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• W1994633260 abstract "Mitochondrial protein kinase C isozymes have been reported to mediate both cardiac ischemic preconditioning and ischemia/reperfusion injury. In addition, cardiac preconditioning improves the recovery of ATP levels after ischemia/reperfusion injury. We have, therefore, evaluated protein kinase C modulation of the F1F0 ATPase in neonatal cardiac myocytes. Exposure of cells to 3 or 100 nm 4β-phorbol 12-myristate-13-acetate induced co-immunoprecipitation of δ protein kinase C (but not α, ϵ, or ζ protein kinase C) with the d subunit of the F1F0 ATPase. This co-immunoprecipitation correlated with 40 ± 3% and 72 ± 9% inhibitions of oligomycin-sensitive F1F0 ATPase activity, respectively. We observed prominent expression of δ protein kinase C in cardiac myocyte mitochondria, which was enhanced following a 4-h hypoxia exposure. In contrast, hypoxia decreased mitochondrial ζPKC levels by 85 ± 1%. Following 4 h of hypoxia, F1F0 ATPase activity was inhibited by 75 ± 9% and δ protein kinase C co-immunoprecipitated with the d subunit of F1F0 ATPase. In vitro incubation of protein kinase C with F1F0 ATPase enhanced F1F0 activity in the absence of protein kinase C activators and inhibited it in the presence of activators. Recombinant δ protein kinase C also inhibited F1F0 ATPase activity. Protein kinase C overlay assays revealed δ protein kinase C binding to the d subunit of F1F0 ATPase, which was modulated by diacylglycerol, phosphatidylserine, and cardiolipin. Our results suggest a novel regulation of the F1F0 ATPase by the δ protein kinase C isozyme. Mitochondrial protein kinase C isozymes have been reported to mediate both cardiac ischemic preconditioning and ischemia/reperfusion injury. In addition, cardiac preconditioning improves the recovery of ATP levels after ischemia/reperfusion injury. We have, therefore, evaluated protein kinase C modulation of the F1F0 ATPase in neonatal cardiac myocytes. Exposure of cells to 3 or 100 nm 4β-phorbol 12-myristate-13-acetate induced co-immunoprecipitation of δ protein kinase C (but not α, ϵ, or ζ protein kinase C) with the d subunit of the F1F0 ATPase. This co-immunoprecipitation correlated with 40 ± 3% and 72 ± 9% inhibitions of oligomycin-sensitive F1F0 ATPase activity, respectively. We observed prominent expression of δ protein kinase C in cardiac myocyte mitochondria, which was enhanced following a 4-h hypoxia exposure. In contrast, hypoxia decreased mitochondrial ζPKC levels by 85 ± 1%. Following 4 h of hypoxia, F1F0 ATPase activity was inhibited by 75 ± 9% and δ protein kinase C co-immunoprecipitated with the d subunit of F1F0 ATPase. In vitro incubation of protein kinase C with F1F0 ATPase enhanced F1F0 activity in the absence of protein kinase C activators and inhibited it in the presence of activators. Recombinant δ protein kinase C also inhibited F1F0 ATPase activity. Protein kinase C overlay assays revealed δ protein kinase C binding to the d subunit of F1F0 ATPase, which was modulated by diacylglycerol, phosphatidylserine, and cardiolipin. Our results suggest a novel regulation of the F1F0 ATPase by the δ protein kinase C isozyme. The mitochondrial F1F0 ATP synthase produces greater than 90% of cellular ATP in the myocardium, yet relatively little is known regarding how this enzyme complex is regulated (1Di Pancrazio F. Mavelli I. Isola M. Losano G. Pagliaro P. Harris D.A. Lippe G. Biochim. Biophys. Acta. 2004; 1659: 52-62Crossref PubMed Scopus (43) Google Scholar, 2Harris D.A. Das A.M. Biochem. J. 1991; 280: 561-573Crossref PubMed Scopus (281) Google Scholar). It has an F1 domain in the mitochondrial matrix (3Carbajo R.J. Kellas F.A. Runswick M.J. Montgomery M.G. Walker J.E. Neuhaus D. J. Mol. Biol. 2005; 351: 824-838Crossref PubMed Scopus (57) Google Scholar), and an F0 domain, which is a proton channel, that traverses the inner mitochondrial membrane (IM). 