Ca2+-dependent binding of cytosolic components to insulin-secretory granules results in Ca2+-dependent protein phosphorylation

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Abstract
Incubation of a rat islet cell tumor homogenate with [.gamma.-32P]ATP resulted in the Ca2+-dependent phosphorylation of 100,000-, 57,000-, 29,000-, 26,000 and 14,000-MW proteins. The Ca2+ concentration required was in the low-.mu.M range. Isolated insulin granules did not exhibit Ca2+-dependent protein phosphorylation, whereas a soluble protein fraction showed the Ca2+-dependent phosphorylation of a 57,000-MW protein. Combination of insulin granules with the soluble protein fraction resulted in the additional Ca2+-dependent phosphorylations of 100,000-, 29,000- and 10,000-MW 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 100,000-, 29,000- and 10,000-MW proteins but rendered the Ca2+-dependent phosphorylation of the 57,000-MW soluble protein dependent on exogenous calmodulin. The components of the soluble protein fraction responsible for the interaction with insulin granules bound to intact granules in a Ca2+-dependent manner. After phosphorylation, the 29,000-MW protein remained attached to granules, whereas the 100,000- and 10,000-MW proteins dissociated from granules. These Ca2+-dependent phenomena may be of regulatory importance in the secretory mechanism.