Calcium-stimulated phosphorylation of MAP-2 in pancreatic beta TC3-cells is mediated by Ca super(2+)/calmodulin-dependent kinase II

An understanding of the role of CaM kinase II in the pancreatic beta -cell is dependent on the identification of its cellular targets. One of the best substrates of CaM kinase II in vitro that could function in secretory events is the microtubule-associated protein, MAP-2. By immunoblot analysis, a high molecular weight protein with electrophoretic properties characteristic of MAP-2, was identified in rat insulinoma beta TC3 cells and isolated rat islets. In immunoprecipitation experiments employing alpha -toxin-permeabilized beta TC3 cells, elevation of intracellular Ca super(2+) or addition of forskolin, an adenylate cyclase activator, induced significant phosphorylation of MAP-2 in situ. The effect of Ca super(2+) was rapid, concentration-dependent and closely correlated with activation of CaM kinase II under similar experimental conditions. H-89, a specific and potent inhibitor of cAMP-dependent protein kinase (PKA), prevented forskolin-induced MAP-2 phosphorylation but had little effect on MAP-2 phosphorylation stimulated by elevated Ca super(2+). Phosphopeptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the beta TC3 cells with increased free Ca super(2+), was strikingly similar to that generated in vitro by CaM kinase II, most notably with regard to the increased phosphate incorporated into one prominent site. These data provide evidence that MAP-2 is phosphorylated by CaM kinase II in the pancreatic beta -cell in situ, and that this event may provide an important link in the mediation of Ca super(2+)-dependent insulin secretion.