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• W2025505655 abstract "Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b558 comprising gp91phox and p22phox, and three cytosolic proteins: p47phox and p67phox, each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47phox, retaining the N-terminal and the two SH3 domains, and the N terminus of p67phox. When either truncated p47phox or p67phox is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47phox and p67phox are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47phox, but not the full-length one, can bind to p22phox, a binding required for the oxidase activation. The N-terminal SH3 domain of p47phox is responsible for the binding not only to p22phox, but also to the p47phox C terminus. Thus the SH3 domain is accessible in the active p47phox, but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions. Anionic amphiphiles, such as arachidonate, activate the superoxide-producing phagocyte NADPH oxidase in a cell-free system with human neutrophil membrane, which contains cytochrome b558 comprising gp91phox and p22phox, and three cytosolic proteins: p47phox and p67phox, each harboring two SH3 domains, and the small GTPase Rac. Here we show that, even without the amphiphiles, the oxidase is activated in vitro by a C terminally truncated p47phox, retaining the N-terminal and the two SH3 domains, and the N terminus of p67phox. When either truncated p47phox or p67phox is replaced by the respective full-length one, the activation absolutely requires the amphiphiles. The results indicate that both p47phox and p67phox are the primary targets of the amphiphiles, and that their C-terminal regions play negative regulatory roles. We also find that the truncated p47phox, but not the full-length one, can bind to p22phox, a binding required for the oxidase activation. The N-terminal SH3 domain of p47phox is responsible for the binding not only to p22phox, but also to the p47phox C terminus. Thus the SH3 domain is accessible in the active p47phox, but is normally masked in the full-length one probably via intramolecularly interacting with the C terminus. The present findings support our previous proposal of regulatory SH3 domain-mediated interactions. Increasing attention has currently been paid to specific protein-protein interactions in intracellular signal transduction, which are mediated by modular binding domains of signaling proteins (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar,2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar). Among the domains to be characterized at an earlier stage is the Src homology 3 (SH3) 1The abbreviations used are: SH3, Src homology 3; PRR, proline-rich region; GST, glutathione S-transferase; GTPγS, guanosine 5′-3-O-(thio)triphosphate; GDPβS, guanosine 5′-2-O-(thio)diphosphate; PAGE, polyacrylamide gel electrophoresis. domain found in various proteins including the Src family tyrosine kinases. The domain directly binds, via its target-binding surface, to a proline-rich region (PRR) of its partners, thereby mediating protein-protein interactions (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar, 2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar, 3Birge R.B. Hanafusa H. Science. 1993; 262: 1522-1524Crossref PubMed Scopus (66) Google Scholar, 4Chen J.K. Schreiber S.L. Angew. Chem. Int. Ed. Engl. 1995; 34: 953-969Crossref Scopus (56) Google Scholar). Unlike the case of SH2 domains, whose interactions with tyrosine-containing peptides are promoted by phosphorylation of the SH2 domain-binding site, the regulatory mechanism for SH3 domain-mediated associations largely remains elusive. Specific interactions via SH3 domains play a crucial role in assembly and activation of the phagocyte NADPH oxidase (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 6Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar, 7de Mendez I. Garrett M.C. Adams A.G. Leto T.L. J. Biol. Chem. 1994; 269: 16326-16332Abstract Full Text PDF PubMed Google Scholar, 8Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 9Leusen J.H.W. