Endocrinology, Vol 136, 3784-3793, Copyright © 1995 by Endocrine Society

ARTICLES

Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells

K Matsumoto, K Fukunaga, J Miyazaki, M Shichiri and E Miyamoto
Department of Pharmacology, Kumamoto University School of Medicine, Japan.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) may play a key role in the regulation of insulin secretion. We obtained evidence for the presence of CaM kinase II and its substrate, a 84-kilodalton (kDa) protein, in mouse insulinoma MIN6 cells. CaM kinase II from MIN6 cells has one subunit of 55 kDa, determined by sodium dodecyl sulfate- polyacrylamide gel electrophoresis, is autophosphorylated in a Ca2+/CaM- dependent manner, and phosphorylates several substrates that serve for rat brain CaM kinase II. In the membrane fraction of MIN6 cells, we identified a 84-kDa protein that was immunoreactive with the antirat brain synapsin I antibody. One-dimensional phosphopeptide mapping by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed the sites of the phosphorylation by cAMP- dependent protein kinase (cAMP kinase) and that by CaM kinase II to be site 1 (10 kDa) and site 2 (30 kDa), respectively, therefore, the same as for rat brain synapsin I. In this context, we tentatively termed it synapsin I-like protein. In 32P-labeled cells, nonfuel insulin secretagogues, such as ionomycin, KCl, and tolbutamide, and a fuel secretagogue, glucose, stimulated autophosphorylation of CaM kinase II and the phosphorylation of synapsin I-like protein. These secretagogues potentiated the Ca(2+)-independent activity of CaM kinase II and secretion of insulin from MIN6 cells. The 84-kDa protein is apparently a newly identified member of the synapsin family. We suggest that CaM kinase II regulates insulin secretion via phosphorylation of synapsin I- like protein.

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