Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2.
Adenylyl cyclase 2 was expressed in Sf9 cells by recombinant baculovirus infection. Phorbol 12-myristate 13-acetate (PMA) treatment of cells expressing adenylyl cyclase 2 (AC2) increased basal activity. This increase was blocked by staurosporine, a protein kinase C inhibitor. PMA treatment increased Vmax without affecting Km. Greatest increase in basal activity was seen at physiologically relevant Mg2+ concentrations. PMA treatment did not alter sensitivity to guanine nucleotide stimulatory factor (Gs) but enhanced stimulation at all concentrations of activated Gs alpha subunit tested. AC2 was tagged at the N terminus with an 8-amino acid epitope. Epitope-tagged AC2 was purified to apparent homogeneity in a single step by using an antiepitope antibody-affinity column. The eluate was resolved by SDS/PAGE. Silver staining of the gel showed a 106-kDa band. The purified protein was recognized by antipeptide antibody against a region common to all mammalian adenylyl cyclases. The epitope-tagged enzyme expressed in Sf9 cells was also stimulated by PMA. When cells were labeled with 32P and treated with PMA, a 3-fold increase in 32P incorporation of purified epitope-tagged AC2 was observed. We conclude that activation of protein kinase C results in phosphorylation and stimulation of AC2, a cell-surface G protein effector enzyme. Thus, covalent modification of cell-surface effectors may provide an independent mode for signal transmission through G protein pathways.
Jacobowitz
O
O
Department of Pharmacology, Mount Sinai School of Medicine, City University of New York, NY 10029.
Iyengar
R
R
D000262:Adenylyl Cyclases / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000470:Alkaloids / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism; D007106:Immune Sera; D007527:Isoenzymes / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008322:Mammals; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D015398:Signal Transduction; D018411:Spodoptera; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014162:Transfection
D000470:Alkaloids; D017931:DNA Primers; D007106:Immune Sera; D007527:Isoenzymes; D010455:Peptides; D011994:Recombinant Proteins; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate