Effects of Ca2+, calmodulin and cyclic AMP on the phosphorylation of endogenous proteins by homogenates of rt islets of langerhans.
Activation of Ca2+ -calmodulin- and cyclic AMP-dependent protein kinases has been suggested to be involved in stimulus-secretion coupling in the pancreatic beta-cell. To study the properties of suc kinases and their endogenous protein substrates homogenates of rat islets of Langerhans were incubated with [gamma-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 Ca2+ plus calmodulin or by cyclic AMP. The major effect of Ca2+ 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 micrometers free Ca2+ and 0.7 micrometers 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 proteins of similar molecular weight could be enhanced to a lesser extent in the absence of Ca2+ 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.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000494:pharmacology*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D000242:Cyclic AMP / Q000494:pharmacology*; D007515:Islets of Langerhans / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D008970:Molecular Weight; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014268:Trifluoperazine / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D010750:Phosphoproteins; D014268:Trifluoperazine; D000255:Adenosine Triphosphate; D000242:Cyclic AMP; D011494:Protein Kinases; D002118:Calcium