Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation.
1. Incubation of a rat islet cell tumour homogenate with [gamma-32P]ATP resulted in the Ca2+-dependent phosphorylation of 100000-, 57000-, 29000-, 26000- and 14000-Mr proteins. The Ca2+ concentration required was in the low-microM range. 2. Isolated insulin granules did not exhibit Ca2+-dependent protein phosphorylation, whereas a soluble protein fraction showed the Ca2+-dependent phosphorylation of a 57000-Mr protein. Combination of insulin granules with the soluble protein fraction resulted in the additional Ca2+-dependent phosphorylations of 100000-, 29000- and 10000-Mr proteins. The latter phosphorylations were not enhanced by exogenous calmodulin, but nevertheless were inhibited by trifluoperazine. Removal of endogenous calmodulin from the soluble protein fraction before incubation with insulin granules did not abolish the Ca2+-dependent phosphorylations of the 100000-, 29000- and 10000-Mr proteins but rendered the Ca2+-dependent phosphorylation of the 57000-Mr soluble protein dependent on exogenous calmodulin. 3. The components of the soluble protein fraction responsible for the interaction with insulin granules bound to intact granules in a Ca2+-dependent manner. 4. After phosphorylation, the 29000-Mr protein remained attached to granules, whereas the 100000- and 10000-Mr proteins dissociated from granules. 5. These Ca2+-dependent phenomena may be of regulatory importance in the secretory mechanism.
D007516:Adenoma, Islet Cell / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D002147:Calmodulin / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000187:drug effects / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D010190:Pancreatic Neoplasms / Q000378:metabolism; D010766:Phosphorylation; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D014268:Trifluoperazine / Q000494:pharmacology
D002147:Calmodulin; D007328:Insulin; D011506:Proteins; D014268:Trifluoperazine; D002118:Calcium