Phorbol ester-induced stimulation and phosphorylation of adenylyl cyclase 2

Proc Natl Acad Sci U S A. 1994 Oct 25;91(22):10630-4. doi: 10.1073/pnas.91.22.10630.


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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Adenylyl Cyclases / biosynthesis
  • Adenylyl Cyclases / isolation & purification
  • Adenylyl Cyclases / metabolism*
  • Alkaloids / pharmacology
  • Amino Acid Sequence
  • Animals
  • Baculoviridae
  • Base Sequence
  • Cell Line
  • Chromatography, Affinity
  • DNA Primers
  • Electrophoresis, Polyacrylamide Gel
  • GTP-Binding Proteins / metabolism
  • Immune Sera
  • Isoenzymes / biosynthesis
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Kinetics
  • Mammals
  • Molecular Sequence Data
  • Peptides / immunology
  • Phosphorylation
  • Protein Kinase C / antagonists & inhibitors
  • Protein Kinase C / metabolism*
  • Rabbits / immunology
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / metabolism
  • Signal Transduction
  • Spodoptera
  • Staurosporine
  • Tetradecanoylphorbol Acetate / pharmacology*
  • Transfection


  • Alkaloids
  • DNA Primers
  • Immune Sera
  • Isoenzymes
  • Peptides
  • Recombinant Proteins
  • Protein Kinase C
  • GTP-Binding Proteins
  • Adenylyl Cyclases
  • Staurosporine
  • Tetradecanoylphorbol Acetate