Ca(2+)-calmodulin-dependent phosphorylation of islet secretory granule proteins.
The effect of Ca2+ and calmodulin on phosphorylation of islet secretory granule proteins was studied. Secretory granules were incubated in a phosphorylation reaction mixture containing [32P]ATP and test reagents. The 32P-labeled proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 32P content was visualized by autoradiography, and the relative intensities of specific bands were quantitated. When the reaction mixture contained EGTA and no added Ca2+, 32P was incorporated into two proteins with molecular weights of 45,000 and 13,000. When 10(-4) M Ca2+ was added without EGTA, two additional proteins (58,000 and 48,000 Mr) were phosphorylated, and the 13,000-Mr protein was absent. The addition of 2.4 microM calmodulin markedly enhanced the phosphorylation of the 58,000- and 48,000-Mr proteins and resulted in the phosphorylation of a major protein whose molecular weight (64,000 Mr) is identical to that of one of the calmodulin binding proteins located on the granule surface. Calmodulin had no effect on phosphorylation in the absence of Ca2+ but was effective in the presence of calcium between 10 nM and 50 microM. Trifluoperazine and calmidazolium, calmodulin antagonists, produced a dose-dependent inhibition of the calmodulin effect. 12-O-tetradecanoylphorbol 13-acetate, a phorbol ester that activates protein kinase C, produced no increase in phosphorylation, and 1-(5-isoquinoline sulfonyl)-2-methyl piperazine dihydrochloride, an inhibitor of protein kinase C, had no effect. These results indicate that Ca(2+)-calmodulin-dependent protein kinases and endogenous substrates are present in islet secretory granules.
Watkins
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DT
Department of Anatomy, University of Connecticut Health Center, Farmington 06030.
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001345:Autoradiography; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology*; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D005399:Fishes; D007515:Islets of Langerhans / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*
D002147:Calmodulin; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D011506:Proteins; D004533:Egtazic Acid; D000255:Adenosine Triphosphate; D002118:Calcium