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. Kaulfersch W. Segar R.A. Roos D. Verhoeven A.J. J. Exp. Med. 1994; 180: 2329-2334Crossref PubMed Scopus (90) Google Scholar, 10McPhail L.C. J. Exp. Med. 1994; 180: 2011-2015Crossref PubMed Scopus (62) Google Scholar, 11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar, 13de Mendez I. Adams A.G. Sokolic R.A. Malech H.L. Leto T.L. EMBO J. 1996; 15: 1211-1220Crossref PubMed Google Scholar, 14Sumimoto H. Ito T. Hata K. Mizuki K. Nakamura R. Kage Y. Sakaki Y. Nakamura M. Takeshige K. Hamasaki N. Mihara K. Membrane Proteins: Structure, Function and Expression Control. Kyushu University Press, Fukuoka, Japan1997: 235-245Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). The oxidase, dormant in resting cells, is activated during phagocytosis to catalyze reduction of molecular oxygen to superoxide, a precursor of microbicidal oxidants (16Smith R.M. Curnutte J.T. Blood. 1991; 77: 673-686Crossref PubMed Google Scholar, 17Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 18Jones O.T.G. BioEssays. 1994; 16: 919-923Crossref PubMed Scopus (66) Google Scholar, 19Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Åhlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar, 20DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (460) Google Scholar). The significance of this enzyme in host defense is made evident by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease, whose phagocytes lack the superoxide-producing system (16Smith R.M. Curnutte J.T. Blood. 1991; 77: 673-686Crossref PubMed Google Scholar, 17Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 18Jones O.T.G. BioEssays. 1994; 16: 919-923Crossref PubMed Scopus (66) Google Scholar, 19Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Åhlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar, 20DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (460) Google Scholar). Recent studies, furthermore, have suggested that oxidants produced by the NADPH oxidase may be also involved in Ras-mediated mitogenic signaling in fibroblasts (21Irani K. Xia Y. Zweier J.L. Sollott S.J. Der C.J. Fearon E.R. Sundaresan M. Finkel T. Goldschmidt-Clermont P.J. Science. 1997; 275: 1649-1652Crossref PubMed Scopus (1441) Google Scholar), oxygen sensing in airway chemoreceptors (22Wang D. Youhgson C. Wang V. Yeger H. Dinauer M.C. Vega-Saez de Miera E. Rudy B. Cutz E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13182-13187Crossref PubMed Scopus (158) Google Scholar), and activation of c-Jun N-terminal kinase in kidney epithelial cells (23Cui X.-L. Douglas J.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3771-3776Crossref PubMed Scopus (166) Google Scholar). The catalytic core of the oxidase, which transfers electrons from NADPH to oxygen molecule, is the membrane-integrated flavocytochromeb558, composed of the two subunit gp91phox and p22phox (16Smith R.M. Curnutte J.T. Blood. 1991; 77: 673-686Crossref PubMed Google Scholar, 17Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 18Jones O.T.G. BioEssays. 1994; 16: 919-923Crossref PubMed Scopus (66) Google Scholar, 19Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Åhlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar, 20DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (460) Google Scholar). Activation of the oxidase requires translocation of three cytosolic proteins, p47phox, p67phox, and the small GTPase Rac1/2, to the membrane where they assemble with the cytochrome. Both p47phox and p67phox harbor two SH3 domains, which mediate specific interactions between the oxidase factors. The C-terminal SH3 domain of p67phox interacts with the PRR of p47phox (6Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar, 8Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar), while the N-terminal one of p47phox does with p22phox (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). The latter interaction is required for both translocation of p47phox and activation of the NADPH oxidase (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 8Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 9Leusen J.H.W. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. Kaulfersch W. Segar R.A. Roos D. Verhoeven A.J. J. Exp. Med. 1994; 180: 2329-2334Crossref PubMed Scopus (90) Google Scholar, 10McPhail L.C. J. Exp. Med. 1994; 180: 2011-2015Crossref PubMed Scopus (62) Google Scholar, 11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). A monoclonal antibody specific for the p47phox SH3 domains interacts with p47phox in the presence of arachidonic acid, an activator of the oxidase, but not with the resting form of p47phox (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar). It is likely that the N-terminal SH3 domain of p47phox is normally inaccessible, and, upon activation, becomes unmasked to interact with p22phox (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In the oxidase system, thus, the SH3 domain-mediated interactions are apparently regulated in contrast with the Grb2/Sos SH3 domain-mediated contacts, which are constitutive (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar, 2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar). In addition to the whole cell activation by various phagocytic or non-phagocytic stimuli, the NADPH oxidase can be activated in a cell-free system reconstituted with cytochromeb558 and the three cytosolic proteins, p47phox, p67phox, and Rac in the GTP-bound form (17Chanock S.J. el Benna J. Smith R.M. Babior B.M. J. Biol. Chem. 1994; 269: 24519-24522Abstract Full Text PDF PubMed Google Scholar, 18Jones O.T.G. BioEssays. 1994; 16: 919-923Crossref PubMed Scopus (66) Google Scholar, 19Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Åhlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar, 20DeLeo F.R. Quinn M.T. J. Leukocyte Biol. 1996; 60: 677-691Crossref PubMed Scopus (460) Google Scholar). In the system, the activation is totally dependent on the addition of such anionic amphiphile activators as arachidonic acid and sodium dodecyl sulfate (SDS) (24Bromberg Y. Pick E. J. Biol. Chem. 1985; 260: 13539-13545Abstract Full Text PDF PubMed Google Scholar). Here we have developed an in vitro system in which the NADPH oxidase is activated without using the amphiphile activators. A mutant p47phox, allowing the N-terminal SH3 domain unmasked, is capable of both binding to p22phox and activating the oxidase without the amphiphiles when used with the N terminus of p67phox. These findings indicate the regulatory intramolecular association of the SH3 domain in p47phox, which is directly linked to the oxidase activation. The DNA fragments encoding the full-length p47phox (p47-F; amino acid residues 1–390), p47-N (1–153), p47-(SH3)2 (154–286), p47-F(W193R) (the full-length p47phox with a substitution of Trp193 for Arg), the full-length p67phox (p67-F, 1–526), p67-N (1–242), p67-SH3(C) (455–526), and p22-C (the cytoplasmic domain of p22phox, residues 132–195) were obtained as described previously (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar). The DNAs for the p47-ΔC (1–286) and p47-ΔN (154–390) were amplified by polymerase chain reaction from a cloned cDNA encoding human p47phox or p67phox. For the mutant p47-F carrying the Trp263→ Arg substitution, namely p47-F(W263R), the mutation was introduced into p47-F by polymerase chain reaction-mediated site-directed mutagenesis. All the polymerase chain reaction products were subcloned into the pGEX-2T expression vector (Pharmacia Biotech, Uppsala, Sweden). All the plasmids were subjected to DNA sequencing for the confirmation of precise construction. The GST fusion proteins were expressed in Escherichia coli and purified by glutathione-Sepharose-4B beads (Pharmacia Biotech). Both membrane and cytosolic fractions of human neutrophils were prepared by sequential centrifugations as described previously (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar). To prepare Rac2-enriched fractions, the cytosolic fraction was applied to a 2′,5′-ADP Sepharose CL-6B column. The flow-through fraction was applied to a DEAE Sepharose CL-6B column, and the Rac2-enriched fraction was eluted with 0.2m NaCl. The fraction contained Rac2 but was free of p47phox and p67phox as confirmed by immunodetection (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). The assay mixture was composed of 100 mm potassium phosphate (pH 7.0), 75 μm cytochrome c, 10 μm FAD, 10 μm GTPγS, 1.0 mmEGTA, and 1.0 mm NaN3. The neutrophil membrane was incubated for 2 min at room temperature with recombinant p47phox, recombinant p67phox, and the Rac2-enriched fraction in the presence or absence of 100 μm SDS. The reaction was then initiated by the addition of NADPH (1.0 mm) to the reaction mixture. The NADPH-dependent superoxide producing activity was measured by determining the rate of superoxide dismutase-inhibitable ferricytochrome c reduction at 550–540 nm with a dual-wavelength spectrophotometer (Hitachi 557), and was expressed as micromole/min/mg of membrane proteins (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). In vitro interaction between p47phox and p22phox was estimated by far Western blot as described previously (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar). Briefly, the GST-p22-C (1.0 μg) were subjected to SDS-PAGE and transferred to nitrocellulose membrane, which was incubated with 10 μg of GST-p47-F, GST-p47-ΔC, or GST-p47-(SH3)2 in 20 mm Tris-HCl (pH 7.5), 150 mm NaCl, and 1% bovine serum albumin. The filter was then probed with an anti-GST monoclonal antibody (25Tanaka S. Hattori S. Kurata T. Nagashima K. Fukui Y. Nakamura S. Matsuda M. Mol. Cell. Biol. 1993; 13: 4409-4415Crossref PubMed Scopus (97) Google Scholar), a generous gift from Drs. Yoichi Tachibana (Nippon Zeon Corp., Tokyo, Japan) and Michiyuki Matsuda (International Medical Center of Japan, Tokyo, Japan), rather than a polyclonal anti-p47phox antibody, because the latter may block the interaction with p22phox. The monoclonal anti-GST antibody did not recognize the GST fusion protein transferred to nitrocellulose membrane after SDS-PAGE under the condition used (25Tanaka S. Hattori S. Kurata T. Nagashima K. Fukui Y. Nakamura S. Matsuda M. Mol. Cell. Biol. 1993; 13: 4409-4415Crossref PubMed Scopus (97) Google Scholar). Complexes were detected using alkaline phosphatase-conjugated anti-mouse IgG antibodies. The p47-F (amino acid residues 1–390), p47-ΔC (1–286), and the C-terminal region of p47phox, namely p47-C (286–390), were cloned into a modified GAL4 activation domain-fusion vector pGAD424g (12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar) to obtain pGAD-p47-F, pGAD-p47-ΔC, and pGAD-p47-C, respectively. Deletion mutants of pGAD-p47-C, namely p47-CΔP1 and p47-CΔP2, lacked amino acid residues 299–346 and 360–390, respectively (12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar). The C-terminal SH3 domain of p67phox (455–526), namely p67-SH3(C), and the C-terminal cytoplasmic tail of p22phox (132–195), namely p22-C, were cloned into a modified GAL4 DNA-binding domain fusion vector pGBT9g (12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar) to obtain pGBT-p67-SH3(C) and pGBT-p22-C, respectively. All plasmids were subjected to DNA sequencing for the confirmation of precise construction. Pairs between the pGAD and pGBT plasmids were cotransformed into competent yeast SFY526 cells with lacZ reporter gene. After selection for Leu+ and Trp+ phenotypes, the transformants were tested for their ability to grow on plates lacking histidine. Activation of lacZ reporter was examined by β-galactosidase filter assay as described previously (12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar). The protein p47phox, an activating factor of the phagocyte NADPH oxidase, comprises four portions: the N-terminal region, the N-terminal and C-terminal SH3 domains, and the C-terminal tail (Fig. 1 A). To investigate roles of the individual regions, we isolated deletion mutant proteins as GST fusions, and tested their abilities to activate the oxidase stimulated by SDS in a cell-free system reconstituted with human neutrophil membrane, the full-length p67phox, and the Rac2-enriched faction (Fig. 1 A). In the cell-free system, the wild-type full-length p47phox (p47-F) activated the NADPH oxidase in a dose-dependent manner (Fig. 1 B), and the maximal activity was obtained at the concentration of 13.0 μg/ml (about 0.2 nmol/ml). At the concentration of 0.1 nmol/ml, the p47phox lacking the C terminus (p47-ΔC) fully activated the oxidase, while neither the N terminally deleted one (p47-ΔN) nor the one without both termini (p47-(SH3)2) was capable of supporting superoxide production (Fig. 1 A). The latter two proteins were completely inactive even at 4-fold higher concentrations (data not shown). Thus the N-terminal region is essential for the oxidase activation. To clarify the role of the C-terminal SH3 domain, we introduced the substitution of Arg for Trp263, the most conserved residue in SH3 domains that directly interacts with a proline of target peptides (1Pawson T. Nature. 1995; 373: 573-580Crossref PubMed Scopus (2234) Google Scholar, 2Cohen G.B. Ren R. Baltimore D. Cell. 1995; 80: 237-248Abstract Full Text PDF PubMed Scopus (926) Google Scholar, 4Chen J.K. Schreiber S.L. Angew. Chem. Int. Ed. Engl. 1995; 34: 953-969Crossref Scopus (56) Google Scholar). The mutation resulted in decreased ability to support superoxide production at submaximal (Fig.1 A) and saturated (Fig. 1 B) concentrations, indicating that the SH3 domain is not essential, but is required for the full activation. This is in contrast with that the N-terminal SH3 domain was an absolute requisite for the oxidase activation (Fig.1 A), which agrees with previous results by us and others (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). Taken together, the N-terminal region and both SH3 domains are required for fully activating the oxidase. Another oxidase activating protein p67phox also harbors two SH3 domains (Fig.2), both of which seem indispensable for superoxide production in stimulated cells (7de Mendez I. Garrett M.C. Adams A.G. Leto T.L. J. Biol. Chem. 1994; 269: 16326-16332Abstract Full Text PDF PubMed Google Scholar). However, the domains and the region between them are not required in a cell-free system: the N-terminal region of p67phox is sufficient for the oxidase activation (7de Mendez I. Garrett M.C. Adams A.G. Leto T.L. J. Biol. Chem. 1994; 269: 16326-16332Abstract Full Text PDF PubMed Google Scholar), which was confirmed in our cell-free activation system (Fig. 2). The region (amino acid residues 1–242) contains the Rac-binding site (26Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-533Crossref PubMed Scopus (349) Google Scholar), and the GTP-dependent interaction between p67phox and Rac seems essential for the oxidase activation (26Diekmann D. Abo A. Johnston C. Segal A.W. Hall A. Science. 1994; 265: 531-533Crossref PubMed Scopus (349) Google Scholar, 27Nishimoto Y. Freeman J.L.R. Motalebi S.A. Hirshberg M. Lambeth J.D. J. Biol. Chem. 1997; 272: 18834-18841Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). The activation accomplished by the C terminally deleted p47phox (p47-ΔC) and the N terminus of p67phox (p67-N) was essentially the same as that by their respective full-length proteins, in both the maximal activity (Fig. 2) and the dose dependence on the proteins (data not shown). Among the four portions of p67phox, the N-terminal region is thus sufficient for the cell-free oxidase activation, in combination with the C terminally deleted p47phox (p47-ΔC) as well as with full-length one (p47-F). In the cell-free system with p47-F and p67-F, the oxidase activation was totally dependent on the anionic amphiphile SDS (Fig. 3 A). On the other hand, to our surprise, the NADPH-dependent superoxide production was observed even in the absence of the amphiphile, when both p47-ΔC and p67-N were used instead of the full-length ones (Fig.3 A). The activation required not only the truncated proteins (Fig. 3 B) but also the neutrophil membrane and Rac2, but was diminished in the presence of GDPβS, an agent keeping Rac2 in an inactive form (Fig. 3 C). These properties confirm that the superoxide production is indeed catalyzed by the phagocyte NADPH oxidase. In combination with the full-length p67phox (p67-F), the C terminally deleted p47phox (p47-ΔC) failed to activate the oxidase in the absence of the amphiphile activator (Fig.4). Similarly, without the activator, combination of p47-F and p67-N did not lead to superoxide production (Fig. 4). These results indicate that the activator directly interacts with both p47phox and p67phox, and that the p47phox C terminus and the p67phox region from the first to the second SH3 domains play negative regulatory roles in the oxidase activation. To investigate the molecular event that enables p47phox to activate the oxidase, we compared the nature of the C terminally deleted p47phox (p47-ΔC) with that of the full-length one (p47-F). It is well established that, upon cell stimulation, p47phox interacts with the C-terminal cytoplasmic tail of p22phox (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 8Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 9Leusen J.H.W. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. Kaulfersch W. Segar R.A. Roos D. Verhoeven A.J. J. Exp. Med. 1994; 180: 2329-2334Crossref PubMed Scopus (90) Google Scholar, 10McPhail L.C. J. Exp. Med. 1994; 180: 2011-2015Crossref PubMed Scopus (62) Google Scholar, 11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 13de Mendez I. Adams A.G. Sokolic R.A. Malech H.L. Leto T.L. EMBO J. 1996; 15: 1211-1220Crossref PubMed Google Scholar, 14Sumimoto H. Ito T. Hata K. Mizuki K. Nakamura R. Kage Y. Sakaki Y. Nakamura M. Takeshige K. Hamasaki N. Mihara K. Membrane Proteins: Structure, Function and Expression Control. Kyushu University Press, Fukuoka, Japan1997: 235-245Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). The induced interaction is mediated by the N-terminal SH3 domain of p47phox and is essential for the oxidase activation (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). An in vitrobinding assay using purified proteins revealed that