosphoinositide-binding activity that is normally suppressed by The phagocyte oxidase (phox) homology (PX) domain (5), interacting intramolecularly with the C-terminal SH3 domain. also known as the phox and Bem1p 2 (PB2) domain (6, 7), occurs in the phox proteins p47phox and p40phox in mammals, the polarity establishment protein Bem1p in budding yeast, and a variety of This paper was submitted directly (Track II) to the PNAS office. eukaryotic proteins involved in membrane trafficking. We have Abbreviations: PtdIns, phosphatidylinositol; phox, phagocyte oxidase; PX, phox homology; determined the NMR structure of the PX domain of p47phox and SH3, Src homology 3; PtdIns(4)P, PtdIns 4-monophosphate; PtdIns(4,5)P2, PtdIns 4,5- bisphosphate; PtdIns(3)P, PtdIns 3-monophosphate; PtdIns(3,4)P2, PtdIns 3,4-bisphosphate; demonstrated that it interacts with the C-terminal Src homology PMA, phorbol 12-myristate 13-acetate. 3 (SH3) domain of this protein (8). The p47phox PX domain ␤ ¶To whom correspondence should be addressed at: Medical Institute of Bioregulation, consists of an antiparallel -sheet formed by three strands and Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail: four helices, and an inspection of the molecular surface of the [email protected]. 4474–4479 ͉ PNAS ͉ April 15, 2003 ͉ vol. 100 ͉ no. 8 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0735712100 Downloaded by guest on September 27, 2021 Stimulus-induced phosphorylation of p47phox disrupts the in- Activation of the NADPH Oxidase in a Whole-Cell System. We trans- tramolecular interaction to render the PX domain in a state duced both gp91phox and p67phox genes into the leukemic cell line accessible to phosphoinositides, which promotes membrane K562 cells as described (7). The pREP4 vector encoding the translocation of this protein and thus plays a crucial role in wild-type full-length p47phox (amino acids 1–390), a full-length activation of the phagocyte NADPH oxidase. This work provides mutant p47phox carrying the R90K or S303͞304͞328A substitu- a previously unknown example of protein phosphorylation elic- tion, or a PX-truncated p47phox (p47-⌬PX, amino acids 129–390) iting protein–phosphoinositide interaction for its recruitment to was transfected by electroporation to the doubly transduced membranes. K562 cells. The K562 cells stably expressing the wild-type or mutant p47phox were selected, and the stable expression in Ͼ99% Experimental Procedures of cells used was confirmed by flow cytometer (FACScan, Plasmid Construction and Expression and Purification of Recombinant Becton Dickinson) using anti-p47phox antibody (Transduction Proteins. The DNA fragment encoding the full-length p47phox or Laboratories, Lexington, KY). Superoxide production by the ϫ 5 ͞ PX domain (p47-PX; amino acids 1–128) was amplified by PCR K562 cells (1 10 cells) in response to 200 ng ml of phorbol using a cloned human p47phox cDNA. Mutations leading to the 12-myristate 13-acetate (PMA) was determined as superoxide indicated amino acid substitutions were introduced by PCR- dismutase-inhibitable chemiluminescence detected with an mediated site-directed mutagenesis (7). The DNAs were ligated enhancer-containing luminol-based detection system to pGEX-2T (Amersham Biosciences) and͞or pREP4 (Invitro- (DIOGENES, National Diagnostics) as described (7, 28). gen) and sequenced for confirmation of their identities. Proteins phox fused to glutathione S-transferase (GST) were expressed in Membrane Translocation of p47 . The K562 cells expressing the phox Escherichia coli strain BL21 and purified by glutathione- wild-type or mutant p47 protein were suspended at a con- centration of 1 ϫ 107 cells per ml in PBS (137 mM NaCl͞2.68 mM Sepharose 4B (Amersham Biosciences) as described