The role of calmodulin in the regulation of protein phosphorylation and insulin release in hamster insulinoma cells

The role of calmodulin in the regulation of protein phosphorylation and insulin release was studied utilizing a Syrian hamster insulinoma and rat pancreatic islets. Cytosol prepared from single cell suspensions of insulinoma cells contains several proteins that are phosphorylated in vitro in a Ca²⁺-dependent, cyclic nucleotide-independent manner. The predominant Ca²⁺-dependent phosphoprotein displayed an apparent Mr of 98,000 (P-98) on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Phosphorylation of P-98 was enhanced by addition of Ca²⁺ or purified calmodulin and was inhibited by EGTA or trifluoperazine. The phosphorylation reaction required Mg²⁺ ATP and the ³²P-labeled phosphoproteins displayed the properties expected of a phosphoester of serine or threonine. We conclude that the insulinoma cytosol contains a calmodulin-dependent protein kinase. We also present evidence suggesting that the enzyme is distinct from phosphorylase kinase. Trifluoperazine, at concentrations that inhibited Ca²⁺-dependent phosphorylation, blocked glucose-stimulated insulin release from isolated rat islets. Trifluoperazine also inhibited insulin release from insulinoma cells induced by 40 mM K⁺ or 1 mM ouabain without affecting the increased cellular ⁴⁵Ca²⁺ uptake observed with these agents. In contrast, glucagon-stimulated insulin release, a cAMP-mediated event, was unaffected by trifluoperazine. The data suggest that cAMP and Ca²⁺ regulate insulin release by distinct mechanisms and that calmodulin may be involved in Ca²⁺-mediated insulin release possibly through an effect on a calmodulin-dependent protein kinase.