Calcium-stimulated phosphorylation of MAP-2 in pancreatic betaTC3-cells is mediated by Ca2+/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 betaTC3 cells and isolated rat islets. In immunoprecipitation experiments employing alpha-toxin-permeabilized betaTC3 cells, elevation of intracellular Ca2+ or addition of forskolin, an adenylate cyclase activator, induced significant phosphorylation of MAP-2 in situ. The effect of Ca2+ 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 Ca2+. Phosphopeptide mapping revealed that the phosphorylation pattern observed in situ upon incubation of the betaTC3 cells with increased free Ca2+, 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 Ca2+-dependent insulin secretion.
Krueger
K A
KA
Department of Biochemistry and Molecular Biology, University of North Texas Health Science Center, Fort Worth, Texas 76107-2699, USA.
Bhatt
H
H
Landt
M
M
Easom
R A
RA
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D005576:Colforsin / Q000494:pharmacology; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007340:Insulinoma; D007515:Islets of Langerhans / Q000378:metabolism*; D007546:Isoquinolines / Q000494:pharmacology; D007700:Kinetics; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D010190:Pancreatic Neoplasms; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000302:isolation & purification; D010766:Phosphorylation; D051381:Rats; D013449:Sulfonamides; D014407:Tumor Cells, Cultured
D004791:Enzyme Inhibitors; D007546:Isoquinolines; D008869:Microtubule-Associated Proteins; D010748:Phosphopeptides; D013449:Sulfonamides; D005576:Colforsin; D017868:Cyclic AMP-Dependent Protein Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D000262:Adenylyl Cyclases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide; D002118:Calcium