Cytosolic Phospholipase A2α Is Targeted to the p47 -PX Domain of the Assembled NADPH Oxidase via a Novel Binding Site in Its C2 Domain
2008; Elsevier BV; Volume: 283; Issue: 46 Linguagem: Inglês
10.1074/jbc.m804674200
ISSN1083-351X
AutoresZeev Shmelzer, Maria Karter, Miriam Eisenstein, Thomas L. Leto, Nurit Hadad, David Ben-Menahem, Daniel Gitler, Shirly Banani, Baruch Wolach, Meir Rotem, Rachel Lévy,
Tópico(s)Nitric Oxide and Endothelin Effects
ResumoWe have previously demonstrated a physical interaction between cytosolic phospholipase A2α (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain. We have previously demonstrated a physical interaction between cytosolic phospholipase A2α (cPLA2) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67phox or p47phox, that cPLA2 specifically binds to p47phox and that p47phox is sufficient to anchor cPLA2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA2 and p47phox demonstrated that the cPLA2-C2 domain and the p47phox-PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA2-C2 domain mutations were designed to explore its interaction p47phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA2-C2 domain caused a slight inhibition of the affinity binding to p47phox, whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47phox-PX domain caused a significant inhibition of the affinity binding to cPLA2. Thus, Ile67 of the cPLA2-C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47phox-PX domain. The NADPH oxidase is a multicomponent electron carrier that transfers electrons from NADPH to molecular oxygen to form superoxide, a precursor of microbicidal oxidants. NADPH oxidase subunits include four cytoplasmic components, p47phox, p67phox, p40phox, and Rac2, and a hetero-dimeric transmembrane glycoprotein flavocytochrome b558 composed of gp91phox and p22phox (for reviews see Refs. 1Leto T.L. Gallin J. Snyderman R. Fearon D. Haynes B. Nathan C. Inflammation: Basic Principles and Clinical Correlates. Lippincott, Williams, and Wilkins Press, Baltimore, MD1999: 769-786Google Scholar, 2Groemping Y. Rittinger K. Biochem. J. 2005; 386: 401-416Crossref PubMed Scopus (438) Google Scholar). Essential roles for at least four of the phox components is evident from studies on chronic granulomatous disease (CGD) 2The abbreviations used are: CGD, chronic granulomatous disease; cPLA2, cytosolic phospholipase A2α; SH3, Src homology 3; NCF1, neutrophil cytosolic factor 1; CBR, calcium binding region; MAPK, mitogen-activated protein kinase; MNK1, MAPK interacting kinase; FRET, Förster resonance energy transfer; PX, phox homology; PMA, phorbol 12-myristate 13-acetate; fMLP, formylmethionylleucylphenylalanine; GST, glutathione S-transferase; aa, amino acid(s). 2The abbreviations used are: CGD, chronic granulomatous disease; cPLA2, cytosolic phospholipase A2α; SH3, Src homology 3; NCF1, neutrophil cytosolic factor 1; CBR, calcium binding region; MAPK, mitogen-activated protein kinase; MNK1, MAPK interacting kinase; FRET, Förster resonance energy transfer; PX, phox homology; PMA, phorbol 12-myristate 13-acetate; fMLP, formylmethionylleucylphenylalanine; GST, glutathione S-transferase; aa, amino acid(s). patients, who suffer from inherited defects in NADPH oxidase-dependent microbial killing due to defect in the genes encoding these oxidase proteins (3Roos D. de Boer M. Kuribayashi F. Meischl C. Weening R.S. Segal A.W. Ahlin A. Nemet K. Hossle J.P. Bernatowska-Matuszkiewicz E. Middleton-Price H. Blood. 1996; 87: 1663-1681Crossref PubMed Google Scholar). In resting cells, p47phox, p67phox, and p40phox exist as a tight cytosolic complex dissociated from the membrane-bound flavocytochrome. Upon stimulation, the cytosolic components translocate to the plasma membrane and associate with the flavocytochrome b558 to form the assembled active oxidase. Based on studies of p47phox-deficient cells from chronic CGD patients (4Heyworth P.G. Curnutte J.T. Nauseef W.M. Volpp B.D. Pearson D.W. Rosen H. Clark R.A. J. Clin. Invest. 1991; 87: 352-356Crossref PubMed Scopus (306) Google Scholar), p47phox appears to have a key role in translocation of the cytosolic subunits. p47phox possesses a phox homology (PX) domain, tandem SH3 domains, a series of basic residues, and phosphorylation targets that is also referred to as an autoinhibitory region, and a proline-rich region in the C terminus. In resting cells, p47phox is found in an autoinhibited state due to intramolecular interactions among the PX domain, the tandem Src homology 3 (SH3) domains, and polybasic sequences of p47phox, thereby preventing its binding to membranes (5Ueyama T. Kusakabe T. Karasawa S. Kawasaki T. Shimizu A. Son J. Leto T. Miyawaki A. Saito N. J. Immunol. 2008; 181: 629-640Crossref PubMed Scopus (48) Google Scholar). In stimulating cells, the restrictive conformation of the autoinhibitory region of p47phox is released through phosphorylation of several critical serine residues within its polybasic region (6Ago T. Nunoi H. Ito T. Sumimoto H. J. Biol. Chem. 1999; 274: 33644-33653Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Conformational changes in p47phox following phosphorylation result in unfolding and exposing the now interactive SH3 domains that direct its translocation to the membranes by binding to specific targets in p22phox and its PX domain that specifically binds phosphatidylinositol 3,4-bisphosphate and phosphatidic acid on the plasma membrane (7Sato T.K. Overduin M. Emr S.D. Science. 2001; 294: 1881-1885Crossref PubMed Scopus (202) Google Scholar, 8Kanai F. Liu H. Field S.J. Akbary H. Matsuo T. Brown G.E. Cantley L.C. Yaffe M.B. Nat. Cell Biol. 2001; 3: 675-678Crossref PubMed Scopus (493) Google Scholar, 9Karathanassis D. Stahelin R.V. Bravo J. Perisic O. Pacold C.M. Cho W. Williams R.L. EMBO J. 2002; 21: 5057-5068Crossref PubMed Scopus (256) Google Scholar). Neither p40phox nor p67phox is able to translocate to membranes in the absence of p47phox, as evidenced by the cytoplasmic location of p40phox and p67phox in stimulated cells from CGD patients who lack a functional p47phox (10Dusi S. Donini M. Rossi F. Biochem. J. 1996; 314: 409-412Crossref PubMed Scopus (110) Google Scholar). However, it appears that p40phox functions in retention of p47phox and p67phox on phagosome membranes through interactions of its PX domain with phosphatidylinositol 3-phosphate, as well as interactions of its PB1 and SH3 domains with p47phox and p67phox, respectively (11Ueyama T. Tatsuno T. Kawasaki T. Tsujibe S. Shirai Y. Sumimoto H. Leto T. Saito N. Mol. Biol. Cell. 2007; 18: 441-454Crossref PubMed Scopus (74) Google Scholar).Cytosolic phospholipase A2α (cPLA2), which hydrolyzes phospholipids containing arachidonate at the sn-2 position (12Clark J.D. Milona N. Knopf J.L. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 7708-7712Crossref PubMed Scopus (420) Google Scholar), has been implicated as the major enzyme in the formation of eicosanoids. cPLA2 has two functionally distinct domains: an N-terminal C2 domain necessary for Ca2+-dependent phospholipid binding, and a C-terminal Ca2+-independent catalytic region (13Nalefski E.A. Sultzman L.A. Martin D.M. Kriz R.W. Towler P.S. Knopf J.L. Clark J.D. J. Biol. Chem. 1994; 269: 18239-18249Abstract Full Text PDF PubMed Google Scholar). It was shown that cPLA2 translocates from the cytosol to the nuclear membrane and to the endoplasmic reticulum by an increase in cytoplasmic [Ca2+] in a variety of cells (14Glover S. de Carvalho M.S. Bayburt T. Jonas M. Chi E. Leslie C.C. Gelb M.H. J. Biol. Chem. 1995; 270: 15359-15367Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). The C2 domain of cPLA2 has been cocrystallized with calcium (15Dessen A. Tang J. Schmidt H. Stahl M. Clark J.D. Seehra J. Somers W.S. Cell. 1999; 97: 349-360Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar), and the structure revealed that it binds two calcium ions at one end of the domain between three loops, called calcium binding regions (CBRs): CBR1, CBR2, and CBR3 (16Essen L.O. Perisic O. Lynch D.E. Katan M. Williams R.L. Biochemistry. 