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• W2030903683 abstract "Ethanol can enhance Gsα-stimulated adenylyl cyclase (AC) activity. Of the nine isoforms of AC, type 7 (AC7) is the most sensitive to ethanol. The potentiation of AC7 by ethanol is dependent on protein kinase C (PKC). We designed studies to determine which PKC isotype(s) are involved in the potentiation of Gαs-activated AC7 activity by ethanol and to investigate the direct phosphorylation of AC7 by PKC. AC7 was phosphorylated in vitro by the catalytic subunits of PKCs. The addition of ethanol to AC7-transfected HEK 293 cells increased the endogenous phosphorylation of AC7, as indicated by a decreased “back-phosphorylation” of AC7 by PKCin vitro. The potentiation of Gαs-stimulated AC7 activity by either phorbol 12,13-dibutyrate or ethanol, in HEL cells endogenously expressing AC7, was not through the Ca2+-sensitive conventional PKCs. However, the potentiation of AC7 activity by ethanol or phorbol 12,13-dibutyrate was found to be reduced by the selective inhibitor of PKCδ (rottlerin), a PKCδ-specific inhibitory peptide (δV1-1), and the expression of the dominant negative form of PKCδ. Immunoprecipitation data indicated that PKCδ could bind and directly phosphorylate AC7. The results indicate that the potentiation of AC7 activity by ethanol involves phosphorylation of AC7 that is mediated by PKCδ. Ethanol can enhance Gsα-stimulated adenylyl cyclase (AC) activity. Of the nine isoforms of AC, type 7 (AC7) is the most sensitive to ethanol. The potentiation of AC7 by ethanol is dependent on protein kinase C (PKC). We designed studies to determine which PKC isotype(s) are involved in the potentiation of Gαs-activated AC7 activity by ethanol and to investigate the direct phosphorylation of AC7 by PKC. AC7 was phosphorylated in vitro by the catalytic subunits of PKCs. The addition of ethanol to AC7-transfected HEK 293 cells increased the endogenous phosphorylation of AC7, as indicated by a decreased “back-phosphorylation” of AC7 by PKCin vitro. The potentiation of Gαs-stimulated AC7 activity by either phorbol 12,13-dibutyrate or ethanol, in HEL cells endogenously expressing AC7, was not through the Ca2+-sensitive conventional PKCs. However, the potentiation of AC7 activity by ethanol or phorbol 12,13-dibutyrate was found to be reduced by the selective inhibitor of PKCδ (rottlerin), a PKCδ-specific inhibitory peptide (δV1-1), and the expression of the dominant negative form of PKCδ. Immunoprecipitation data indicated that PKCδ could bind and directly phosphorylate AC7. The results indicate that the potentiation of AC7 activity by ethanol involves phosphorylation of AC7 that is mediated by PKCδ. Intracellular signaling via cAMP generates downstream effects that range from changes in the function of ion channels to changes in intracellular energy metabolism to changes in gene transcription (for review see Ref. 1Smit M.J. Iyengar R. Cooper D.M.F. Advances in Second Messenger and Phosphoprotein Research: Adenylyl Cyclases. 32. Lippincott-Raven, Philadelphia, PA1998: 1-21Google Scholar). It is therefore not surprising that the generation of intracellular cAMP is a tightly regulated process that involves the α and βγ subunits of G-proteins, intracellular Ca2+acting independently or in concert with calmodulin, and phosphorylation events that are postulated to involve protein kinase A and protein kinase C (PKC) 1The abbreviations used are: PKC, protein kinase C; BSA, bovine serum albumin; AC, adenylyl cyclase; AC7, adenylyl cyclase type 7; HEL, human erythroleukemia cells; HEK 293, human embryonic kidney cells; NFDM, nonfat dairy milk; RACK, receptor for activated C-kinase; PDBu, phorbol 12,13-dibutyrate; PGE1, prostaglandin E1; Sf9, Spodoptera frugiperda ovarian