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• W2012604352 abstract "β-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O⨪2). Neutrophils and neutrophilic HL60 cells contain both βI and βII-PKC, isotypes that are derived by alternate splicing. βI-PKC-positive and βI-PKC null HL60 cells generated equivalent amounts of O⨪2 in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of βII-PKC from βI-PKC null cells inhibited ligand-initiated O⨪2generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47phox, with βII-PKC but not with RACK1, a binding protein for βII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of β-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O⨪2 generation. Therefore, βII-PKC but not βI-PKC is essential for activation of O⨪2generation and plays a positive role in signaling for NADPH oxidase activation in association with p47phox. In contrast, RACK1 is involved in negative signaling for O⨪2 generation. RACK1 binds to βII-PKC but not with the p47phox ·βII-PKC complex. RACK1 may divert βII-PKC to other signaling pathways requiring β-PKC for signal transduction. Alternatively, RACK1 may sequester βII-PKC to down-regulate O⨪2 generation. β-Protein kinase (PKC) is essential for ligand-initiated assembly of the NADPH oxidase for generation of superoxide anion (O⨪2). Neutrophils and neutrophilic HL60 cells contain both βI and βII-PKC, isotypes that are derived by alternate splicing. βI-PKC-positive and βI-PKC null HL60 cells generated equivalent amounts of O⨪2 in response to fMet-Leu-Phe and phorbol myristate acetate. However, antisense depletion of βII-PKC from βI-PKC null cells inhibited ligand-initiated O⨪2generation. fMet-Leu-Phe triggered association of a cytosolic NADPH oxidase component, p47phox, with βII-PKC but not with RACK1, a binding protein for βII-PKC. Thus, RACK1 was not a component of the signaling complex for NADPH oxidase assembly. Inhibition of β-PKC/RACK1 association by an inhibitory peptide or by antisense depletion of RACK1 enhanced O⨪2 generation. Therefore, βII-PKC but not βI-PKC is essential for activation of O⨪2generation and plays a positive role in signaling for NADPH oxidase activation in association with p47phox. In contrast, RACK1 is involved in negative signaling for O⨪2 generation. RACK1 binds to βII-PKC but not with the p47phox ·βII-PKC complex. RACK1 may divert βII-PKC to other signaling pathways requiring β-PKC for signal transduction. Alternatively, RACK1 may sequester βII-PKC to down-regulate O⨪2 generation. HL60 cells differentiated to a neutrophil phenotype N-formyl-methionyl-leucyl-phenylalanine superoxide anion protein kinase C polyacrylamide gel electrophoresis phorbol myristate acetate 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethylammoniumbromide/cholesterol phosphatidylserine diglyceride polyvinylidene difluoride bovine serum albumin density units anti-sense missense recombinant human βII Ligand-initiated activation of superoxide anion (O⨪2) generation by phagocytic cells such as neutrophils and neutrophilic-differentiated HL60 cells (dHL60 cells),1 involves translocation of cytosolic components p47phox and p67phox to the membrane and interaction with membrane-associated cytochrome b558 (1Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 2Robinson J.M. Badwey J.A. Histochemistry. 