1997; 36: 2753-2762Crossref PubMed Scopus (131) Google Scholar). The calcium ions neutralize the negative electrostatic potential surrounding the hydrophobic residues located at the tips of CBRs 1 and 3, thereby facilitating their interactions with the hydrophobic portions of the lipid head groups of PC-enriched membranes (16Essen L.O. Perisic O. Lynch D.E. Katan M. Williams R.L. Biochemistry. 1997; 36: 2753-2762Crossref PubMed Scopus (131) Google Scholar). Phosphorylation of cPLA2 is important for regulating release of arachidonic acid in cells, but this process is not clearly understood. The catalytic domain of cPLA2 contains several functionally important phosphorylation sites (for review see Ref. 17Ghosh M. Tucker D. Burchett S. Leslie C. Prog. Lipid Res. 2006; 45: 487-510Crossref PubMed Scopus (304) Google Scholar), Ser505, Ser727, and Ser515, which are phosphorylated by mitogen-activated protein kinases (MAPKs), mitogen-activated protein kinase interacting kinase (MNK1), or a related MAPK-activated protein kinase, and calmodulin kinase II, respectively. It is suggest that, depending on the cell type and agonist used for activation, phosphorylation may function to regulate cPLA2 catalytic activity and membrane binding.We have previously demonstrated an essential requirement for cPLA2 in activation of the assembled phagocyte NADPH oxidase (18Dana R. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 1998; 273: 441-445Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar), the oxidase-associated H+ channel (19Lowenthal A. Levy R. J. Biol. Chem. 1999; 274: 21603-21608Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar), and oxidase-associated diaphorase activity (20Pessach I. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 2001; 276: 33495-33503Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The absolute requirement of cPLA2 for oxidase activation is in line with other studies (21Li Q. Cathcart M.K. J. Biol. Chem. 1997; 272: 2404-2411Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 22Bae Y.S. Kim Y. Kim J.H. Lee T.G. Suh P.G. Ryu S.H. J. Immunol. 2000; 164: 4089-4096Crossref PubMed Scopus (40) Google Scholar, 23O'Dowd Y.M. El-Benna J. Perianin A. Newsholme P. Biochem. Pharmacol. 2004; 67: 183-190Crossref PubMed Scopus (48) Google Scholar) utilizing inhibitors and antisense molecules. In contrast, phagocytes from cPLA2-deficient mice showed a normal stimulated superoxide release (24Rubin B.B. Downey G.P. Koh A. Degousee N. Ghomashchi F. Nallan L. Stefanski E. Harkin D.W. Sun C. Smart B.P. Lindsay T.F. Cherepanov V. Vachon E. Kelvin D. Sadilek M. Brown G.E. Yaffe M.B. Plumb J. Grinstein S. Glogauer M. Gelb M.H. Singer A. Borregaard N. Reithmeier R. Lichtenberger C. Reinisch W. Lambeau G. Arm J. Tischfield J. Dienstag J.L. Schiff E.R. Mitchell M. Casey Jr., D.E. Gitlin N. Lissoos T. Gelb L.D. Condreay L. Crowther L. Rubin M. Brown N. J. Biol. Chem. 2005; 280: 7519-7529Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar) that could be attributed to the effect of other compensating isoenzymes, a response frequently observed in knockout animal models. Our most recent study (25Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar) demonstrated that in peripheral blood neutrophils and granulocyte-like PLB-985 cells, cPLA2 translocates to the plasma membrane by interacting with the assembled oxidase complex in addition to its translocation to nuclear membranes. Thus, the ability of cPLA2 to colocalize in two different compartments in the same cells enables it to participate in both eicosanoid production and to regulate NADPH oxidase activation. The activation and