Identification of Exo2 as the catalytic subunit of protein kinase A reveals a role for cyclic AMP in Ca(2+)-dependent exocytosis in chromaffin cells.
Digitonin-permeabilized chromaffin cells secrete catecholamines by exocytosis in response to micromolar Ca2+ concentrations, but lose the ability to secrete in response to Ca2+ as the cells lose soluble proteins through the plasma membrane pores. We have previously shown [Morgan and Burgoyne (1992) Nature, 355, 833-836] that cytosol can retard this loss of secretory competence and that two distinct stimulatory activities (Exo1 and Exo2) are present in cytosol. Here we report that Exo2 behaved as a single peak of activity through purification on hydroxyapatite, ammonium sulfate precipitation and gel filtration and the activity correlated with a single polypeptide of approximately 44 kDa on SDS gels. Protein sequencing of this band revealed it to be the catalytic subunit of cyclic AMP-dependent protein kinase (PKA). Both cyclic AMP and the commercially available catalytic subunit of PKA stimulated exocytosis in a dose-dependent manner which was absolutely dependent on the presence of micromolar Ca2+. These data show that PKA (Exo2) regulates Ca(2+)-dependent exocytosis in bovine adrenal chromaffin cells.
Morgan
A
A
Department of Physiology, University of Liverpool, UK.
Wilkinson
M
M
Burgoyne
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D002384:Catalysis; D002417:Cattle; D002478:Cells, Cultured; D002838:Chromaffin System / Q000166:cytology / Q000378:metabolism*; D002850:Chromatography, Gel; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis / Q000502:physiology*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012756:Sheep
C073170:Exo2 protein, Ovis aries; D009419:Nerve Tissue Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D002118:Calcium