CALCIUM-STIMULATED PHOSPHORYLATION OF MAP-2 IN PANCREATIC BETA-TC3-CELLS IS MEDIATED BY CA2+ CALMODULIN-DEPENDENT KINASE-II/

An understanding of the role of CaM kinase II in the pancreatic beta-c ell 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 immu no-blot 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 experim ents employing alpha-toxin-permeabilized beta TC3 cells, elevation of intracellular Ca2+ or addition of forskolin, an adenylate cyclase acti vator, induced significant phosphorylation of MAP-2 in situ. The effec t of Ca2+ was rapid, concentration-dependent and closely correlated wi th 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 littl e effect on MAP-2 phosphorylation stimulated by elevated Ca2+. Phospho peptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the beta TC3 cells with increased free Ca2+, w as strikingly similar to that generated in vitro by CaM kinase Il, mos t notably with regard to the increased phosphate incorporated into one prominent site, These data provide evidence that MAP-2 is phosphoryla ted by CaM kinase II in the pancreatic beta-cell in situ, and that thi s event may provide an important Link in the mediation of Ca2+-depende nt insulin secretion.