cells; EtOH, ethanol; IP, immunoprecipitation; PAA, protein A-agarose; TBS, Tris-buffered saline; WT, wild-type; DN, dominant negative 1The abbreviations used are: PKC, protein kinase C; BSA, bovine serum albumin; AC, adenylyl cyclase; AC7, adenylyl cyclase type 7; HEL, human erythroleukemia cells; HEK 293, human embryonic kidney cells; NFDM, nonfat dairy milk; RACK, receptor for activated C-kinase; PDBu, phorbol 12,13-dibutyrate; PGE1, prostaglandin E1; Sf9, Spodoptera frugiperda ovarian cells; EtOH, ethanol; IP, immunoprecipitation; PAA, protein A-agarose; TBS, Tris-buffered saline; WT, wild-type; DN, dominant negative (2Houslay M.D. Milligan G. Trends Biochem. Sci. 1997; 22: 217-224Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 3Sunahara R.K. Dessauer C.W. Gillman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar). The current evidence indicates that each of the nine adenylyl cyclase (AC) isoforms differs in its repertoire of regulatory controls (2Houslay M.D. Milligan G. Trends Biochem. Sci. 1997; 22: 217-224Abstract Full Text PDF PubMed Scopus (401) Google Scholar, 3Sunahara R.K. Dessauer C.W. Gillman A.G. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 461-480Crossref PubMed Scopus (727) Google Scholar), and all of the isoforms can be regulated coincidentally by multiple signals to modulate the production of cAMP. It was therefore not entirely unexpected that the activity of the various isoforms of AC were also found to be differentially sensitive to the effect of ethanol (4Yoshimura M. Tabakoff B. Alcohol Exp. Clin. Res. 1995; 19: 1435-1440Crossref PubMed Scopus (72) Google Scholar). We and others have shown that ethanol acutely potentiates Gαs-activated AC activity (5Saito T. Lee J.M. Tabakoff B. J. Neurochem. 1985; 44: 1037-1044Crossref PubMed Scopus (86) Google Scholar, 6Bode D.C. Molinoff P.B. J. Pharmacol. Exp. Ther. 1988; 246: 1040-1047PubMed Google Scholar), and studies with HEK 293 cells transfected with the various isoforms of AC have demonstrated a broad range of AC sensitivity to ethanol. Some isoforms were found to be insensitive to ethanol (types 1, 3, 8a, and 8b), others were moderately sensitive (types 2, 5, 6, 8c, and 9), and Type 7 AC was at least two to three times more sensitive to the stimulatory actions of ethanol in comparison with all other tested ACs (4Yoshimura M. Tabakoff B. Alcohol Exp. Clin. Res. 1995; 19: 1435-1440Crossref PubMed Scopus (72) Google Scholar). Agonist-stimulated AC7 activity in transfected HEK 293 cells can be significantly potentiated by 10–20 mm ethanol (which corresponds to tissue concentrations of 46–92 mg/100 ml ethanol), and the potentiation by ethanol of AC7 activity is not mediated through the inhibition of phosphodiesterase activity or an adenosine receptor-mediated event (7Yoshimura M. Tabakoff B. Alcohol Exp. Clin. Res. 1999; 23: 1457-1461PubMed Google Scholar). Ethanol has been shown to further potentiate AC activity when AC is activated through a variety of different membrane receptor systems, including dopamine D1a, β-adrenergic, and prostaglandin receptors (4Yoshimura M. Tabakoff B. Alcohol Exp. Clin. Res. 1995; 19: 1435-1440Crossref PubMed Scopus (72) Google Scholar, 5Saito T. Lee J.M. Tabakoff B. J. Neurochem. 1985; 44: 1037-1044Crossref PubMed Scopus (86) Google Scholar, 8Rabbani M. Nelson E.J. Hoffman P.L. Tabakoff B. Alcohol Exp. Clin. Res. 1999; 23: 77-86Crossref PubMed Scopus (18) Google Scholar). These data indicate that the ability of ethanol to potentiate cAMP accumulation is independent of transmitter receptors and is dependent primarily on the AC isoform present in the cell. cAMP signaling has been shown to be important in the behavioral effects of ethanol through studies with Drosophila (9Moore M.S. DeZazzo J. Luk A.Y. Tully T. Singh C.M. Heberlein U. Cell. 1998; 