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

U. K. Schubart, J. Erlichman, N. Fleischer

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)4120-4124
Number of pages5
JournalJournal of Biological Chemistry
Volume255
Issue number9
StatePublished - Dec 1 1980

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ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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