PHOSPHORYLATION OF CHROMOGRANIN-A AND CATECHOLAMINE SECRETION STIMULATED BY ELEVATION OF INTRACELLULAR CA2-MEDULLARY CELLS( IN CULTURED BOVINE ADRENAL)

We have recently isolated a new endogenous substrate of 70 kDa for Ca2 +/calmodulin dependent protein kinase II (CaM kinase II) from bovine a drenal medullary cells (Yanagihara, N., Toyohira, Y., Yamamoto, H., Oh ta, Y., Tsutsui, M., Miyamoto, E., and Izumi, F. (1994) Mol. Pharmacol . 46, 423-430). Here we report the sequence analysis of the 70-kDa pro tein and examine its phosphorylation by various protein kinases in vit ro and by depolarization of the cultured cells, Protein sequencing and immunoblotting revealed that the 70-kDa protein is chromogranin A (Cg A) or a closely related protein. Partially purified CgA was phosphoryl ated by cyclic AMP-dependent protein kinase and protein kinase C as we ll as CaM kinase II, Tryptic phosphopeptide mapping patterns of CgA di ffered among these protein kinases. In P-32-labeled bovine adrenal med ullary cells, 56 mM K+ increased the phosphorylation of CgA and catech olamine 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 ele vation of the intracellular Ca2+ stimulates the phosphorylation of CgA associated with catecholamine secretion in cultured adrenal medullary cells.