93: 997-1007Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar) and mice (10Dar M.S. Brain Res. 1997; 749: 263-274Crossref PubMed Scopus (50) Google Scholar,11Szabo G. Hoffman P.L. Tabakoff B. Life Sci. 1988; 42: 615-621Crossref PubMed Scopus (10) Google Scholar). Mutations in the components of the cAMP-generating or degrading systems in Drosophila significantly altered the responses of the fly to the intoxicating (incoordinating) effects of ethanol (9Moore M.S. DeZazzo J. Luk A.Y. Tully T. Singh C.M. Heberlein U. Cell. 1998; 93: 997-1007Abstract Full Text Full Text PDF PubMed Scopus (377) Google Scholar). A similar relationship between ethanol-induced incoordination and changes in cAMP generating systems was found in both mice and rats (10Dar M.S. Brain Res. 1997; 749: 263-274Crossref PubMed Scopus (50) Google Scholar, 12Meng Z.H. Pennington S.N. Dar M.S. Neuroscience. 1998; 85: 919-930Crossref PubMed Scopus (18) Google Scholar). Activation of cAMP generation in the brains of mice, by the use of forskolin, altered the development of tolerance to the sedative effects of ethanol (11Szabo G. Hoffman P.L. Tabakoff B. Life Sci. 1988; 42: 615-621Crossref PubMed Scopus (10) Google Scholar). Clinical studies with humans have also linked the cAMP signaling system to behavioral manifestations of physical dependence on ethanol. Alcohol-dependent individuals, even when abstinent for substantive periods of time, have been shown to have lower G-protein-activated AC activity in their platelets than control subjects (13Tabakoff B. Hoffman P.L. Lee J.M. Saito T. Willard B. De Leon-Jones F. N. Engl. J. Med. 1988; 318: 134-139Crossref PubMed Scopus (189) Google Scholar). More recently it was found that low platelet AC activity was characteristic of individuals classified as family history positive for alcoholism, regardless of whether the subjects were themselves diagnosed alcoholics (14Menninger J.A. Baron A.E. Tabakoff B. Alcohol Exp. Clin. Res. 1998; 22: 1955-1961Crossref PubMed Google Scholar, 15Ratsma J.E. Gunning W.B. Leurs R. Schoffelmeer A.N. Alcohol Exp. Clin. Res. 1999; 23: 600-604Crossref PubMed Google Scholar). Because ethanol has been demonstrated to enhance receptor/G-protein-coupled generation of cAMP in platelets, the depressed AC activity in the platelets of individuals at risk to develop alcoholism may parallel what is occurring in the brain and may contribute to the addiction process (16Nestler E.J. Nat. Rev. Neurosci. 2001; 2: 119-128Crossref PubMed Scopus (1497) Google Scholar). We have recently shown that in human erythroleukemia (HEL) platelet precursor cells, which contain a preponderance of AC7 mRNA (17Hellevuo K. Yoshimura M. Kao M. Hoffman P.L. Cooper D.M.F. Tabakoff B. Biochem. Biophys. Res. Commun. 1993; 192: 311-318Crossref PubMed Scopus (67) Google Scholar), both ethanol and phorbol esters (PDBu and phorbol 12-myristate 13-acetate) could significantly potentiate AC activity generated in response to the activation of the prostanoid receptor by the agonist PGE1 (8Rabbani M. Nelson E.J. Hoffman P.L. Tabakoff B. Alcohol Exp. Clin. Res. 1999; 23: 77-86Crossref PubMed Scopus (18) Google Scholar). This potentiation of AC activity by either ethanol or PDBu could be diminished by PKC inhibitors such as staurosporine and chelerythrine (8Rabbani M. Nelson E.J. Hoffman P.L. Tabakoff B. Alcohol Exp. Clin. Res. 1999; 23: 77-86Crossref PubMed Scopus (18) Google Scholar). In the current work, we report on a series of studies that determine the PKC isotype(s) involved in the enhancement of Gαs-activated AC7 activity by ethanol, and we investigate the phosphorylation of AC7 by PKC. [2-3H]Adenine was obtained fromAmersham Biosciences. 