2The abbreviations used are: IM, inner mitochondrial membrane; Hx, hypoxia; 4β-PMA, 4β-phorbol 12-myristate-13-acetate; Nx, normoxia; DG, diacylglycerol; PS, phosphatidylserine; NCM, neonatal cardiac myocytes; NCP, nitrocellulose paper; CL, cardiolipin; PKC, protein kinase C; IR, ischemia/reperfusion. 2The abbreviations used are: IM, inner mitochondrial membrane; Hx, hypoxia; 4β-PMA, 4β-phorbol 12-myristate-13-acetate; Nx, normoxia; DG, diacylglycerol; PS, phosphatidylserine; NCM, neonatal cardiac myocytes; NCP, nitrocellulose paper; CL, cardiolipin; PKC, protein kinase C; IR, ischemia/reperfusion. The F0 domain allows proton re-entry into the mitochondrial matrix down a concentration gradient to provide the energy for ATP synthesis (4Walker, J. E., and Dickson, V. K. (2006) Biochim. Biophys. Acta 1757, 286–296Google Scholar). The F1 domain consists of three α, three β, and γ, δ, and ϵ subunits. The interfaces between α- and β-subunits are the site of nucleotide binding and ATP synthesis. The F1 and F0 domains are connected by a central stalk consisting of the γ, δ, and ϵ subunits and by a peripheral stalk that is made up of the OSCP, F6, b, and d subunits (5Gaballo A. Zanotti F. Papa S. Curr. Protein Pept. Sci. 2002; 3: 451-460Crossref PubMed Scopus (23) Google Scholar). The central stalk rotates along with a ring of 10–14 “c” subunits during ATP synthesis, and the peripheral stalk acts as a stator to prevent the α- and β-subunits from rotating with the central stalk and c-subunits. This latter function is crucial for the enzyme proton pumping activity. In vivo, the electrochemical/proton gradient across the IM provides the driving force for F1F0 ATP synthase activity. Loss of this gradient first leads to inhibition of F1F0 ATP synthase activity and eventually the enzyme operates in reverse (F1F0 ATPase). This ATP hydrolysis contributes significantly to ATP loss during cardiac ischemia/reperfusion (IR) injury (6Solaini G. Harris D.A. Biochem. J. 2005; 390: 377-394Crossref PubMed Scopus (201) Google Scholar). There are 2 endogenous inhibitory proteins of the F1F0 ATP synthase/ATPase complex known as Inhibitor of F1 (IF1) (6Solaini G. Harris D.A. Biochem. J. 2005; 390: 377-394Crossref PubMed Scopus (201) Google Scholar, 7Green D.W. Grover G.J. Biochim. Biophys. Acta. 2000; 1458: 343-355Crossref PubMed Scopus (90) Google Scholar) and calcium-sensitive binding inhibitor (Ca BI) (2Harris D.A. Das A.M. Biochem. J. 1991; 280: 561-573Crossref PubMed Scopus (281) Google Scholar, 6Solaini G. Harris D.A. Biochem. J. 2005; 390: 377-394Crossref PubMed Scopus (201) Google Scholar). IF1 inhibits predominantly the hydrolysis of ATP by F1F0 ATPase. Its binding to the F1 catalytic domain is favored under conditions of low pH and decreased IM potential, such as would occur in ischemia (6Solaini G. Harris D.A. Biochem. J. 2005; 390: 377-394Crossref PubMed Scopus (201) Google Scholar, 7Green D.W. Grover G.J. Biochim. Biophys. Acta. 2000; 1458: 343-355Crossref PubMed Scopus (90) Google Scholar, 8Ylitalo K. Ala-Rämi A. Vuorinen K. Peuhkurinen K. Lepojärvi M. Kaukoranta P. Kiviluoma K. Hassinen I. Biochim. Biophys. Acta. 2001; 1504: 329-339Crossref PubMed Scopus (32) Google Scholar, 9Zanotti F. Raho G. Gaballo A. Papa S. J. Bioenerg. Biomembr. 2004; 36: 447-457Crossref PubMed Scopus (30) Google Scholar). It has been proposed to be a type of “fail safe” mechanism, which limits the destruction of ATP in pathological states (6Solaini G. Harris D.A. Biochem. J. 2005; 390: 377-394Crossref PubMed Scopus (201) Google Scholar, 9Zanotti F. Raho G. Gaballo A. Papa S. J. Bioenerg. Biomembr. 