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.

Abstract

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.