3-Isobutyl-1-methylxanthine, phorbol 12,13-dibutyrate (PDBu), staurosporine, chelerythrine, rottlerin, thapsigargin, and Gö 6976 were purchased fromCalbiochem (La Jolla, CA). Anti-PKC monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY). Dr. W.-J. Tang (University of Chicago) kindly provided the anti-AC II family antibody, C6C. PGE1 was obtained from the Cayman Chemical Company (Ann Arbor, MI). Cicaprost was a generous gift from Schering AG (Berlin, Germany). All of the other products were purchased from Sigma. Concentrated solutions of drugs, including PGE1 and cicaprost, were prepared in Me2SO, and the final concentration of Me2SO in the assay mixtures was never greater than 0.8%. Control assays were always performed containing the appropriate amounts of Me2SO. Dr. Daria Mochly-Rosen (18.Mochly-Rosen, D., Ron, D., Kauvar, L. M., and Napolitano, E. W. (July 21, 1998) U. S. Patent 5,783,405.Google Scholar) kindly provided the following PKC-derived inhibitory peptides: δV1-1 (δPKC8–16, AFNSYELGS), εV1–2 (εPKC14–21, EAVSLKPT), and a control nonsense octapeptide (LSETKPAV). These peptides were conjugated to aDrosophila antennapedia peptide (RQIKIWFQNRRMKWKK) to make them more cell permeant. Catalytic subunits of PKC purified from rat brain were prepared by trypsin digestion by Dr. Michael D. Browning (University of Colorado Health Sciences Center, Denver, CO). Dr. Trevor Biden (Garvan Institute of Medical Research, Sydney, Australia) kindly provided the replication deficient adenovirus (Ad5 DL312) vectors carrying either the wild-type or dominant negative PKCδ. Full-length recombinant human PKCδ and PKCε were purchased fromCalbiochem (La Jolla, CA). A BamHI site was introduced upstream of the initiation codon of the human AC7 cDNA (19Nomura N. Miyajima N. Sazuka T. Tanaka A. Kawarabayashi Y. Sato S. Nagase T. Seki N. Ishikawa K. Tabata S. DNA Res. 1994; 1: 27-35Crossref PubMed Scopus (269) Google Scholar) by in vitro mutagenesis using an oligonucleotide, CGTGCCAAGGATCCGGAGGATGCCAG. The mutation was confirmed by DNA sequencing. A 3.6-kb fragment containing the human AC7 coding sequence was prepared from pBlueScript II SK-containing the AC7 cDNA by digestion with BamHI and XbaI. The fragment was inserted into a pcDNA3.1 His (Invitrogen) mammalian expression vector after digestion of this vector with the restriction enzymes BamHI and XbaI. The constructed vector provides an N-terminal fusion of hexahistidine tag and T7 epitope with AC7 under the control of the cytomegalovirus promoter. The recombinant baculovirus for AC7 was constructed as follows. cDNA for human AC7 (19Nomura N. Miyajima N. Sazuka T. Tanaka A. Kawarabayashi Y. Sato S. Nagase T. Seki N. Ishikawa K. Tabata S. DNA Res. 1994; 1: 27-35Crossref PubMed Scopus (269) Google Scholar) was subcloned into the BamHI andBglII sites of an Invitrogen pBlueBacHis2 vector (frame C). Sf9 cells were cotransfected with the AC7 plasmid and wild-type viral DNA, and positive recombinant viral plaques were isolated. When expressed in the insect cells, this vector generated a fusion protein including AC7 that contains N-terminal (His)6, T7, and Xpress™ epitopes. The fusion junction was verified by automated sequencing. The control vector was generated from the T7-AC7 vector by creating a stop codon in the multiple cloning site of the vector and the control vector produced only the fusion tag. HEL cells (American Type Culture Collection, Manassas, VA) were grown in suspension culture in RPMI 1640 medium (Invitrogen) containing 10% heat-inactivated, charcoal-stripped fetal calf serum (Gemini-Bio-Products, Calabasas, CA). HEL cells were maintained at 37 °C (5% CO2) and used for experiments at a density range of between 0.2 and 0.4 × 106cell/ml. HEK 293 cells were grown in 20 mm HEPES-minimal essential medium containing 10% fetal bovine serum at 37 °C (5% CO2). pcDNA3.1-T7.His-AC7 and the control pcDNA3.1-T7.His