translocation of cPLA2 by PMA (25Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar, 26Hazan I. Dana R. Granot Y. Levy R. Biochem. J. 1997; 326: 867-876Crossref PubMed Scopus (77) Google Scholar), which does not induce an increase in cytoplasmic [Ca2+], together with its translocation to the plasma membrane suggest the existence of alternative pathways for inducing translocation of cPLA2 that are distinct from the C2 domain phospholipid-binding mechanism. In agreement with our results, it was recently reported (27Girotti M. Evans J.H. Burke D. Leslie C.C. J. Biol. Chem. 2004; 279: 19113-19121Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar) that during phagocytosis of zymosan, cPLA2 translocates in a Ca2+-independent manner to the forming phagosomes in kinetics similar to acquisition of the plasma membrane and prior to phagolysosome fusion. The aim of the present study was to explore the nature of the interaction between cPLA2 and the assembled NADPH oxidase, which anchors cPLA2 to the plasma membrane upon stimulation of neutrophils or granulocyte-like PLB cells.EXPERIMENTAL PROCEDURESNeutrophil Purification—Neutrophils from healthy volunteers or from CGD patients were separated by Ficoll/Hypaque centrifugation, dextran sedimentation, and hypotonic lysis of erythrocytes (18Dana R. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 1998; 273: 441-445Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). p47phox-deficient CGD patients and p67phox-deficient CGD patients lacking expression of these cytosolic oxidase proteins due to mutations in the genes for neutrophil cytosolic factor 1 on chromosome 7q11.23 and in the gene for neutrophil cytosolic factor 2, located on 1q25, respectively, were enrolled in the study. The study was approved by the institutional Human Research Committee of the Soroka University Medical Center.Cell Culture and Differentiation—PLB-985 leukemic cell lines and gp91phox-deficient PLB-985 cells lacking normal expression of normal gp91phox (X-CGD), provided by M. C. Dinauer (James Whitcomb Riley Hospital for Children, Indianapolis, IN), were grown in stationary suspension culture in RPMI 1640 and differentiated toward the granulocyte phenotype with 10-6m retinoic acid as described earlier (18Dana R. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 1998; 273: 441-445Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar).Retroviral Transduction of gp91phox-deficient PLB-985 Cells—Retroviral gp91phox was expressed in gp91phox-deficient PLB-985 cells as done in our previous study (20Pessach I. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 2001; 276: 33495-33503Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).Cell Stimulation—Cells (5 × 106 cells/ml) in Hanks' balanced salt solution buffer were incubated with 1 mg/ml opsonized zymosan prepared as described before (28Hazan-Halevy I. Seger R. Levy R. J. Biol. Chem. 2000; 275: 12416-12423Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), 50 ng/ml PMA or 5 × 10-7m fMLP for 3 min at 37 °C.Superoxide Anion Measurements—The production of superoxide anion (O2.) by intact cells was measured as the superoxide dismutase inhibitable reduction of ferricytochrome c (18Dana R. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 1998; 273: 441-445Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar).Isolation of Membrane and Cytosol Fractions—Isolation of membrane and cytosol fractions was performed exactly as described earlier (29Levy R. Rotrosen D. Nagauker O. Leto T.L. Malech H.L. J. Immunol. 