Abstract
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.
Highlights
Tein kinases that are involved in diverse cellular processes such as synaptic transmission, metabolism, synaptic plasticity, gene expression, stimulus-secretion coupling, and cell growth and differentiation
Phosphorylation of the 70-kDa Protein and chromogranin A (CgA) by CaM Kinase II—Since the 70-kDa protein is phosphorylated by CaM kinase II [17], we examined whether CgA partially purified from chromaffin granules was phosphorylated by CaM kinase II
The anti-CgA antibody recognized the 70-kDa protein (Fig. 3, lane 3) as well as CgA (Fig. 3, lane 4). These results indicate that the 70-kDa protein is CgA
Summary
(Received for publication, August 31, 1995, and in revised form, February 27, 1996). Nobuyuki Yanagihara‡, Yasuharu Oishi§, Hideyuki Yamamoto§, Masato Tsutsui¶, Jun Kondohʈ, Tutomu Sugiura**, Eishichi Miyamoto§, and Futoshi Izumi. In 32P-labeled bovine adrenal medullary cells, 56 mM K؉ increased the phosphorylation of CgA and catecholamine secretion in similar time- and concentrationdependent 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. The protein was phosphorylated in vitro by multifunctional protein kinases, and the phosphorylation of CgA was associated with catecholamine secretion in cultured adrenal medullary cells stimulated by 56 mM Kϩ
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