vector were transfected in HEK 293 cells using Effectene reagent (Qiagen, Valencia, CA) while maintaining the cells in the regular growth medium. After incubation with the transfection reagents for 48 h, the cells were maintained in serum containing minimal essential medium for another 24 h prior to harvesting the cells. AC7 was expressed in Sf9 cells using the baculovirus expression system. The Sf9 cells were cultured in a spinner vessel containing Grace's insect cell medium (Invitrogen) and 10% fetal bovine serum at 27 °C (Tissue Culture Core/University of Colorado Cancer Center). Sf9 cells (300 × 106) were then infected with the positive recombinant baculovirus for 60 min in serum-free Grace's insect medium followed by an additional 48 h incubation in Grace's insect medium containing 10% fetal bovine serum. AC7-expressing Sf9 cells were identified based on Western blot analysis of total Sf9 cell protein using a monoclonal anti-T7 antibody (Novagen, Madison, WI). No signals in the molecular mass range of full-length AC7 were detected by the anti-T7 antibody in solubilized protein preparations from control vector-infected Sf9 cells. For the “back-phosphorylation” experiments, T7-tagged AC7-transfected HEK 293 or control transfected HEK 293 cells were pretreated with drugs or other reagents prior to harvesting as indicated in the figure legends and the text. For harvesting, the cells were resuspended in ice-cold membrane lysis buffer containing 20 mmHEPES, pH 7.4, 2 mm Na2EDTA, 150 mmNaCl, 0.2 mm Na3VO4, 10 mm β-glycerophosphate, 2 mm NaF, andCalbiochem Protease Inhibitor Mixture Set III (l mmAEBSF, 0.8 μm aprotinin, 50 μmbestatin, 15 μm E-64, 20 μm leupeptin, and 10 μm pepstatin A). The cell suspension was sonicated briefly on ice and centrifuged at 500 × g for 10 min at 4 °C. The resulting supernatant was then centrifuged at 90,000 × g for 45 min at 4 °C, and the pellet was solubilized as described below. For the isolation of HEL or HEK 293 cell membranes in experiments not involving back-phosphorylation, a minimum of 15 × 106cells were washed twice with ice-cold phosphate-buffered saline. The pelleted cells were resuspended at 5–6 × 106cells/ml of lysis buffer containing 50 mm Tris, pH 7.6, 2 mm MgCl2, 0.1 mmNa3VO4, 10 mm β-glycerophosphate, 1 mm benzamidine, 10 mm dithiothreitol, andCalbiochem Protease Inhibitor Mixture Set III. The cell suspension was drawn through a 22-gauge syringe 10 times, while the preparation was kept on ice and then centrifuged at 500 × g for 3 min at 4 °C. The resulting supernatant was then centrifuged at 90,000 × g for 60 min at 4 °C. Both the pellet (membrane protein) and supernatant (cytosolic protein) were saved. For immunoprecipitation (phosphorylation) experiments and Western blot analysis, the membrane protein was solubilized in 1.0% SDS. The samples were then incubated for 20 min at 80 °C and sonicated briefly. The protein concentration was determined using the BCA method (Pierce). Membranes from T7-AC7-expressing Sf9 cells or control vector-expressing Sf9 cells were harvested by centrifugation (2000 × g, for 5 min at 4 °C), suspended in 20 ml (per 100 ml of culture pellet) of 20 mm HEPES, pH 7.8, 500 mm NaCl, 5 mm EDTA, 1 mm EGTA, 2 mm dithiothreitol, and protease inhibitors (as described above). The samples were freeze-thawed twice (from liquid nitrogen into a 42 °C water bath), and the DNA was sheared by passing the preparation through an 18-gauge needle four times. The cell debris was removed by centrifugation at 500 × g for 10 min at 4 °C. The supernatant was then centrifuged at 100,000 ×g for 40 min at 4 °C, and the membrane pellet was resuspended in 20 mm HEPES, pH 7.8, 200 mmsucrose, 1 mm dithiothreitol plus protease inhibitors, as described above. The suspension was then recentrifuged