1990; 145: 2595-2601PubMed Google Scholar).Coimmunoprecipitation—Immunoprecipitation was performed as described earlier (25Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar). The detection of cPLA2 or the NADPH oxidase components after SDS-PAGE electrophoresis was analyzed as described previously (26Hazan I. Dana R. Granot Y. Levy R. Biochem. J. 1997; 326: 867-876Crossref PubMed Scopus (77) Google Scholar).Bacterial Expression and Purification of Recombinant Proteins—PCR was used to subclone the different cPLA2 regions: C2, Lid, or catalytic domain B, in-frame into the expression vector pGEX-4T-2 using primers containing the EcoRI or XhoI restriction endonucleases sites (underlined): 5′-TATAGAATTCTGTCATTTATAGATCCTTAC-3′,5′-TATACTCGAGATTAAACTTCAAGAGACATTTCTAG-3′,5′-TATAGAATTCGATTTTTTATGGGAACAG-3′, 5′-TATACTCGAGATTACTGTCCACTACATGAA-3′, 5′-TATAGAATTCCGCGAATATATGAGCCTC-3′, and 5′-TATACTCGAGCTATGCTTTGGGTTTATT-3′, respectively. The template used was pMT2 plasmid containing the cloned gene from the cDNA library. GST catalytic domain A construct has been described earlier (26Hazan I. Dana R. Granot Y. Levy R. Biochem. J. 1997; 326: 867-876Crossref PubMed Scopus (77) Google Scholar). The GST fusion proteins were overexpressed and purified as described previously (30Leto T.L. Adams A.G. de Mendez I. Proc. Natl. Acad. Sc.i U. S. A. 1994; 91: 10650-10654Crossref PubMed Scopus (242) Google Scholar). To isolate the p47phox N-terminal protein, the GST protein was cleaved from GST-p47phox N-terminal protein by the addition of 5 μg of factor Xa (New England Biolabs).Overlay Assay—Recombinant p47phox and p67phox were a kind gift from Prof. Edgar Pick (Tel-Aviv University, Israel). The different cPLA2 fusion proteins or cell lysates of 5 × 107 (25Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar) were separated on SDS-PAGE gel without β-mercaptoethanol and boiling. Protein renaturation was performed by incubation in 25% isopropanol solution for 30 min before immunoblotting (20Pessach I. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 2001; 276: 33495-33503Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar).Affinity Binding Assay—GST or GST fusion proteins were added to lysates of resting or stimulated neutrophils or to recombinant p47phox N-terminal in phosphate-buffered saline, and were tumbled end-over-end for 1 h at room temperature. The samples were washed six times with phosphate-buffered saline, boiled in SDS sample buffer, and separated SDS-PAGE before immunoblotting.FRET Analysis—Glass-adherent neutrophils or granulocyte-like PLB cells were fixed with 3.7% formaldehyde in phosphate-buffered saline, either resting or after stimulation for 3 min with 50 ng/ml PMA. The cells were co-immunostained with anti-cPLA2 mouse monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and either p67phox or p47phox polyclonal rabbit antibodies (31Leto T.L. Garrett M.C. Fujii H. Nunoi H. J. Biol. Chem. 1991; 266: 19812-19818Abstract Full Text PDF PubMed Google Scholar) and with Cy3-conjugated (anti-rabbit IgG) and Cy5-conjugated (anti-goat IgG) secondary antibodies, respectively (Jackson ImmunoResearch Laboratories). Primary and secondary antibodies were diluted 1:500/1:4000 for neutrophils and 1:400/1:500 for granulocyte-like PLB cells, respectively. Cells were imaged with a Zeiss LSM 510 laser scanning confocal microscope using the 543 nm Green HeNe laser line and a 560-615 nm band-pass filter for Cy3 and the 633 nm Red HeNe laser line and a 650 long pass filter for Cy5. Under these conditions, bleed-through between the Cy3 and Cy5 channels was insignificant. Cy3-sensitized emission of Cy5 was measured directly in the Cy5 channel while exciting Cy3 with the 543 nm line. A relative FRET index was calculated by normalizing the intensity of Cy3-sensitized Cy5 emission by the direct Cy3 emission intensity. FRET was further validated by donor recovery after acceptor photobleaching (32Kenworthy A.K. Methods. 