1995; 103: 163-180Crossref PubMed Scopus (93) Google Scholar). Protein kinase C (PKC), a phospholipid-dependent family of serine/threonine kinases, acts in the signal transduction pathway for O⨪2 generation and is critical for assembly of an active NADPH oxidase (3Curnutte J.T. Erickson R.W. Ding J. Badwey J.A. J. Biol. Chem. 1994; 269: 10813-10819Abstract Full Text PDF PubMed Google Scholar, 4Majumdar S. Rossi M.W. Fujiki T. Phillips W.A. Disa S. Queen C.F. Johnston Jr., R.B. Rosen O.M. Corkey B.E. Korchak H.M. J. Biol. Chem. 1991; 266: 9285-9294Abstract Full Text PDF PubMed Google Scholar, 5Park 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 (73) Google Scholar, 6Inanami O. Johnson J.L. McAdara J.K. El Benna J. Faust L.R.P. Newburger P.E. Babior B.M. J. Biol. Chem. 1998; 273: 9539-9543Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 7Park H.-S. Kim I.S. Park J.-W. Biochem. Biophys. Res. Commun. 1999; 259: 38-42Crossref PubMed Scopus (15) Google Scholar). Phosphorylation of p47phox allows a conformational change, translocation of the p47phox to the membrane, and assembly of an active NADPH oxidase (7Park H.-S. Kim I.S. Park J.-W. Biochem. Biophys. Res. Commun. 1999; 259: 38-42Crossref PubMed Scopus (15) Google Scholar). p47phox contains multiple phosphorylation sites including classical PKC substrate sites; p47phox is phosphorylated by β-PKC in vitro and is phosphorylated in ligand-activated phagocytic cells (1Babior B.M. Blood. 1999; 93: 1464-1476Crossref PubMed Google Scholar, 2Robinson J.M. Badwey J.A. Histochemistry. 1995; 103: 163-180Crossref PubMed Scopus (93) Google Scholar, 3Curnutte J.T. Erickson R.W. Ding J. Badwey J.A. J. Biol. Chem. 1994; 269: 10813-10819Abstract Full Text PDF PubMed Google Scholar, 8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). PKC is a family of structurally related isotypes, with differing cofactor requirements but similar substrate specificity (9Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1215) Google Scholar, 10Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar, 11Mosior M. Newton A.C. J. Biol. Chem. 1995; 270: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 12Yan X. Curley K. Lawrence D.S. Biochem. J. 2000; 349: 709-715Crossref PubMed Scopus (10) Google Scholar). Classical PKC isotypes α-PKC, β-PKC, and γ-PKC are phosphatidylserine (PS)-, diglyceride (DG)-, and Ca2+-dependent; novel PKC isotypes δ-PKC, ε-PKC, and θ- and η-PKC also require PS and DG but are Ca2+-independent. The atypical PKC isotypes, ζ-PKC, and λ-PKC, require PS but are DG- and Ca2+-independent (9Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1215) Google Scholar, 10Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar, 11Mosior M. Newton A.C. J. Biol. Chem. 1995; 270: 25526-25533Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 12Yan X. Curley K. Lawrence D.S. Biochem. J. 2000; 349: 709-715Crossref PubMed Scopus (10) Google Scholar). PKC isotypes differ in their tissue distribution and localization within the cell, suggesting that each isotype plays a specific role in specific signal transduction pathways. dHL60 cells and neutrophils possess multiple PKC isotypes including α-PKC, βI-PKC, βII-PKC, δ-PKC, and ζ-PKC (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar,13Wooten M.W. Seibenhener M.L. Soh Y. Cytobios. 1993; 76: 19-29PubMed Google Scholar, 14Sargeant S. McPhail L.C. J. Immunol. 