at 100,000 × g for 40 min at 4 °C, and the final pellet was solubilized in 1.0% SDS and stored at −80 °C. For the immunoprecipitation of T7-tagged AC7, all of the steps were carried out at 4 °C, except when noted. In each sample, 150–200 μg of solubilized total Sf9 or HEK 293 cell membrane protein (AC7 and control vector-transfected) in 1% SDS was diluted with the IP buffer (40 mm Tris-HCl, pH 7.8, 100 mm NaCl, 5 mm Na2EDTA, 0.4 mm MgCl2, 2 mm methionine, 10 mm NaF, 1 mm Na3VO4, 20 mm sodium pyrophosphate, 25 mmNa2-β-glycerophosphate, and protease inhibitors as described above, and 1.2% Nonidet P-40) to a final SDS concentration of 0.08–0.12% (a minimum 10-fold Nonidet P-40/SDS ratio). The samples were incubated for 5 min with washed (IP buffer) protein A-agarose (PAA) beads (ImmunoPure immobilized protein A; Pierce) at a ratio of 10–20%:80–90% beads to solubilized protein in IP buffer, respectively, to remove material that bound nonspecifically to the beads. The beads were pelleted by centrifugation (10,000 ×g), and the supernatants were collected and shaken with 6 μg of monoclonal anti-T7 antibody (Novagen) for 2 h at room temperature and then incubated with washed (IP buffer) PAA beads for 1 h. The antigen/antibody/PAA conjugates were pelleted (10,000 × g) and washed once with IP buffer. The beads were then washed twice at 22 °C with phosphorylation buffer (50 mm Tris-HCl, pH 8.0, 5 mm MgCl2, 1 mm EGTA, 0.1% Triton X-100) and incubated for 2–5 min at 37 °C in 40 μl of phosphorylation buffer including 0.3 μm [γ-32P]ATP (6000 Ci/mmol; PerkinElmer Life Sciences) and 250 nm of the constitutively active PKC catalytic subunit purified from rat brain (20Lin Y.F. Browning M.D. Dudek E.M. Macdonald R.L. Neuron. 1994; 13: 1421-1431Abstract Full Text PDF PubMed Scopus (56) Google Scholar). The reaction was terminated with 10 μl of 200 mm Na2EDTA, pH 8.0. The beads were pelleted and washed two times with IP buffer. The samples were then reduced and alkylated to produce a tight band of AC during the PAGE. The sample was reduced by incubation in 40 mm dithiothreitol at 80 °C for 15 min. After a 15-min incubation at room temperature with 65 mm N-ethylmaleimide, loading dye was added, and the sample was briefly boiled before the PAGE. The precast 8% Tris-glycine polyacrylamide gels were from Novex/Invitrogen (Carlsbad, CA). After the electrophoresis, the gel was dried, and Kodak X-Omat Blue film was used for the autoradiography. For the phosphorylation by full-length recombinant PKC, T7-tagged AC7-transfected HEK 293 cells or control transfected HEK 293 cells were lysed in ice-cold membrane lysis buffer and a Triton X-100-insoluble plasma membrane fraction (representing a membrane microdomain thought to have characteristics similar to lipid rafts and enriched in signaling molecules) was isolated. This membrane fraction was isolated as described above under “HEK 293, HEL, and Sf9 Cell Membrane Preparations” with the following additional modification; in the final step the non-nuclear cell lysate was centrifuged at 90,000 × g in the presence of 0.5% Triton X-100 for 60 min at 4 °C. This membrane pellet was solubilized in 1% SDS, and the T7-tagged AC7 was immunoprecipitated with an anti-T7 monoclonal antibody with the addition of PAA beads (as described above). The immunoprecipitates from T7-AC7 and control transfected HEK 293 cells were then incubated for 30 min at 30 °C with recombinant PKCδ (0.8 specific activity units) or PKCε (1.3 specific activity units) and 10 μCi of [γ-32P]ATP (3000 Ci/mmol from PerkinElmer Life Sciences; final ATP concentration equaled 40 μm) in phosphorylation buffer (40 mm Tris-HCl, pH 7.4, 10 mm MgCl2, 0.2 mm CaCl2, 1 mm dithiothreitol, 25 mm β-glycerol phosphate, 1 mmNa3VO4, 2 μg/ml phosphatidylserine, 0.2 μg/ml diolein, 10 