2004; 36: 447-457Crossref PubMed Scopus (30) Google Scholar). CaBI inhibits both ATPase and ATP synthase activities and is released from the enzyme in the presence of elevated Ca2+ (2Harris D.A. Das A.M. Biochem. J. 1991; 280: 561-573Crossref PubMed Scopus (281) Google Scholar, 6Solaini G. Harris D.A. Biochem. J. 2005; 390: 377-394Crossref PubMed Scopus (201) Google Scholar). Collectively, these are the only established mechanisms for inhibiting F1F0 synthase or ATPase activities. However, IF1- and CaBI-mediated inhibition of F1F0 activities cannot completely account for the regulation of this F1F0 enzyme complex (2Harris D.A. Das A.M. Biochem. J. 1991; 280: 561-573Crossref PubMed Scopus (281) Google Scholar). Recently, mitochondrial mechanisms involving protein kinase C (PKC) isozymes in cardiac preconditioning (PC) and IR injury have received considerable attention (10Nguyen T. Ogbi M. Guo D. Johnson J.A. Curr. Enzyme Inhib. 2007; 3: 143-159Crossref Scopus (7) Google Scholar). For example, ϵPKC has been reported to activate mitochondrial ATP-sensitive K+ channels (11Liang B.T. Am. J. Physiol. Heart Circ. Physiol. 1997; 273: H847-H853Crossref PubMed Google Scholar), inhibit the opening of the mitochondrial permeability transition pore (12Baines C.P. Song C. Zheng Y. Wang G. Zhang J. Wang O. Guo Y. Bolli R. Cardwell E.M. Ping P. Circ. Res. 2003; 92: 873-880Crossref PubMed Scopus (403) Google Scholar), induce the phosphorylation of the BAD protein to inhibit apoptosis in diabetic hearts (13Malhotra A. Begley R. Kang B.P. Rana I. Liu J. Yang G. Mochly-Rosen D. Meggs L.G. Am. J. Physiol. Heart Circ. Physiol. 2005; 289: H1343-H1350Crossref PubMed Scopus (40) Google Scholar), regulate anti-apoptotic activity through the regulation of Bcl-2 family of proteins (14Manat R. Singal T. Dhalla N.S. Tappia P.S. Am. J. Physiol. Heart Circ. Physiol. 2006; 291: H854-H860Crossref PubMed Scopus (28) Google Scholar), and enhance the activity of cytochrome c oxidase in PC (15Ogbi M. Johnson J.A. Biochem. J. 2006; 393: 191-199Crossref PubMed Scopus (97) Google Scholar, 16Guo D. Nguyen T. Ogbi M. Tawfik H. Ma G. Yu Q. Caldwell R.W. Johnson J.A. Am. J. Physiol. Heart Circ. Physiol. 2007; 293: H2219-H2230Crossref PubMed Scopus (57) Google Scholar). The role of δPKC is more controversial with reports indicating it plays significant roles in PC (17Wang Y. Hirai K. Ashraf M. Circ. Res. 1999; 85: 731-741Crossref PubMed Scopus (181) Google Scholar, 18Mayr M. Metzler B. Chung Y.L. McGregor E. Mayr U. Troy H. Hu Y. Leitges M. Pachinger O. Griffiths J.R. Dunn M.J. Xu Q. Am. J. Physiol. Heart Circ. Physiol. 2004; 287: H946-H956Crossref PubMed Scopus (99) Google Scholar) and IR injury (19Inagaki K. Hahn H.S. Dorn II G.W. Mochly-Rosen D. Circulation. 2003; 108: 869-875Crossref PubMed Scopus (184) Google Scholar, 20Churchill E.N. Muriel C.L. Chen C.H. Mochly-Rosen D. Szweda L.I. Circ. Res. 2005; 97: 78-85Crossref PubMed Scopus (111) Google Scholar). Wang et al. (17Wang Y. Hirai K. Ashraf M. Circ. Res. 1999; 85: 731-741Crossref PubMed Scopus (181) Google Scholar) reported diazoxide-induced PC, which correlated with δPKC translocation to mitochondria. Also, Mayr et al., (18Mayr M. Metzler B. Chung Y.L. McGregor E. Mayr U. Troy H. Hu Y. Leitges M. Pachinger O. Griffiths J.R. Dunn M.J. Xu Q. Am. J. Physiol. Heart Circ. Physiol. 2004; 287: H946-H956Crossref PubMed Scopus (99) Google Scholar, 21Mayr M. Chung Y.L. Mayr U. McGregor E. Troy H. Gottfried B. Leitges M. Dunn M.J. Griffiths J.R. Xu Q. Am. J. Physiol. 2004; 287: H937-H945Crossref PubMed Scopus (71) Google Scholar) reported that δPKC knock-out mice demonstrated decreased glycolysis and an increased lipid metabolism under baseline conditions and were unable to induce a PC response. In contrast, Mochly-Rosen and co-workers (19Inagaki K. Hahn H.S. Dorn II G.W. Mochly-Rosen D. Circulation. 2003; 108: 869-875Crossref PubMed Scopus (184) Google Scholar) demonstrated in rodent studies that δPKC activation induces apoptosis and also phosphorylates pyruvate dehydrogenase kinase 2, which in turn may phosphorylate and delay the reactivation of pyruvate dehydrogenase following IR injury (20Churchill E.N. Muriel C.L. Chen C.H. Mochly-Rosen D. Szweda L.I. Circ. Res. 2005; 97: 78-85Crossref PubMed Scopus (111) Google Scholar). However, there have been no reports demonstrating PKC isozyme-selective modulation of the F1F0 synthase/ATPase complex under normoxic or low oxygen conditions. We present here the first evidence for modulation of F1F0 ATPase by δPKC and propose that δPKC interacts with the d subunit of the F1F0 ATPase (dF1F0). We hypothesize that this putative interaction regulates F1F0 activities under normoxic conditions, as well as mediates inhibition of F1F0 ATP synthase or ATPase activities during prolonged hypoxia. Such inhibition could contribute to cardiac IR injury by delaying the recovery of aerobic ATP production. Alternatively, the δPKC-dF1F0 interaction may be cardioprotective if δPKC limits IR-induced reverse-mode F1F0 ATPase activity. Primary Neonatal Cardiac Myocytes (NCMs)—NCMs were isolated from the hearts of 1-day-old Sprague-Dawley rats as previously described (22Johnson J.A. Gray M.O. Chen C. Karliner J. Mochly-Rosen D. Circ. Res. 1996; 79: 1086-1099Crossref PubMed Scopus (80) Google Scholar, 23Johnson J.A. Mochly-Rosen D. Circ. Res. 1995; 76: 654-663Crossref PubMed Scopus (93) Google Scholar). This study was conducted in accordance with the institutional, state, and federal guidelines for the humane care and use of laboratory animals. Induction of Hypoxia in NCMs—NCMs were placed in a PlasLabs Anaerobic chamber at 37 °C, 1% CO2, < 0.5% O2, and the balance N2 as previously described (15Ogbi M. Johnson J.A. Biochem. J. 2006; 393: 191-199Crossref PubMed Scopus (97) Google Scholar). Incubations were conducted in glucose-free M199 medium (24Gray M.O. Karliner J.S. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 30945-30951Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). Normoxic incubations were conducted in a water-jacketed incubator gassed with 1% CO2 and 99% air at 37 °C in M199 containing 5 mm glucose. Cell Lysis and Isolation of Mitochondria Using Percoll/Optiprep Density Gradients—Fifteen 100-mm dishes of NCMs were used for each treatment group. These procedures have been extensively described in previous studies (15Ogbi M. Johnson J.A. Biochem. J. 2006; 393: 191-199Crossref PubMed Scopus (97) Google Scholar, 16Guo D. Nguyen T. Ogbi M. Tawfik H. Ma G. Yu Q. Caldwell R.W. Johnson J.A. Am. J. Physiol. Heart Circ. Physiol. 2007; 293: H2219-H2230Crossref PubMed Scopus (57) Google Scholar, 25Ogbi M. Chew C.S. Pohl J. Stuchlik O. Ogbi S. Johnson J.A. Biochem. J. 2004; 382: 923-932Crossref PubMed Scopus (60) Google Scholar). Immunoprecipitation Experiments—The antisera against the d subunit of the F1F0 ATPase (dF1F0) from Molecular Probes (50 μg) was coupled with Bio-Rad Affi-gel (1 ml) according to the manufacturer's instructions. The immunoprecipitation was performed as previously described (15Ogbi M. Johnson J.A. Biochem. J. 2006; 393: 191-199Crossref PubMed Scopus (97) Google Scholar, 16Guo D. Nguyen T. Ogbi M. Tawfik H. Ma G. Yu Q. Caldwell R.W. Johnson J.A. Am. J. Physiol. Heart Circ. Physiol. 