Phosphorylation of chromogranin A and catecholamine secretion stimulated by elevation of intracellular Ca2+ in cultured bovine adrenal medullary cells.
We have recently isolated a new endogenous substrate of 70 kDa for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) from bovine adrenal medullary cells (Yanagihara, N., Toyohira, Y., Yamamoto, H., Ohta, Y., Tsutsui, M., Miyamoto, E., and Izumi, F. (1994) Mol. Pharmacol. 46, 423-430). Here we report the sequence analysis of the 70-kDa protein and examine its phosphorylation by various protein kinases in vitro and by depolarization of the cultured cells. Protein sequencing and immunoblotting revealed that the 70-kDa protein is chromogranin A (CgA) or a closely related protein. Partially purified CgA was phosphorylated by cyclic AMP-dependent protein kinase and protein kinase C as well as CaM kinase II. Tryptic phosphopeptide mapping patterns of CgA differed among these protein kinases. In 32P-labeled bovine adrenal medullary cells, 56 mM K+ increased the phosphorylation of CgA and catecholamine secretion in similar time- and concentration-dependent manners, both of which were inhibited by 20 mM MgSO4, an inhibitor of voltage-dependent Ca2+ channels. These findings suggest that CgA serves as a substrate for several multifunctional protein kinases and that the elevation of the intracellular Ca2+ stimulates the phosphorylation of CgA associated with catecholamine secretion in cultured adrenal medullary cells.
Yanagihara
N
N
Department of Pharmacology, University of Occupational and Environmental Health, School of Medicine, Kitakyushu 807, Japan.
Oishi
Y
Y
Yamamoto
H
H
Tsutsui
M
M
Kondoh
J
J
Sugiura
T
T
Miyamoto
E
E
Izumi
F
F
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D053379:Chromogranin A; D002864:Chromogranins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D010710:Phosphates / Q000378:metabolism; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism*; D014357:Trypsin
D002395:Catecholamines; D053379:Chromogranin A; D002864:Chromogranins; D010446:Peptide Fragments; D010710:Phosphates; D010748:Phosphopeptides; D004299:Dopamine beta-Hydroxylase; D011494:Protein Kinases; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D014357:Trypsin; D011188:Potassium; D002118:Calcium