Interaction between protein kinase C and Exo1 (14-3-3 protein) and its relevance to exocytosis in permeabilized adrenal chromaffin cells.
The roles of protein kinase C (PKC) and Exo1 in exocytosis from digitonin-permeabilized adrenal chromaffin cells were explored by using exogenous purified proteins in a run-down/reconstitution system. The stimulatory action of Exo1 on exocytosis from run-down cells was found to be completely dependent on the continuous presence of exogenous MgATP, suggesting that it acts on the slow phase of exocytosis [Holz, Bittner, Peppers, Senter & Eberhard (1989), J. Biol. Chem. 264, 5412-5419]. Partially purified rat brain PKC was found to be able to stimulate Ca(2+)-dependent exocytosis from run-down cells in a dose-dependent manner. This effect was indeed due to PKC and not a contaminant in the PKC fraction, since the PKC activator phorbol 12-myristate 13-acetate (PMA), under conditions in which control secretion was not affected, potentiated the effect of the exogenous PKC in stimulating secretion. Furthermore, although either PKC or Exo1 alone could stimulate exocytosis from run-down cells, the effect of combining the fractions was synergistic, as had previously been observed using PMA treatment combined with Exo1 incubation [Morgan & Burgoyne (1992) Nature (London) 355, 833-836]. The observed synergy between PKC and Exo1 was not due to PKC-mediated phosphorylation of Exo1, and Exo1 was found not to affect PKC activity in enzyme assays. We conclude that PKC and Exo1 act synergistically in the slow phase of Ca(2+)-dependent exocytosis from adrenal chromaffin cells. Furthermore, PKC does not directly affect Exo1, but rather enhances the activity of Exo1 by a putative phosphorylation of another, unidentified, component of the exocytotic machinery which facilitates the action of Exo1 in exocytosis.
Morgan
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Department of Physiology, University of Liverpool, U.K.
Burgoyne
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RD
D048948:14-3-3 Proteins; D000313:Adrenal Medulla / Q000166:cytology / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002417:Cattle; D002478:Cells, Cultured; D002837:Chromaffin Granules / Q000378:metabolism*; D004789:Enzyme Activation; D005089:Exocytosis; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010751:Phosphopyruvate Hydratase / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D009419:Nerve Tissue Proteins; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C; D010751:Phosphopyruvate Hydratase; D013755:Tetradecanoylphorbol Acetate