1997; 159: 2877-2885PubMed Google Scholar, 15Majumdar S. Kane L., H. Rossi M.W. Volpp B.D. Nauseef W.M. Korchak H.M. Biochim. Biophys. Acta. 1993; 1176: 276-286Crossref PubMed Scopus (121) Google Scholar). Depletion of β-PKC by antisense pretreatment was previously shown to inhibit phosphorylation of p47phox, translocation of p47phox to the membrane, and generation of O⨪2 in response to cell activation by ligands such as fMet-Leu-Phe or to the PKC activator phorbol myristate acetate (PMA) (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The ability of a β-PKC specific inhibitor to reduce ligand-initiated O⨪2generation also indicated that β-PKC is essential for activation of O⨪2 generation (16Dekker L.V. Leitges M. Altschuler G. Mistry N. McDermott A. Roes J. Segal A.W. Biochem. J. 2000; 347: 285-289Crossref PubMed Scopus (144) Google Scholar). However, these studies did not distinguish between a role for βI-PKC or βII-PKC, isotypes that are identical except for the C-terminal V5 variable region (9Hug H. Sarre T.F. Biochem. J. 1993; 291: 329-343Crossref PubMed Scopus (1215) Google Scholar, 10Mellor H. Parker P.J. Biochem. J. 1998; 332: 281-292Crossref PubMed Scopus (1345) Google Scholar). The antisense oligonucleotide targeted the transcriptional start site, which is common to both these isoforms, and the inhibitor inhibited both βI-PKC and βII-PKC (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 16Dekker L.V. Leitges M. Altschuler G. Mistry N. McDermott A. Roes J. Segal A.W. Biochem. J. 2000; 347: 285-289Crossref PubMed Scopus (144) Google Scholar). Formation of a signaling complex that can target β-PKC to substrates such as p47phox and p47phox to the cell membrane is essential for specificity and efficiency of signal transduction (17Schillace R.V. Scott J.D. J. Clin. Invest. 1999; 103: 761-765Crossref PubMed Scopus (93) Google Scholar). However β-PKC plays a role in signaling for multiple cell responses. β-PKC is essential for both proliferation (18Gamard C.J. Blobe G.C. Hannun Y.A. Obeid L.M. Cell Growth Differ. 1994; 5: 405-409PubMed Google Scholar) and for O⨪2generation in HL60 cells (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar), events that occur at the nucleus and plasmalemma, respectively. β-PKC also associates with the cytoskeleton (19Niggli V. Djafarzadeh S. Keller H. Exp. Cell Res. 1999; 250: 558-568Crossref PubMed Scopus (35) Google Scholar). Therefore spatial considerations are a key element in defining a role for β-PKC in signal transduction for a particular response. β-PKC must be directed to different locations in the cell for each function, suggesting a role for scaffold proteins or PKC-binding proteins in β-PKC-based signaling for activation of O⨪2 generation (20Sim A.T.R. Scott J.D. Cell Calcium. 1999; 26: 209-217Crossref PubMed Scopus (86) Google Scholar, 21Mochly-Rosen D. Kauvar L.M. Semin. Immunol. 2000; 12: 55-61Crossref PubMed Scopus (34) Google Scholar, 22Newton A.C. Curr. Biol. 1996; 6: 806-809Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Receptor for Activated CKinase (RACKs) are a family of cytoskeleton and membrane-associated anchor molecules that bind activated, Ca2+/DG-dependent PKC isotypes α-, β-, and γ-PKC as well as phospholipase Cγ (23Mochly-Rosen D. Gordon A.S. FASEB J. 1998; 12: 35-42Crossref PubMed Scopus (507) Google Scholar, 24Mochly-Rosen D. Khaner H. Lopez J. Smith B.L. J. Biol. Chem. 1991; 266: 14866-14868Abstract Full Text PDF PubMed Google Scholar, 25Souroujon M.C. Mochly-Rosen D. Nat. Biotechnol. 1998; 16: 919-924Crossref PubMed Scopus (201) Google Scholar, 26Smith B.L. Mochly-Rosen D. Biochem. Biophys. Res. Commun. 1992; 188: 1235-1240Crossref PubMed Scopus (46) Google Scholar, 27Ron D. Mochly-Rosen D. J. Biol. Chem. 1994; 269: 21395-21398Abstract Full Text PDF PubMed Google Scholar, 28Ron D. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 492-496Crossref PubMed Scopus (176) Google Scholar, 29Disatnik M-H. Hernandez-Sotomayor S.M.T. Jones G. Carpenter G. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 559-563Crossref PubMed Scopus (68) Google Scholar, 30Ron D. Chen C-H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (639) Google Scholar). PKC isotypes possess a pseudo-Rack binding site in the Ca2+ binding domain of α, β, and γ-PKC (28Ron D. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 492-496Crossref PubMed Scopus (176) Google Scholar). The conformational change in PKC induced by cofactors frees the RACK binding site and allows the PKC to bind to RACK. Thus cofactors simultaneously activate and target PKC isotypes. A peptide based on a sequence in annexin I disrupts the binding of PKC to RACK (24Mochly-Rosen D. Khaner H. Lopez J. Smith B.L. J. Biol. Chem. 1991; 266: 14866-14868Abstract Full Text PDF PubMed Google Scholar, 25Souroujon M.C. Mochly-Rosen D. Nat. Biotechnol. 1998; 16: 919-924Crossref PubMed Scopus (201) Google Scholar, 26Smith B.L. Mochly-Rosen D. Biochem. Biophys. Res. Commun. 1992; 188: 1235-1240Crossref PubMed Scopus (46) Google Scholar, 27Ron D. Mochly-Rosen D. J. Biol. Chem. 1994; 269: 21395-21398Abstract Full Text PDF PubMed Google Scholar). When peptide I is injected into Xenopusoocytes, it inhibits insulin-induced translocation of β-PKC and oocyte maturation (25Souroujon M.C. Mochly-Rosen D. Nat. Biotechnol. 1998; 16: 919-924Crossref PubMed Scopus (201) Google Scholar, 26Smith B.L. Mochly-Rosen D. Biochem. Biophys. Res. Commun. 1992; 188: 1235-1240Crossref PubMed Scopus (46) Google Scholar). RACK1 is a binding protein for βII-PKC (31Ron D. Jiang Z. Yao L. Vagts A. Diamond I. Gordon A. J. Biol. Chem. 1999; 274: 27039-27046Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In this study we have assessed the roles of the PKC isotypes βI-PKC and βII-PKC in O⨪2 generation and, secondly, the role of RACK1 in β-PKC-based signaling for O⨪2 generation. A βI-PKC null subclone of HL60 cells (32Hocevar B.A. Fields A.P. J. Biol. Chem. 1991; 266: 28-33Abstract Full Text PDF PubMed Google Scholar) and depletion of βII-PKC by an antisense strategy was used to demonstrate that βII-PKC, but not βI-PKC, is necessary for activation of O⨪2 generation. rhβII-PKC bound to a number of endogenous HL60 proteins in a cofactor-dependent manner, including RACK1 and p47phox. Depletion of RACK1 by an antisense strategy and electroporation of cells with a peptide that inhibits the RACK1-βII-PKC interaction enhanced fMet-Leu-Phe- and PMA-induced O⨪2 generation. Therefore RACK1 is not essential in signaling for activation of the NADPH oxidase but may down-regulate βII-PKC-based signaling either by diverting the βII-PKC to another signaling pathway or by sequestering βII-PKC as part of a down-regulation step in O⨪2 generation. Human promyelocytic HL60 leukemia cells were obtained from the American Type Culture Collection. The cells were grown in suspension culture in RPMI 1640 medium supplemented with 2 mml-glutamine, 1% minimal essential medium vitamin solution, 1% nonessential amino acids, 0.1% gentamicin, and 10% heat-inactivated fetal bovine serum. The cell cultures were maintained at 37 °C in a 5% CO2-humidified atmosphere. The initial culture was positive for α-PKC, βI-PKC, βII-PKC, δ-PKC, and ζ-PKC. A clone that was protein null for βI-PKC but positive for α-PKC, βII-PKC, δ-PKC, and ζ-PKC was selected (32Hocevar B.A. Fields A.P. J. Biol. Chem. 1991; 266: 28-33Abstract Full Text PDF PubMed Google Scholar). An antisense oligonucleotide was designed against the translation start site of human RACK1 using the commercial primer analysis software Oligo (National Biosciences). A 20-mer sequence was chosen that was without significant self-complimentarity and was optimized for maximalTm to promote high affinity binding to mRNA; aTm of 64.6 °C was calculated at 150 mm salt and 37 °C. The 20-mer oligonucleotides had the following sequences: RACK1 antisense (RACK1 AS): 5′-T GCC ACG AAG GGT CAT CTG C-3′; RACK1 sense: 5′-G CAG ATG ACC CTT CGT GGC A-3′. A scrambled missense oligonucleotide of the RACK1 AS was used as a control. The unique nature of these sequences was confirmed by searching the GenBankTM data base. For depletion of β-PKC, a 19-mer oligonucleotide having the sequence β-PKC antisense (βAS), 5′-AGC CGG GTC AGC CAT CTT G-3′, and a scrambled missense oligonucleotide β-PKC missense (βMS) were used as previously described (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Antisense and scrambled control oligonucleotides to RACK1 and β-PKC were synthesized by the PENN Nucleic Acid Facility as the phosphorothioate derivatives and purified by high performance liquid chromatography. In all oligonucleotides, the internucleoside linkages were completely phosphorothioate-modified. Delivery of the oligonucleotides was enhanced with the cationic lipid 1,2-dimyristoyloxypropyl-3-dimethylhydroxyethyl ammonium bromide/cholesterol (DMRIE-C) (1:1 (m/m)) (Life Technologies, Inc.). HL60 cells were cultured in the presence of 1.3% Me2SO for 4 days to initiate differentiation to a neutrophil-like phenotype before treatment with the oligonucleotide. On day 4, the cells were washed and resuspended in Opti-MEM I reduced serum medium (Life Technologies, Inc.) at a cell concentration of 25 × 106 cells/well. Oligonucleotides RACK1 AS, RACK1 MS, βAS, or βMS were suspended in Optimem at a final concentration of 100–1000 nm and mixed with the cationic lipid DMRIE-C (4 μg/ml). The cationic lipid/oligonucleotide mixture was added to the cells and incubated at 37 °C for 4 h. An equal volume of RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum plus Me2SO (1.3% final concentration) was then added, and the cells were cultured for 20 h. On day 5, the cells were washed and resuspended in fresh Opti-MEM medium and treated again with the cationic lipid/oligonucleotide mixture. After a 4-h incubation, an equal volume of RPMI 1640 medium containing 20% heat-inactivated fetal bovine serum plus Me2SO (1.3% final concentration) was then added, and the cells were cultured for an additional 24 h. The cells were harvested and suspended in Hepes buffer (pH 7.5) having the composition 150 mm Na+, 5 mm K+, 1.29 mm Ca2+, 1.2 mmMg2+, 155 mm Cl−, and 10 mm Hepes (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Lysates of dHL60 cells (1 × 106 cells/sample) were prepared by heating the cells at 95 °C for 5 min in 2× SDS-PAGE sample buffer. The samples were briefly sonicated (12 s) to reduce viscosity. The dHL60 cell lysates were run on a 4–12% gradient SDS-PAGE, transferred to a PVDF membrane, and blocked for 1 h at room temperature with Tris-buffered saline (pH 7.5) containing 0.1% Tween 20 and 1% BSA, 3% casein. To identify the different PKC isotypes, the membrane was incubated with a panel of PKC antibodies followed by incubation with peroxidase-conjugated goat anti-rabbit IgG. For detection of RACK1, the membrane was incubated with a monoclonal antibody to RACK1, followed by incubation with peroxidase-conjugated goat anti-mouse IgM. Immunoreactive bands were visualized by Pierce SuperSignal Ultra chemiluminescence substrate. The software SigmaProscan (Jandel/SPSS) was used for densitometric analysis. The generation of superoxide anion (O⨪2) by dHL60 cells was measured as superoxide dismutase inhibitable cytochrome c reduction by either a continuous recording method (33Korchak H.M. Vienne K. Rutherford L.E. Wilkenfeld C. Finkelstein