Effects of Ca 2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rat islets of Langerhans

Abstract

Activation of Ca 2+-calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic β-cell. To study the properties of such kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [γ- 32P]ATP. Phosphorylated proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis and detected by autoradiography. The phosphorylation of certain proteins could be enhanced by Ca 2+ plus calmodulin or by cyclic AMP. The major effect of Ca 2+ and calmodulin was to stimulate the phosphorylation of a protein (P53) of molecular weight 53 100±500 ( n = 15). Maximum phosphorylation of protein P53 occurred within 2 min with 2 μM free Ca 2+ and 0.7 μM calmodulin. Incorporation of label into protein P53 was inhibited by trifluoperazine or W7 but not by cyclic AMP-dependent protein kinase inhibitor. Phosphorylation of a protein of similar molecular weight could be enhanced to a lesser extent in the absence of Ca 2+ but in the presence of cyclic AMP and 3-isobutylmethylxanthine: this phosphorylation was blocked by cyclic AMP-dependent protein kinase inhibitor. Cyclic AMP also stimulated incorporation of label into polypeptides of molecular weights 55 000 and 70–80 000. The results are consistent with the hypothesis that protein phosphorylation mechanisms may play a role in the regulation of insulin secretion.