μm PDBu, and 0.02% Triton X-100). The reaction mixture was pelleted to separate the T7-AC7/anti-T7 antibody/PAA immunocomplex from the supernatant containing PKC and [γ-32P]ATP. The pellet was washed with 40 mm Tris-HCl, pH 8.0, and the supernatant was precipitated with trichloroacetic acid. Aliquots from both the pelleted fraction and the supernatant were dithiothreitol- andN-ethylmaleimide-treated, boiled in gel loading buffer, and loaded onto an 8% Tris-glycine gel for electrophoretic separation. The gel was dried and then exposed to x-ray autoradiography film. Recombinant purified PKCδ or PKCε (2 μg) was 32P-labeled through autophosphorylation for 30 min at 30 °C in the presence of 10 μCi of [γ-32P]ATP (3000 Ci/mmol from PerkinElmer Life Sciences; final ATP concentration equaled 40 μm) in phosphorylation buffer. After this reaction, the32P-labeled PKC was trichloroacetic acid-precipitated (10%) on ice, and the free 32P was removed with three washes in 95% ice-cold ethanol. 32P-Labeled PKC was resuspended at 100 ng/μl in 50% glycerol buffer (100 mmNaCl, 2 mm EGTA, 2 mm EDTA, 0.05% Triton X-100, and 5 mm TCEP adjusted to pH 7.8 with HEPES). The Triton X-100-insoluble plasma membrane fraction (100 μg) from control or AC7-transfected HEK cells was suspended in IP binding buffer A (20 mm HEPES, pH 7.8, 100 mm NaCl, 1 mm Na2EDTA, 0.4 mmMgCl2, 1 mm CaCl2, 2 mmmethionine, 1 mm Na3VO4, 20 mm sodium pyrophosphate, 25 mmNa2-β-glycerophosphate, 1.2% Nonidet P-40, 0.5 μg/ml γ-globulin free BSA, and protease inhibitors as described above). The membrane protein mixture was then added to washed PAA beads for 5 min at 4 °C to remove the nonspecific binding proteins. The beads were discarded, and the supernatant was incubated overnight at 4 °C with 4 μg of anti-T7 antibody. The solution containing the T7-AC7·anti-T7 immunocomplex was added to washed PAA beads and incubated for 120 min at 4 °C. The beads containing the immunocomplex were pelleted and washed once in IP binding buffer A to remove residual SDS. The beads were then resuspended in IP binding buffer A, and 100 ng of 32P-PKCδ or ε was added and incubated at 4 °C for 120 min. The beads containing the immunocomplex and bound 32P-PKC were pelleted and washed twice in IP binding buffer A. The supernatant and washes were combined and counted along with the pellets. 32P-PKC binding to PAA beads in the absence of T7 antibody or membrane protein was used for determination of nonspecific binding of 32P-PKC and was subtracted as background from the pelleted samples. For Western blotting, whole cell Sf9, HEK 293 cell lysates, and HEL cell lysates (in 1% SDS) were reduced and alkylated as described under “Immunoprecipitation, phosphorylation, and back-phosphorylation of AC7” prior to gel electrophoresis. Precast 8% Tris-glycine polyacrylamide gels (Novex/Invitrogen) were used to separate the protein bands. After the electrophoresis, the samples were transferred to MSI micronSep nitrocellulose (Osmonics, Westborough, MA) using a Xcell II Blot Module (Novex/Invitrogen) according to the manufacturer's recommendations. The blots containing Sf9 or HEK 293 cell protein were blocked with 3% nonfat dairy milk (NFDM) in TBS, pH 8.0, containing 0.1% Tween 20 (TBST) for 1 h at room temperature and then incubated for 1 h in 3% BSA in TBST containing 1:3000 dilution of monoclonal anti-T7 antibody (Novagen) or a 1:5000 dilution of the C6C anti-AC II family antibody (21Chakrabarti S. Rivera M. Yan S.Z. Tang W.J. Gintzler A.R. Mol. Pharmacol. 