M.C. Weissmann G. J. Biol. Chem. 1984; 259: 4076-4082Abstract Full Text PDF PubMed Google Scholar) or end point analysis. Cells were activated by 1 μm fMet-Leu-Phe in the presence of 5 μg/ml cytochalasin B or by 1 μg/ml PMA in the absence of cytochalasin B. dHL60 cells (50 × 106 cells/ml) were stimulated with either buffer alone or fMet-Leu-Phe (1 μm) for 1 min. The reaction was stopped by the addition of cold immunoprecipitation buffer. Immunoprecipitation buffer consisted of 10 mm Hepes (pH 7.4) containing 150 mm NaCl, 5 mm EDTA, 1 mm sodium orthovanadate, 2 mmphenylmethanesulfonyl fluoride, 0.2% Nonidet P-40, 0.027 trypsin inhibitory units/ml of aprotinin, 2 μg/ml leupeptin, and 5 mg/ml BSA. The samples were then vortexed for 20 min to solubilize the membrane fraction, and the supernatant was collected after microcentrifuging for 5 min. A rabbit polyclonal antibody to p47phox or to βII-PKC was added, and the samples were incubated for 2 h at 4 °C. Protein A-agarose was added, and the samples were incubated for 1 h at 4 °C with shaking. The reaction tubes were then microcentrifuged for 30 s, and the supernatants were discarded. The protein A-agarose pellet was washed four times with immunoprecipitation buffer, and the sample was eluted by incubation for 20 min at 65 °C in 2× SDS-PAGE sample buffer. The ability of rhβII-PKC to bind to endogenous proteins from dHL60 cells was assayed by an overlay procedure (30Ron D. Chen C-H. Caldwell J. Jamieson L. Orr E. Mochly-Rosen D. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 839-843Crossref PubMed Scopus (639) Google Scholar). Lysates of dHL60 cells were separated by SDS-PAGE and transferred to PVDF membranes. Membrane strips were incubated with overlay block buffer consisting of 50 mm Tris-HCl (pH 7.5), 0.1% polyethylene glycol, 0.2 m NaCl, and 3% BSA for 1 h. rhβII-PKC (0.1 ng) was bound to the membrane strip for 30 min at room temperature in an overlay incubation buffer consisting of 50 mm Tris-HCl (pH 7.5), 0.1% polyethylene glycol, 0.2m NaCl, 12 mm β-mercaptoethanol, 0.1% BSA, 5 μg/ml leupeptin, 10 μg/ml soy bean trypsin inhibitor, 20 mm phenylmethanesulfonyl fluoride in the presence or absence of 0.1 mm CaCl2, 50 μg/ml PS, and 1 mg/ml DG. The membranes were washed three times for 15 min with phosphate-buffered saline/Tween (140 mm NaCl, 8 mm Na2HPO4, 1.5 mmKH2PO4, 3 mm KCl, 0.05% Tween 20 (pH 7.0)) and probed with antibodies to βII-PKC or RACK1. A method of electroporation was chosen that allows efficient incorporation of molecules of molecular mass <1000 Da and transient passage of molecules >1000Da. We chose a one-pulse protocol to optimize preservation of intracellular metabolites. The cells were electroporated once in a Bio-Rad Gene Pulser at 400 V and a capacitance of 500 microfarads. dHL60 cells were suspended in 800 ml of ice-cold Hepes buffer containing 1 mm ATP and 1 mm NADPH in the presence or absence of 200 μm peptide I. Previous studies using the fluorescent probe Dextran-Indo1 conjugate indicated that the attained intracellular concentration of peptides under these electroporation conditions is ∼10 μm (15Majumdar S. Kane L., H. Rossi M.W. Volpp B.D. Nauseef W.M. Korchak H.M. Biochim. Biophys. Acta. 1993; 1176: 276-286Crossref PubMed Scopus (121) Google Scholar). Results are expressed as mean ± S.E. (n). Data were analyzed by Student's ttest. Cytochalasin B, cytochrome c, protease inhibitors (leupeptin, soy bean trypsin inhibitor, and aprotinin), BSA, PMA, fMet-Leu-Phe, and phenylmethanesulfonyl fluoride were purchased from Sigma. PMA was stored as a concentrated stock solution in Me2SO