p47-ΔC directly bound to p22phox as strongly as the p47phox composed solely of the SH3 domains (p47-(SH3)2) did, whereas the full-length p47phox could not (Fig.5 A). The result completely agrees with that obtained by an in vivo binding assay in the yeast two-hybrid system (Fig. 5 B). In the C terminally deleted protein, thus, the N-terminal SH3 domain exists in a state accessible to the target p22phox. This raised a question how the SH3 domain is normally masked. We have previously shown that the SH3 domains of p47phox can interact with the C terminus of this protein, an interaction which seems to keep the SH3 domains inaccessible (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar). To determine the precise regions involved in this interaction, we performed binding experiments in the two-hybrid system. The N-terminal SH3 domain seems responsible for the interaction with the C terminus (p47-C; amino acid residues 286–390), since a mutation in this domain (Trp193 → Arg) abrogated the binding (Fig.5 C) but one in the other SH3 domain (Trp263 → Arg) did not (data not shown). The results are consistent with the observation that the N-terminal SH3 domain can interact with p47phox in vitro (15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). As shown in Fig.5 C, the SH3 domain interacted with p47-CΔP2 that lacked the PRR (Pro361-Gln-Pro-Ala-Val-Pro-Pro-Arg-Pro369), the target for the C-terminal SH3 domain of p67phox (6Finan P. Shimizu Y. Gout I. Hsuan J. Truong O. Butcher C. Bennett P. Waterfield M.D. Kellie S. J. Biol. Chem. 1994; 269: 13752-13755Abstract Full Text PDF PubMed Google Scholar, 8Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 12Ito T. Nakamura R. Sumimoto H. Takeshige K. Sakaki Y. FEBS Lett. 1996; 385: 229-232Crossref PubMed Scopus (51) Google Scholar). Another deletion (the deleted residues 299–346), giving p47-CΔP1, abolished the interaction with the p47phox SH3 domain, but did not affect the contact with p67phox (Fig. 5 C). Thus the intramolecular target of the p47phox N-terminal SH3 domain seems different from the site for the p67phox SH3 domain. The p67phox-binding site is likely to be exposed in the folded inactive form of p47phox, as indicated by the two hybrid interaction between the full-length p47phox and the C-terminal SH3 domain of p67phox (Fig. 5 B). Here we presented that the phagocyte NADPH oxidase is activated by the C terminally deleted p47phox (p47-ΔC) and the N terminus of p67phox (p67-N), even in the absence of in vitroactivators, anionic amphiphiles, such as arachidonic acid and SDS. The observation that the anionic amphiphile-independent activation requires both active forms of p47phox and p67phox indicates that the two proteins are the primary targets of the activators in the cell-free system. On the other hand, the action of the amphiphiles on either cytochrome b558 or Rac, if any, is not essential for the oxidase activation. The extent of the oxidase activation using both truncated proteins is comparable to that elicited by the amphiphiles: about a half of the superoxide producing activity is obtained in the present system. To our knowledge, a similar level of the activation has not been accomplished in any reported cell-free systems using purified cytosolic factors without the amphiphiles. Two recent studies have demonstrated arachidonic acid- or SDS-independent cell-free activation of the NADPH oxidase using crude neutrophil cytosol (28McPhail L.C. Qualliotine-Mann D. Waite K.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7931-7935Crossref PubMed Scopus (89) Google Scholar, 29Park J.-W. Hoyal C.R. El Benna J. Babior B.M. J. Biol. Chem. 1997; 272: 11035-11043Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar): one reports that phosphatidic acid fully activates the enzyme in a phosphorylation-dependent manner in a system using cellular membranes and cytosol with diacylglycerol (28McPhail L.C. Qualliotine-Mann D. Waite K.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7931-7935Crossref PubMed Scopus (89) Google Scholar), and the other shows that phosphorylated p47phox is capable of activating the oxidase, but to a lesser extent, when membranes are treated with cytosol and GTPγS (29Park J.