2001; 24: 289-296Crossref PubMed Scopus (442) Google Scholar), by comparing Cy3 emission intensity before and after selective photobleaching of Cy5 with the 633 nm laser line. Mean fluorescence intensities were measured by calculating the average gray level of a circular region of interest encompassing each analyzed cell, correcting each channel for the background (offset), the average autofluorescence (determined in unstained samples), and the low bleed through between channels (calculated using singly stained samples).Neutrophil Permeabilization and Superoxide Production—Neutrophils were electroporated exactly as described earlier (20Pessach I. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 2001; 276: 33495-33503Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar) based on a previous study (34Grinstein S. Furuya W. J. Biol. Chem. 1988; 263: 1779-1783Abstract Full Text PDF PubMed Google Scholar). The effect of the synthetic peptides on binding and NADPH oxidase activity was studied as described (35Rotrosen D. Kleinberg M.E. Nunoi H. Leto T. Gallin J.I. Malech H.L. J. Biol. Chem. 1990; 265: 8745-8750Abstract Full Text PDF PubMed Google Scholar). The cell suspension was supplemented with 800 μm synthetic peptides (Sigma) incubated on ice for 30 min, centrifuged, and resuspended in the same supplemented buffer containing 200 μm peptide and 150 mm cytochrome c, stimulated for immediate measurement of superoxide production at 22 °C.Molecular Modeling—p47phox-PX with bound phosphatidylinositol 3-phosphate was docked to the C2 domain of cPLA2 (36Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) using the protein-protein docking program MolFit (37Katchalski-Katzir E. Shariv I. Eisenstein M. Friesem A.A. Aflalo C. Vakser I.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2195-2199Crossref PubMed Scopus (855) Google Scholar, 38Heifetz A. Katchalski-Katzir E. Eisenstein M. Protein Sci. 2002; 11: 571-587Crossref PubMed Scopus (117) Google Scholar, 39Ben-Zeev E. Eisenstein M. Proteins. 2003; 52: 24-27Crossref PubMed Scopus (49) Google Scholar, 40Berchanski A. Shapira B. Eisenstein M. Proteins. 2004; 56: 130-142Crossref PubMed Scopus (70) Google Scholar). The initial step consisted of MolFit docking of the p47phox-PX in complex with phosphatidylinositol 3-phosphate and cPLA2-C2 domain (36Perisic O. Fong S. Lynch D.E. Bycroft M. Williams R.L. J. Biol. Chem. 1998; 273: 1596-1604Abstract Full Text Full Text PDF PubMed Scopus (240) Google Scholar) (PDB code 1rlw), employing translational interval of 1.05 Å and rotational interval of 12°. The docking models were filtered, selecting models in which the active site in the catalytic domain of intact cPLA2 (15Dessen A. Tang J. Schmidt H. Stahl M. Clark J.D. Seehra J. Somers W.S. Cell. 1999; 97: 349-360Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar) pointed toward the membrane, as deduced from the position of phosphatidylinositol 3-phosphate. In the second docking stage, the interactions of inhibitory peptides identified experimentally in this study were up-weighted. A similar filtering procedure for the position of the active site of cPLA2 was employed as in the first docking stage, and the accepted models were clustered. The final models were energy-minimized. Details of the procedure are given as supplemental text.Mutagenesis of Expression Vectors—pGEX-4T-2 expression vector encoding the cDNA of cPLA2-C2 domain or pGEX-3X expression vector encoding the cDNA of p47phox-PX domain were used as a template, to generate the desired mutations introduced according to the molecular modeling, and generated by the overlap extension PCR (41Ho S. Hunt H. Horton R. Pullen J. Pease L. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6795) Google Scholar). The PCR reactions, using appropriate complementary synthetic oligonucleotides introducing the desired mutation and two additional primers at the ends of the cPLA2-C2 fragment and of p47phox-PX, were performed with Red Load Taq Master/high yield using Thermostable DNA polymerase (Larova, Germany) with appropriate complementary synthetic oligonucleotides that introduced the desired mutation and two additional primers at the ends of the cPLA2-C2 fragment. The mutated products were digested with EcoRI and XhoI or with BamH1 and EcoRI and cloned into pGEX-4T-2 or pGEX-3X expression vectors, respectively, digested with the same enzymes. The vectors were then transformed into Escherichia coli DH-101. The mutated fragments were sequenced using the ABI3100 Genetic Analyzer. The first three mutations in cPLA2-C2 (F35A plus M38A plus L39A) were mutated in combination to generate triple mutation. The I67A mutation was mutated alone or in addition to the three mutations shown above. M59A and H115A in the p47phox-PX were mutated alone or together.Statistical Analysis—The mean differences were analyzed by Student's t test.RESULTS AND DISCUSSIONFlavocytochrome b558 Alone Is Not Sufficient to Anchor cPLA2 to Plasma Membranes after Cell Stimulation—To define the exact binding domains between cPLA2 and the assembled NADPH oxidase we first explored the oxidase subunits responsible for recruitment of cPLA2 to the plasma membrane. To study whether cPLA2 binds the cytochrome b558, cPLA2 translocation to the plasma membranes was examined in undifferentiated X-CGD PLB cells transduced with full-length gp91phox (XCGD-gp91) as reported in our previous studies (20Pessach I. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 2001; 276: 33495-33503Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 25Shmelzer Z. Haddad N. Admon E. Pessach I. Leto T.L. Eitan-Hazan Z. Hershfinkel M. Levy R. J. Cell Biol. 2003; 162: 683-692Crossref PubMed Scopus (79) Google Scholar). As shown in Fig. 1A, membranes were prepared from XCGD-gp91 PLB cells expressing gp91phox protein, and its level did not change after differentiation. Stimulation with PMA did not cause any translocation of cPLA2 to the plasma membrane of undifferentiated XCGD-gp91phox PLB cells that do not express any other cytosolic oxidase subunits, indicating that cPLA2 does not bind the gp91phox itself. During differentiation the cells acquire the NADPH oxidase subunits as reported previously (18Dana R. Leto T.L. Malech H.L. Levy R. J. Biol. Chem. 1998; 273: 441-445Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). In the presence of the cytosolic oxidase subunits in differentiated XCGD-gp91phox PLB cells, stimulation did cause translocation of cPLA2 to the plasma membranes, consistent with assembly of the oxidase, as determined by the translocation of p47phox. In addition, the specific protein kinase C inhibitor GF-109203X attenuated PMA-stimulated translocation of both p47phox and cPLA2 in a similar dose-dependent manner (Fig. 1B), consistent with our previous study reporting that GF-109203X causes the same dose response inhibition of p47phox phosphorylation, oxidase assembly, and activity (26Hazan I. Dana R. Granot Y. Levy R. Biochem. J. 1997; 326: 867-876Crossref PubMed Scopus (77) Google Scholar). Taken together, these results suggest that cPLA2 does not bind directly to flavocytochrome b558 but, rather, binds to the assembled oxidase complex.cPLA2 Is Bound to p47phox in the Assembled Oxidase—To determine which cytosolic component in the assembled oxidase is bound to cPLA2 after stimulation, neutrophils from p67phox-deficient CGD patients were studied, as shown by immunoblot of their cytosol (Fig. 2A). Activation of these cells resulted in translocation of p47phox and cPLA2 to the membrane fractions (Fig. 2A) similar to the translocation of these proteins in neutrophils from healthy donors, who express all oxidase components (Fig. 2C). In contrast, when neutrophils from p47phox-deficient CGD patients were stimulated, there was no translocation of either p67phox or cPLA2 (Fig. 2B). The lack of p67phox translocation is expected, because it is dependent on the translocation of p47phox, which is missing in these cells. Addition of antibodies against cPLA2 to the membrane fractions of activated neutrophils fr
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