and diluted with Hepes Buffer before use. fMet-Leu-Phe was stored as a stock solution in ethanol and diluted in buffer before use. Peptide I, KGDYEKILVALCGGN, was purchased from Coast Scientific. Anti-peptide polyclonal antibodies to α-PKC, βI-PKC, βII-PKC, γ-PKC, and δ-PKC and peroxidase-conjugated goat anti-rabbit IgG and peroxidase-conjugated goat anti-mouse IgG were obtained from Santa Cruz Biotechnology. Peroxidase-conjugated anti-mouse IgM was obtained from Kirkegaard and Perry Laboratories. Mouse monoclonal antibodies to δ-PKC, ζ-PKC, and RACK1 and a rabbit polyclonal antibody to p47phox were purchased from Transduction Laboratories. Protein A-agarose was obtained from Life Technologies, Inc. A clone of HL60 cells that contained the βII-PKC isotype of PKC as well as α-PKC, δ-PKC, and ζ-PKC was selected and probed for immunoreactivity to PKC antibodies (Fig.1 A). In comparison to the parent cell line, which contains βI-PKC, the βI-PKC protein null line contained no detectable amount of βI-PKC (Fig. 1 A). In contrast, both βI-PKC-positive and βI-PKC null cell lines contained equivalent amounts of α-PKC, δ-PKC, and ζ-PKC. Previous studies in which both βI-PKC and βII-PKC were depleted by an antisense strategy demonstrated a role for β-PKC in activation of the NADPH oxidase in dHL60 cells (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). To discriminate between roles for βI-PKC and βII-PKC in ligand-initiated activation of O⨪2generation, we compared fMet-Leu-Phe-triggered O⨪2 generation in βI-PKC null and βI-PKC-positive dHL60 cells. Generation of O⨪2 triggered by 1 μm fMet-Leu-Phe was 11.3 ± 1.9 (n = 12) nmol/106 cells/10 min in βI-PKC null dHL60 cells, a rate that was not significantly different from the rate of 11.2 ± 1.8 (n = 8) nmol/106 cells/10 min observed in βI-PKC-positive dHL60 cells (Fig. 1 B). Generation of O⨪2 in response to 1 μg/ml PMA was also similar in βI-PKC null and βI-PKC-positive dHL60 cells (Fig. 1 B). In βI-PKC-positive dHL60 cells, PMA triggered generation of 22.3 ± 4.1 (n = 5) nmol of O⨪2/106 cells/10 min, whereas in βI-PKC cells, PMA triggered generation of 21.2 ± 1.7 (n = 6) nmol of O⨪2/106 cells/10 min (Fig. 1 B). Therefore, βI-PKC was not essential for optimal PMA- or fMet-Leu-Phe-initiated generation of O⨪2 in dHL60 cells. βI-PKC null dHL60 cells were treated for 2 days with 400 nm of an antisense oligonucleotide to β-PKC (βPKC AS) or with 400 nm control missense oligonucleotide to β-PKC (βPKC MS) as described under “Materials and Methods.” Treatment with βPKC AS resulted in a reduction in the level of βII-PKC to 362 ± 47 (n = 4) density units (DU) as compared with a level of 603 ± 34 (n = 4) DU in control βPKC MS-treated cells (62.2 ± 10.0% control,p < 0.02) (Fig. 2,A and B). Treatment of the dHL60 cells with the antisense or missense oligonucleotides to β-PKC had no significant effect on the levels of α-PKC, δ-PKC, or ζ-PKC (Fig. 2,A and B) and as previously shown (8Korchak H.M. Rossi M.W. Kilpatrick L.E. J. Biol. Chem. 1998; 273: 27292-27299Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). The effect of depletion of βII-PKC on O⨪2 generation was determined in βI-PKC null cells treated with βPKC AS or βPKC MS. fMet-Leu-Phe (1 μm) triggered generation of 5.3 ± 1.5 (n = 6) nmol of O⨪2/106cells/10 min in βPKC AS-treated βI-PKC null cells, which was significantly less than the level of 12.2 ± 2.7 (n = 6) nmol/106 cells/10 min observed in control cells pretreated with βPKC MS (41.7 ± 9.5% control,p < 0.025) (Fig.3 A). The kinetics of fMet-Leu-Phe-activated O⨪2 generation are characterized by a rapid initial rate continuing