-W. Hoyal C.R. El Benna J. Babior B.M. J. Biol. Chem. 1997; 272: 11035-11043Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). The present system is considered quite useful for studying in detail the activation mechanisms of the individual cytosolic proteins. In the absence of the amphiphiles, the oxidase activation is dependent on the state of p47phox, when p67-N (an active form of p67phox) is used with the GTP-bound Rac2. Under the conditions, the oxidase is activated by the addition of the active p47phox (p47-ΔC), but not the full-length one (Fig. 4). Similarly, with p47-ΔC, the state of p67phox is the determinant. When both p47-ΔC and p67-N are present, the activation totally depends on the state of Rac: little superoxide production was observed with Rac2 in the GDP-bound inactive form (Fig. 3 C). We also studied here the mechanism for activating p47phox. The active p47phox (p47-ΔC) interacted with p22phox bothin vivo and in vitro, while the full-length one in the resting state did not (Fig. 5). This interaction is mediated via binding of the p47phox N-terminal SH3 domain to the PRR of p22phox (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar), an interaction which is indispensable for the oxidase activation (11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar). Thus the conformational change of p47phox that is induced by the amphiphiles appears to culminate in unmasking of the N-terminal SH3 domain, leading to the access to p22phox. In the resting state, this domain is masked probably by interacting with a C-terminal region of p47phox. The region required for this interaction is different from the PRR, the target for the C-terminal SH3 domain of p67phox. This implies that the intramolecular interaction in p47phox and its intermolecular binding to p67phox are not mutually exclusive, i.e.both interactions can occur at the same time. Indeed the full-length p47phox, in which the N-terminal SH3 domain is masked, can bind to the p67phox SH3 domain (Fig. 5 B). The intramolecular interaction appears to require the target-binding surface of the p47phox SH3 domain, since a mutation of the surface (W193R) abrogates the binding (Fig. 5 C). On the other hand, the target region in p47phox does not contain a typical PRR. In this context, it should be noted that the SH3 domain of the tyrosine kinase Src interacts intramolecularly, via its target-binding surface, with the region lacking a proline-rich sequence (30Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar). Taken together, the N-terminal SH3 domain of p47phox likely undergoes an intramolecular interaction with the C-terminal region, which could restrict access of the domain to its target (Fig.6). The anionic amphiphile activators seem to disrupt the intramolecular interaction in p47phox, thereby liberating the SH3 domain to engage p22phox. When cells are stimulated, arachidonic acid released from the membrane may promote the conformational change of p47phox, as well as p67phox, leading to the superoxide production. It is known that, during activation, p47phox becomes phosphorylated at multiple sites in the C-terminal region (31el Benna J. Faust L.P. Babior B.M. J. Biol. Chem. 1994; 269: 23431-23436Abstract Full Text PDF PubMed Google Scholar), accompanied by its translocation to the membrane, and it has recently been reported that the phosphorylated p47phox is active in a cell-free activation system (29Park J.-W. Hoyal C.R. El Benna J. Babior B.M. J. Biol. Chem. 1997; 272: 11035-11043Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar). Future studies should test whether the phosphorylation converts the intramolecular interaction of the p47phox SH3 domain to the intermolecular one with p22phox, thereby activating the oxidase. In addition, the present finding that the truncated versions of p47phox and p67phox are both active, also raises a possibility that these proteins might be proteolytically activated in stimulated cells. Some protease inhibitors are shown to inhibit the superoxide production by phagocytes (32Cross A.W. Free Radical Biol. & Med. 1990; 8: 71-93Crossref PubMed Scopus (116) Google Scholar). This study unequivocally shows that the amphiphile activators directly interact not only with p47phox but also with p67phox, the latter interaction which is previously suggested (33Freeman J.L. Lambeth J.D. J. Biol. Chem. 1996; 271: 22578-22582Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 34Koshkin V. Lotan O. Pick E. J. Biol. Chem. 1996; 271: 30326-30329Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The precise event evoked in p67phox, however, is presently unknown. The oxidase activation is repressed by the p67phox region containing both SH3 domains. Pinpointing