Endogenous substrate proteins for Ca2+-calmodulin-dependent, Ca2+-phospholipid-dependent and cyclic AMP-dependent protein kinases in mouse pancreatic islets

Abstract

The occurrence of endogenous substrate proteins for Ca2+-dependent protein kinase, augmented by either phospholipid or calmodulin, and for cyclic AMP-dependent protein kinase was examined in homogenates and subcellular fractions of mouse pancreatic islets. Islet protein phosphorylation was enhanced by Ca2+-calmodulin; the major endogenous substrates in the homogenate were two proteins of Mr 53000 and 100000. The Mr-100000 phosphoprotein was localized to a 27000g-supernatant fraction, whereas the Mr-53000 phosphoprotein was present in a 27000g particulate fraction of mouse islets. In the presence of Ca2+, phosphatidylserine stimulated phosphorylation of 15 proteins, of Mr 17000-190000, in a 27000g-supernatant fraction. No effects of Ca2+ plus phosphatidylserine were observed in a 27000g particulate fraction of mouse islets. Examination of cyclic AMP-dependent protein phosphorylation revealed five substrate proteins, of Mr 23000-72000, present in the 27000g supernatant of mouse islets. No common substrates for either the two Ca2+-dependent phosphorylation systems or for the cyclic AMP-dependent and the Ca2+-calmodulin-dependent phosphorylation systems were noted. On the other hand, the actions of the cyclic AMP-sensitive and the Ca2+-phospholipid-sensitive systems may be overlapping, since two common substrates for them were noted in the 27000g-supernatant fraction. The results are consistent with the hypothesis that protein phosphorylation may play a role in the regulation of insulin secretion by Ca2+ and cyclic AMP. The extensive stimulatory effect of phosphatidylserine furthermore suggests that the Ca2+-phospholipid-sensitive protein kinase may prove to be a prominent phosphorylation system in pancreatic islets.

Identification of calmodulin-dependent protein kinase III and its major Mr 100,000 substrate in mammalian tissues.

“…It is also likely that the 100-kDa protein is identical to phosphorylated proteins of similar molecular weight observed in previous studies of endogenous CaM-dependent protein phosphorylation in pancreas and in a number ofother mammalian tissues. Substrates with Mr 92,000-100,000 have been identified in pancreatic acinar cells (27), in islet cells (23,24,43), and in hamster insulinoma cells (44). These results strongly suggest that the 100-kDa protein-CaM kinase III system is present in both endocrine and exocrine pancreas.…”

Ion Channels and Insulin Secretion

“…This kinase has been described in pancreatic islets, HIT cells, and a transplantable rat insulinoma (224)(225)(226). The partially characterized PKC activity from mouse and rat islets closely resembles the enzyme activity seen in other tissues (225,227,228).…”

Distinct mechanisms for two amplification systems of insulin release

“…If one accepts that, at the low concentrations purposely used, forskolin [11] and TPA [12] rather specifically activate the adenylate cyclase and the protein kinase C respectively, the following conclusion seems warranted. Pancreatic f-cells are equipped with two systems that serve to amplify the release of insulin in response to a primary stimulus.…”

“…Evidence that activation of protein kinase C also amplifies insulin release is more recent, and the few attempts to elucidate how this amplification occurs remain contradictory [7][8][9][10]. The demonstration that distinct islet proteins can be phosphorylated by these two kinases [11][12][13] raises the possibility that different cellular events underlie the changes in insulin release resulting from an activation of these two pathways.…”