for ∼2 min followed by a slow rate of generation that ceases by 5–10 min. In contrast, PMA triggers a sustained generation of O⨪2. The Vmax of fMet-Leu-Phe-induced O⨪2 generation, defined as the maximal rate of O⨪2 generation, was reduced in cells depleted of β-PKC. Calculation of the Vmax demonstrated that in control βPKC MS-treated cells activated by 1 μmfMet-Leu-Phe, the Vmax was 2.70 ± 0.44 (n = 6) nmol/min/106 cells; theVmax of β-PKC-depleted cells treated with βPKC AS was significantly reduced to 1.41 ± 0.74 (n = 6) nmol/min/106 cells (53.0 ± 7.2% control βPKC MS-treated cells (p < 0.01) (Fig.3 B). Similarly, activation of control βPKC MS-pretreated cells by 1 μg/ml PMA triggered generation of 16.6 ± 1.5 (n= 7) nmol of O⨪2/106 cells/10 min, an amount that was significantly greater than the generation of 8.2 ± 1.3 (n = 7) nmol of O⨪2/106 cells/10 min observed in dHL60 cells depleted of β-PKC by treatment with βPKC AS (48.7 ± 6.2% control, p < 0.01) (Fig.3 A). The Vmax of PMA-induced O⨪2 generation was also significantly decreased in β-PKC-depleted cells as compared with controls. TheVmax of O⨪2 generation decreased significantly from a rate of 3.12 ± 0.37 (n = 7) nmol/min/106 cells in control βPKC MS-treated cells to aVmax of 2.06 ± 0.27 (n = 8) nmol/min/106 cells in β-PKC-depleted cells (66.1 ± 8.2% control, p < 0.01) (Fig. 3 B). Thus, depletion of βII-PKC in βI-PKC null dHL60 cells resulted in inhibition of the rate of O⨪2 generation in response to both fMet-Leu-Phe and PMA, indicating an essential role for the βII isotype but not the βI isotype of PKC in signaling for O⨪2generation. β-PKC is capable of phosphorylating multiple proteins in vitro (15Majumdar S. Kane L., H. Rossi M.W. Volpp B.D. Nauseef W.M. Korchak H.M. Biochim. Biophys. Acta. 1993; 1176: 276-286Crossref PubMed Scopus (121) Google Scholar). However, in the intact cell, scaffold proteins may provide added substrate specificity by targeting the kinase to a particular cellular location. RACK1 is a scaffold or escort protein that selectively binds to βII-PKC in the presence of the cofactors PS, DG, and Ca2+. To assess the presence of binding proteins for βII-PKC in dHL60 cells, we tested the ability of rhβII-PKC to bind to endogenous dHL60 proteins using an overlay assay. Lysates of βI-PKC null dHL60 cells were separated on SDS-PAGE and transferred to PVDF membranes, and the membranes were incubated with rhβII-PKC in the presence and absence of the PKC cofactors PS, DG, and Ca2+. Western blotting with an antibody to βII-PKC demonstrated that the exogenous rhβII-PKC bound to several endogenous proteins in a cofactor-dependent manner (Fig. 4 A). In the absence of exogenous rhβII-PKC and cofactors, a band at 80 kDa corresponding to the endogenous βII-PKC was observed (Fig.4 A, lane 1). rhβII-PKC in the absence of cofactors bound strongly only to a protein of 19 kDa (Fig.4 A, lane 2). However when rhβII-PKC was added in the presence of the cofactors PS, DG, and Ca2+, additional binding of rhβII-PKC to bands of 29, 32, 36, 39, 47, and 55 kDa was observed (Fig. 4 A, lane 3). Probing with an antibody to RACK1 (Fig. 4 A, lane 4) showed a strong band at 36 kDa, demonstrating the presence of RACK1 in dHL60 cells. Quantitation by densitometry demonstrated that the band at 80 kDa corresponding to endogenous βII-PKC was" @default.
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• W2012604352 title "Roles for βII-Protein Kinase C and RACK1 in Positive and Negative Signaling for Superoxide Anion Generation in Differentiated HL60 Cells" @default.
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