2007; 293: H2219-H2230Crossref PubMed Scopus (57) Google Scholar, 25Ogbi M. Chew C.S. Pohl J. Stuchlik O. Ogbi S. Johnson J.A. Biochem. J. 2004; 382: 923-932Crossref PubMed Scopus (60) Google Scholar). Briefly, the dF1F0 antisera-coupled Affi-gel was blocked for 1 h in IP buffer (13.3 mm Tris-HCl, pH 7.4, 2 mm EDTA, 1.8 mm EGTA, 0.5% Triton X-100 (w/v), 0.5% SDS (w/v), 1.5 mm NaPPi, 0.005% (w/v) bovine serum albumin, 1.5 nm calyculin A, and 9.1 μg/ml each of aprotinin, leupeptin, phenylmethylsulfonyl fluoride) before immunoprecipitation. Mitochondria (500 μg) from the each treatment group were solubilized in IP buffer in a total volume of 1 ml. After this step, the solubilized mitochondria were incubated with 200 μl of antibody-coupled Affi-gel overnight at 4 °C. Affi-gel immunoprecipitates were washed, and then SDS-PAGE sample buffer was added to Affi-gel pellets to liberate proteins. Affi-gel was then pelleted by centrifugation, and the resulting SDS-PAGE sample buffer supernatants were subjected to SDS-PAGE (12–13.5% acrylamide) and transferred onto nitrocellulose paper (NCP) using standard Western blot techniques. The resulting blots were probed for α, δ, ϵ, and ζPKC isozymes using the Enhanced Chemiluminescence (ECL) detection kit (GE Healthcare). F1F0 ATPase Activity Measurements—The oligomycin-sensitive activity of F1F0 ATPase activity was measured in NCM cell lysates as previously described (26Buchanan S.K. Walker J.E. Biochem. J. 1996; 318: 343-349Crossref PubMed Scopus (70) Google Scholar). Briefly, cells were scraped and lysed by sonication in 400 μl of buffer containing 20 mm Tris-HCl, pH 7.5, 1 mm MgCl2, 5 mm KCl, and 1 mm EGTA. The F1F0 ATPase activity was then measured spectrophotometrically by monitoring the disappearance of NADH, which manifests as a decline in absorbance at 340 nm as NADH is oxidized to NAD+. ATP hydrolysis is coupled to NADH loss in the presence of an ATP-regenerating system as previously described (1Di Pancrazio F. Mavelli I. Isola M. Losano G. Pagliaro P. Harris D.A. Lippe G. Biochim. Biophys. Acta. 2004; 1659: 52-62Crossref PubMed Scopus (43) Google Scholar). The final F1F0 ATPase assay buffer contained 25 mm Tris-HCl, pH 7.5, 83 mm sucrose, 4 mm MgCl2, 25 mm KCl, 1 mm KCN, 1 mm EDTA, 1 mm EGTA, 2 mm ATP, 1.5 mm phosphoenolpyruvate, 5 units of pyruvate kinase, 5 units of lactate dehydrogenase, and 75 μm NADH in a 1-ml cuvette. All assay reagents were obtained from Sigma. The assay was monitored continuously for 1–5 min at 25 °C in the absence and presence of oligomycin (8 μg/ml). All assays were conducted in triplicate. F1F0 ATPase Purification for Overlay and PKC Add-back Assays—Isolation of the F1F0 ATPase holo-enzyme by chromatography was performed as previously described by Buchanan and Walker (26Buchanan S.K. Walker J.E. Biochem. J. 1996; 318: 343-349Crossref PubMed Scopus (70) Google Scholar). Briefly, mitochondria were isolated from the ventricles of 5 adult Sprague-Dawley rat hearts, then washed in phosphate buffer, and solubilized in buffer containing 20 mm ATP, 20 mm MgSO4, 0.001% phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and 1% dodecyl-β-maltoside. The solubilized mitochondria were precipitated with ammonium sulfate. The ammonium sulfate pellet was resuspended in buffer, dialyzed overnight, and then loaded on a Q-Sepharose anion exchange column. The F1F0 ATPase was eluted using a linear NaCl gradient from 0 to 1 m, and peak F1F0 ATPase fractions were identified by F1F0 ATPase activity. Subsequently, the presence of the α, β, and dF1F0 ATPase subunits were confirmed in peak F1F0 ATPase activity fractions by Western blot. Peak fractions were then pooled and used for overlay assays. PKC Overlay Assays—The overlay method to detect PKC-binding partners was previously described by Mochly-Rosen and co-workers (27Schechtman D. Murriel C. Bright R. Mochly-Rosen D. Meth. Mol. Biol. 2003; 233: 345-350PubMed Google Scholar). Required activators and cofactors of PKC such as 1 mm calcium (Ca2+), 240 μg/ml phosphatidylserine (PS), 3.2 μg/ml diacylglycerol (DG, Avanti