the responsible region will help our understanding of the mechanism, and such studies are now in progress in our laboratory. On the basis of the studies on the NADPH oxidase system, we have previously proposed (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar) and advanced here the “masking-unmasking” model for a regulatory mechanism of SH3 domain-mediated interactions: an SH3 domain, that is normally masked via its intramolecular interaction, becomes exposed to intermolecularly interact with the target. It should be strengthened that, in the oxidase system, the induced intermolecular interaction is specific and required for the activation both in vivo and in vitro (5Sumimoto H. Kage Y. Nunoi H. Sasaki H. Nose T. Fukumaki Y. Ohno M. Minakami S. Takeshige K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5345-5349Crossref PubMed Scopus (257) Google Scholar, 8Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (248) Google Scholar, 9Leusen J.H.W. Bolscher B.G.J.M. Hilarius P.M. Weening R.S. Kaulfersch W. Segar R.A. Roos D. Verhoeven A.J. J. Exp. Med. 1994; 180: 2329-2334Crossref PubMed Scopus (90) Google Scholar,11Sumimoto H. Hata K. Mizuki K. Ito T. Kage Y. Sakaki Y. Fukumaki Y. Nakamura M. Takeshige K. J. Biol. Chem. 1996; 271: 22152-22158Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 15de Mendez I. Homayounpour N. Leto T.L. Mol. Cell. Biol. 1997; 17: 2177-2185Crossref PubMed Scopus (85) Google Scholar): p22phox is the bona fide target for the N-terminal SH3 domain of p47phox. A similar molecular event has recently been postulated in the T-cell specific tyrosine kinase Itk, a member of the Tec family of non-receptor tyrosine kinase, that is required for signaling via the T-cell antigen receptor (35Andreotti A.H. Bunnell S.C. Feng G. Berg L.J. Schreiber S.L. Nature. 1997; 375: 93-97Crossref Scopus (229) Google Scholar). The PRR adjacent to the SH3 domain of Itk interacts with the domain intramolecularly. Formation of this complex prevents the SH3 domain and the PRR from interacting with their respective putative substrates, Sam68 and Grb2 (35Andreotti A.H. Bunnell S.C. Feng G. Berg L.J. Schreiber S.L. Nature. 1997; 375: 93-97Crossref Scopus (229) Google Scholar). In p120 Ras-GAP (GTPase-activating protein), containing an SH3 domain flanked by two SH2 domains, it undergoes a conformational change that leads to increased accessibility of the target binding surface of its SH3 domain, when two closely linked phosphotyrosine-containing peptides bind simultaneously to the two SH2 domains (36Hu K.-Q. Settleman J. EMBO J. 1997; 16: 473-483Crossref PubMed Scopus (126) Google Scholar). Furthermore, recent structural studies have revealed that the SH3 domain of the tyrosine kinases Src and Hck interacts intramolecularly not only with the linker region between the SH2 and kinase domains, but also simultaneously with the kinase domain, resulting in inhibition of the enzymatic activity (30Xu W. Harrison S.C. Eck M.J. Nature. 1997; 385: 595-602Crossref PubMed Scopus (1252) Google Scholar, 37Sicheri F. Moarefi I. Kuriyan J. Nature. 1997; 385: 602-609Crossref PubMed Scopus (1047) Google Scholar). Thus such regulatory intramolecular association of SH3 domains, as occurred in p47phox, is currently considered to be much more general than previously expected (38Nguyen J.T. Lim W.A. Nat. Struct. Biol. 1997; 4: 256-260Crossref PubMed Scopus (24) Google Scholar). This type of the regulation would be found in a variety of signaling proteins that carry both an SH3 domain and a PRR, such as the p85 subunit of phosphoinositide 3-kinase (39Liu X. Marengere L.E. Koch C.A. Pawson T. Mol. Cell. Biol. 1993; 13: 5225-5232Crossref PubMed Scopus (155) Google Scholar) and the yeast protein Bem1p (40Chenevert J. Corrado K. Bender A. Pringle J. Herskowitz I. Nature. 1992; 356: 77-79Crossref PubMed Scopus (158) Google Scholar). We thank Drs. Y. Tachibana (Nippon Zeon Corp.) and M. Matsuda (International Medical Center of Japan) for providing the anti-GST monoclonal antibody, Prof. Y. Sakaki ( University of Tokyo) for encouragement, and Drs. D. Kang (Kyushu University) and T. Muta (Kyushu University) for helpful discussions." @default.
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• W2025505655 title "Anionic Amphiphile-independent Activation of the Phagocyte NADPH Oxidase in a Cell-free System by p47 and p67 , Both in C Terminally Truncated Forms" @default.
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