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Phorbol Ester-Induced Stimulation and Phosphorylation of Adenylyl Cyclase 2
Ofer Jacobowitz and Ravi Iyengar
Proceedings of the National Academy of Sciences of the United States of America
Vol. 91, No. 22 (Oct. 25, 1994), pp. 10630-10634
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/2366083
Page Count: 5
You can always find the topics here!Topics: Cell membranes, Proteins, Antibodies, Baculoviridae, Gels, Receptors, Phosphorylation, Enzymes, Infections, Signals
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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 α 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.
Proceedings of the National Academy of Sciences of the United States of America © 1994 National Academy of Sciences