Polar Lipids, Alabaster, AL), and 0–1 mm cardiolipin (CL, Sigma Aldrich) were included in the assay as indicated. Purified F1F0 holo-enzyme (50 μg) was separated by 13.5% SDS-PAGE and transferred onto NCP. The NCP blots were then cut into (0.3 cm × 5.5 cm) strips, washed with distilled water, and blocked with overlay blocking buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 3% bovine serum albumin, and 0.1% polyethylene glycol) for 1 h at room temperature. NCP strips were then incubated with overlay buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 12 mm β-mercaptoethanol, 1.0% polyethylene glycol, and 10 μg/ml protease inhibitors (aprotinin, leupeptin, SBTI, phenylmethylsulfonyl fluoride)) containing purified PKC (500 μg/ml) and cofactors for 1 h at room temperature. The strips were washed four times for 5 min with overlay wash buffer (50 mm Tris-HCl, pH 7.5, 200 mm NaCl, 12 mm β-mercaptoethanol, and 0.1% polyethylene glycol). Bound PKC was detected with PKC isozyme antisera as previously described (28Ron D. Luo J. Mochly-Rosen D. J. Biol. Chem. 1995; 270: 1-8Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Western Blot Analyses—Western blotting was carried out as described previously (22Johnson J.A. Gray M.O. Chen C. Karliner J. Mochly-Rosen D. Circ. Res. 1996; 79: 1086-1099Crossref PubMed Scopus (80) Google Scholar, 23Johnson J.A. Mochly-Rosen D. Circ. Res. 1995; 76: 654-663Crossref PubMed Scopus (93) Google Scholar) and using ECL Detection (GE Healthcare) according to the manufacturer's instructions. Samples were subjected to SDS-PAGE on 12–13.5% acrylamide gels and then transferred onto NCP. For two-dimensional electrophoresis analysis, the samples were separated using 13-cm pH 3–10 NL dry IPG strips (GE Healthcare) followed by SDS-PAGE 12–13.5% acrylamide gels and then transferred onto NCP. The resulting blots were probed for PKC isozymes and F1F0 subunits. PKC antisera were obtained from BD Transduction Laboratories or Santa Cruz Biotechnology while F1F0 antisera were obtained from Invitrogen and were used at recommended dilution concentrations. In Vitro PKC/F1F0 ATPase Add-back Experiments—Rat brain PKC was purified as previously described (29Mochly-Rosen D. Koshland Jr., D.E. J. Biol. Chem. 1987; 262: 2291-2297Abstract Full Text PDF PubMed Google Scholar), and recombinant δPKC expressed in sf9 cells, was obtained from BIOSOURCE, Inc. (Camarillo, CA). F1F0 ATPase was used on the day of purification without freezing. Rat brain PKC (120 units/mg) was added to 50 μg of purified F1F0 ATPase for 5 min at room temperature in the presence or absence of DG and PS. F1F0 ATPase assays were then conducted for 1, 3, or 5 min. For heated (inactivated) PKC groups, PKC was heated at 85 °C for 10 min, placed on ice for at least 3 min, and then added to the F1F0 ATPase. All data were expressed as mean ± S.E. Student's t test or one-way analysis of variance with Bonferroni's posthoc analyses were used for comparison of differences between groups, and a p value ≤ 0.05 was considered to be significant. δPKC Co-immunoprecipitates with the d Subunit of the F1F0 ATPase (dF1F0) following 4β-Phorbol 12-Myristate-13-acetate (4β-PMA) Treatment of NCMs—Cells were treated with either 3 nm 4β-PMA for 1 h or 100 nm 4α- and 4β-PMA for 20 min. Mitochondria were isolated, solubilized, and subjected to immunoprecipitation using anti-dF1F0 antisera. Immunoprecipitates were then subjected to SDS-PAGE and electrotransfer of proteins onto NCP followed by probing of NCP blots with PKC isozyme-selective antisera. Following 3 nm 4β-PMA treatment δPKC (but not α, ϵ, or ζPKC) co-immunoprecipitated with dF1F0 (Fig. 1A). This co-immunoprecipitation did not occur under basal (4α-PMA) conditions. Following 100 nm 4β-PMA treatment, the δPKC-dF1F0 co-immunoprecipitation increased by 3.6 ± 1.2-fold over 3 nm 4β-PMA levels (Fig. 1). These results suggest that 4β-PMA induces a δPKC co-immunoprecipitation with dF1F0 under normoxic (Nx) conditions. 4β-PMA Treatment Inhibits F1F0 ATPase Activity in NCMs—To determine if the above δPKC-dF1F0 co-immunoprecipitation (Fig. 1) correlated with changes in F1F0 ATPase activity, the experiments represented in Fig. 2 were conducted. NCMs were exposed to 0, 3, or 100 nm 4β-PMA and assayed for F1F0 ATPase activity. 3 nm 4β-PMA treatment inhibited the basal oligomycin-sensitive F1F0 ATPase activity by 39.7 + 3.1%. This inhibition was increased to 72.1 ± 9.4% following a 20-min 100 nm 4β-PMA exposure (Fig. 2). These results demonstrated that the 4β-PMA-induced δPKC-dF1F0 co-immunoprecipitation (Fig. 1) correlated with a significant inhibition of F1F0 ATPase activity (Fig. 2).FIGURE 2PMA attenuates F1F0 ATPase activity. NCMs were treated as in Fig. 1. The oligomycin-sensitive F1F0 ATPase activity was measured spectrophotometrically using sonicated NCM lysates (“Experimental Procedures”). Results are expressed as mean ± S.E. from four independent experiments, each conducted in triplicate using samples from four different myocyte preparations. Asterisks indicate statistically significant differences between 4α-PMA and either 3 or 100 nm 4β-PMA (p < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Exposure of NCMs to Hx inhibits F1F0 ATPase Activity—We next determined if limited Hx also inhibited F1F0 ATPase activity. In Fig. 3, NCMs were exposed to 4 h of Hx in an anaerobic chamber (15Ogbi M. Johnson J.A. Biochem. J. 2006; 393: 191-199Crossref PubMed Scopus (97) Google Scholar, 24Gray M.O. Karliner J.S. Mochly-Rosen D. J. Biol. Chem. 1997; 272: 30945-30951Abstract Full Text Full Text PDF PubMed Scopus (343) Google Scholar). In NCMs, 4 h of Hx causes minimal cell death. In support of this, we observed no significant release of rat cardiac troponin I (cTnI) into NCM media following control or 4-h Hx treatments (not shown). However, under the same conditions, we observed a 75.1 + 8.6% inhibition of baseline F1F0 ATPase activity. Therefore, in addition to 4β-PMA inducing F1F0 ATPase inhibition under Nx conditions, a 4-h Hx exposure also decreased F1F0 ATPase activity. δPKC Exists in NCM Mitochondria under Basal and Hx Conditions—We next determined the mitochondrial distributions of δPKC following Nx and Hx conditions to determine if δPKC translocated to or from mitochondria under conditions that inhibit F1F0 ATPase activity. Mitochondria were isolated via Percoll/Optiprep gradients (15Ogbi M. Johnson J.A. Biochem. J. 2006; 393: 191-199Crossref PubMed Scopus (97) Google Scholar, 16Guo D. Nguyen T. Ogbi M. Tawfik H. Ma G. Yu Q. Caldwell R.W. Johnson J.A. Am. J. Physiol. Heart Circ. Physiol. 2007; 293: H2219-H2230Crossref PubMed Scopus (57) Google Scholar, 25Ogbi M. Chew C.S. Pohl J. Stuchlik O. Ogbi S. Johnson J.A. Biochem. J. 2004; 382: 923-932Crossref PubMed Scopus (60) Google Scholar) and subjected to SDS-PAGE and Western blot analyses. The resulting blots were probed with antisera directed against α, δ, or ζPKC isozymes. We found no mitochondrial αPKC in NCMs following basal or 4 h of Hx treatment. However, δ and ζPKC isozymes were found in NCM mitochondria under Nx conditions (Fig. 4). Further, the level of mitochondrial δPKC increased by 20.7 ± 8.2% following 4 h of Hx. In contrast, 4 h of Hx decreased mitochondrial ζPKC by 84.6 ± 1.0%. Hx Induces Co-immunoprecipitation of δPKC with dF1F0 Antisera—NCMs were exposed to Nx or 4 h of Hx. Mitochondria were isolated and dF1F0 was IPed as in Fig. 1. Immunoprecipitates were then subjected to SDS-PAGE and Western blot analyses using PKC isozyme selective antisera (Fig. 5). We observed no significant co-immunoprecipitation of PKC isozymes using dF1F0 antisera under Nx conditions. In contrast, δPKC (but not α, ϵ, or ζPKC) co-immunoprecipitated with dF1F0 following 4 h of Hx (Fig. 5). The Hx-induced increase in δPKC-dF1F0 co-immunoprecipitation was 92.9 ± 7.1%. Therefore, δPKC co-immunoprecipitates with dF1F0 following Hx conditions, which correlates with inhibition of F1F0 ATPase activity (Fig. 3) in NCMs. δPKC Binds to dF1F0 in Overlay Assays—Purified F1F0 ATPase holoenzyme was next, isolated from adult rat heart mitochondria by chromatography (26Buchanan S.K. Walker J.E. Biochem. J. 1996; 318: 343-349Crossref PubMed Scopus (70) Google Scholar), and individual F1F0 ATPase subunits were resolved by SDS-PAGE, transferred to NCP, and subjected to the PKC overlay assay (27Schechtman D. Murriel C. Bright R. Mochly-Rosen D. Meth. Mol. Biol. 2003; 233: 345-350PubMed Google Scholar). When NCP containing resolved F1F0 ATPase subunits was “overlaid” with purified rat brain PKC (mixture of PKC isozymes) (29Mochly-Rosen D. Koshland Jr., D.E. J. Biol. Chem. 1987; 262: 2291-2297Abstract Full Text PDF PubMed Google Scholar), in the presence of the PKC-activating lipids DG and PS, we observed δPKC binding to a protein that co-migrated with dF1F0 immunoreactivity (Fig. 6). There also appeared to be δPKC binding to an unknown protein of ∼35 kDa. It is interesting to note that the γ-subunit of F1F0 ATPase is ∼35 kDa, but further study will be required to confirm the identity of the 35-kDa protein. Addition of 1 mm CaCl2 to the assay appeared to reduce the DG/PS-induced δPKC binding to dF1F0, but had minimal effects on δPKC binding to the ∼35-kDa protein (Fig. 6, lane 4). However, further study will be required to assess the role of calcium in these binding events. The δPKC-dF1F0 binding was also induced when DG/PS was replaced by 200 μm CL. Of interest, CL also induced δPKC binding to the ∼35 kDa protein to an extent similar to that induced by DG/PS (Fig. 6, lane 3 versus 5). Another interesting observation was that CL revealed the presence of at least 4 additional δPKC-binding proteins in our purified F1F0 preparations (Fig. 6A, top). This suggested that δPKC may regulate the F1F0 ATPase via multiple protein-protein interactions with additional F1F0 ATPase subunits or accessory proteins. It is interesting that these latter binding events were not observed in the absence of CL even when DG/PS was present (Fig. 6, lanes 5–7, top versus lanes 3–4, top). Further, the effects of DG/PS and CL on δPKC binding to dF1F0 were not additive (Fig. 6, lanes 3 versus 5). Collectively, these results suggested an in vitro binding interaction between δPKC and dF1F0. Combining Two-dimensional Electrophoresis and PKC Overlay Assay Techniques Confirm δPKC Binding to dF1F0—To obtain further evidence for δPKC-selective binding to dF1F0, we subjected purified F1F0 ATPase (26Buchanan S.K. Walker J.E. Biochem. J. 1996; 318: 343-349Crossref PubMed Scopus (70) Google Scholar) to two-dimensional electrophoresis and electrotransfer onto NCP. In Fig. 7, we probed these two-dimensional blots with antisera against the α-(panel A), β-(panel B), and d-(panel C) subunits of F1F0 ATPase. Fig. 7D is a PKC overlay assay (conducted in the presence of DG/PS and CL) and as is shown, δPKC bound only to a protein that co-migrated with dF1F0 immunoreactivity (F" @default.
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• W1994633260 title "Delta Protein Kinase C Interacts with the d Subunit of the F1F0 ATPase in Neonatal Cardiac Myocytes Exposed to Hypoxia or Phorbol Ester" @default.
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• W1994633260 doi "https://doi.org/10.1074/jbc.m801642200" @default.
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