NUTRIENT STIMULATION RESULTS IN A RAPID CA2-DEPENDENT THREONINE PHOSPHORYLATION OF MYOSIN HEAVY-CHAIN IN RAT PANCREATIC-ISLETS AND RINM5F CELLS()

Activation of protein kinases plays an important role in the Ca2+-depe ndent stimulation of insulin secretion by nutrients, The aim of the pr esent study was to identify kinase substrates with the potential to re gulate secretion because these have been poorly defined. Nutrient stim ulation of the rat insulinoma RINm5F cell line and rat pancreatic isle ts resulted in an increase in the threonine phosphorylation of a 200-k Da protein. This was secondary to the gating of voltage-dependent Ca2 channels because it was reproduced by depolarizing KCI concentrations and blocked by the Ca2+ channel antagonist, verapamil. The peak rises in [Ca2+](i) preceded or were coincident with the maximal threonine p hosphorylation in response to both glyceraldehyde and KCl. In digitoni n-permeabilized RINm5F cells a rise in Ca2+ from 0.1 to 0.15 mu M was sufficient to increase phosphorylation, Protein kinase C, protein kina se A, and Ca2+/calmodulin-dependent kinase II did not appear to be res ponsible for the phosphorylation, yet the Ca2+ dependence of the respo nse suggests possible involvement of other members of the Ca2+/calmodu lin-dependent kinase family. The 200-kDa protein was identified as myo sin heavy chain by immunoprecipitation with a polyclonal nonmuscle myo sin antibody. Phosphopeptide mapping indicated that the site of phosph orylation on myosin heavy chain was the same for both KCl- and glycera ldehyde-stimulated cells. Phosphoamino acid analysis confirmed a low b asal phosphothreonine content of myosin heavy chain, which increased 6 -fold in response to KCl. A lesser (a-fold) increase in serine phospho rylation was also detected using this technique. Although myosin IIA a nd IIB were shown to be present in RINm5F cells and rat islets, myosin IIA was the predominant threonine-phosphorylated species, suggesting that the two myosin species might be independently regulated. Our resu lts identify myosin heavy chain as a novel kinase substrate in pancrea tic beta-cells and suggest that it might play an important role in the regulation of insulin secretion.