1998; 54: 655-662Crossref PubMed Scopus (79) Google Scholar) and washed three times with 3% NFDM TBST. The blots were then incubated for 1 h in 5% NFDM-TBST containing 1:30,000 dilution of goat anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody and subsequently washed three times with TBST and twice with TBS. The blots containing HEL cell protein were blocked with 5% NFDM-TBST for 1 h at room temperature, quickly rinsed in TBS, and then blotted overnight at 4 °C in 3% NFDM-TBST containing 1:1500 dilution of anti-δPKC antibody, 1:5000 dilution of anti-β-actin antibody, or the other appropriately diluted isoform specific PKC antibodies (PKCβ, 1:750; PKCγ, 1:5000; PKCδ, 1:750; PKCε, 1:2500; PKCθ, 1:750; PKCλ, 1:750; PKCζ, 1:1000; and PKCι, 1:750). The blot was then quickly rinsed with fresh 3% NFDM-TBST and washed three times with 0.1% liquid fish gelatin-TBS, 0.1% Tween 20, and 0.1% Triton X-100 at 22 °C. The blot was then incubated with 1:10,000 dilution of goat anti-mouse IgG-horseradish peroxidase-conjugated secondary antibody in 3% NFDM-TBST for 1 h at 22 °C and then washed three times with 0.1% fish gelatin in TBS, 0.1% Tween 20 at 22 °C. The blots were rinsed in TBS prior to immunoreactive band detection using enhanced chemiluminescence (Renaissance, PerkinElmer Life Sciences). For whole cell cAMP synthesis measurements, HEL cells were preloaded with 2 μCi/ml [2-3H]adenine in HEL culture medium for 6 h at 37 °C as previously described (4Yoshimura M. Tabakoff B. Alcohol Exp. Clin. Res. 1995; 19: 1435-1440Crossref PubMed Scopus (72) Google Scholar, 22Hellevuo K. Yoshimura M. Mons N. Hoffman P.L. Cooper D.M.F. Tabakoff B. J. Biol. Chem. 1995; 270: 11581-11589Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). At the end of the incubation, HEL cells were pelleted by centrifugation (200 ×g for 5 min), washed, and resuspended at a cell concentration of 1 × 106/ml in serum-free RPMI 1640, without phenol red, supplemented with 20 mm HEPES, pH 7.4 (assay buffer). Aliquots (0.4 ml) of the cell suspension were added to each well (24-well plates) and allowed to equilibrate for 30 min at 36 °C before the start of an assay. In experiments that utilized the PKC-derived inhibitory peptides, the assay buffer was modified slightly as described below to prevent premature reduction of the chimeric peptide (the PKC-derived/antennapedia peptides contain a Cys-Cys disulfide bond) prior to entry into the cells. Briefly, HEL cells were extensively washed three times (to remove exogenous glutathione) and resuspended at a cell concentration of 1 × 106/ml in glutathione-free, serum-free RPMI 1640 supplemented with 20 mm HEPES, pH 7.4, and 0.05% BSA. PKC-derived inhibitory peptides were added (final concentration of 2.0 μm) to the cell suspension and incubated at 37 °C for 2 h prior to the start of the experiment. cAMP formation was measured by monitoring the conversion of [3H]ATP to [3H]cAMP. The cells were treated with the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (400 μm), for 10 min prior to the addition of agonist. After the addition of agonist (PGE1 or cicaprost), cAMP formation was allowed to continue for 5 min before the reaction was terminated with trichloroacetic acid (final concentration of 10%). Other modulators of AC activity were added at concentrations and time points as described in the text and figure legends. ATP and cAMP were separated by sequential chromatography on Dowex 50 and neutral alumina columns and quantitated using a Beckman LS 6000TA liquid scintillation counter as previously described (4Yoshimura M. T" @default.
• W2030903683 created "2016-06-24" @default.
• W2030903683 creator A5014576325 @default.
• W2030903683 creator A5041404592 @default.
• W2030903683 creator A5056798350 @default.
• W2030903683 creator A5077317216 @default.
• W2030903683 date "2003-02-01" @default.
• W2030903683 modified "2023-10-13" @default.
• W2030903683 title "Ethanol-induced Phosphorylation and Potentiation of the Activity of Type 7 Adenylyl Cyclase" @default.
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