Ca2+/calmodulin-dependent protein kinase II and synapsin I-like protein in mouse insulinoma MIN6 cells.
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.
Matsumoto
K
K
Department of Pharmacology, Kumamoto University School of Medicine, Japan.
Fukunaga
K
K
Miyazaki
J
J
Shichiri
M
M
Miyamoto
E
E
D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000235:genetics / Q000378:metabolism; D000078790:Insulin Secretion; D007340:Insulinoma / Q000737:chemistry* / Q000201:enzymology / Q000473:pathology; D015759:Ionomycin / Q000494:pharmacology; D051379:Mice; D008822:Mice, Transgenic; D010190:Pancreatic Neoplasms / Q000737:chemistry* / Q000201:enzymology / Q000473:pathology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D013379:Substrate Specificity; D016704:Synapsins / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors; D014044:Tolbutamide / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D007328:Insulin; D016704:Synapsins; D015759:Ionomycin; D011189:Potassium Chloride; D014044:Tolbutamide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium