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J Biol Chem, Vol. 273, Issue 35, 22729-22737, August 28, 1998

Nutrient Stimulation Results in a Rapid Ca²⁺-dependent Threonine Phosphorylation of Myosin Heavy Chain in Rat Pancreatic Islets and RINm5F Cells

Justine R. Wilson, Russell I. Ludowyke^¶, and Trevor J. Biden

From the  Garvan Institute of Medical Research and the ^¶ Centre for Immunology, St. Vincent's Hospital, University of New South Wales, Sydney, New South Wales 2010, Australia

Activation of protein kinases plays an important role in the Ca²⁺-dependent stimulation of insulin secretion by nutrients. The aim of the present study was to identify kinase substrates with the potential to regulate secretion because these have been poorly defined. Nutrient stimulation of the rat insulinoma RINm5F cell line and rat pancreatic islets resulted in an increase in the threonine phosphorylation of a 200-kDa protein. This was secondary to the gating of voltage-dependent Ca²⁺ channels because it was reproduced by depolarizing KCl concentrations and blocked by the Ca²⁺ channel antagonist, verapamil. The peak rises in [Ca²⁺]_i preceded or were coincident with the maximal threonine phosphorylation in response to both glyceraldehyde and KCl. In digitonin-permeabilized RINm5F cells a rise in Ca²⁺ from 0.1 to 0.15 µM was sufficient to increase phosphorylation. Protein kinase C, protein kinase A, and Ca²⁺/calmodulin-dependent kinase II did not appear to be responsible for the phosphorylation, yet the Ca²⁺ dependence of the response suggests possible involvement of other members of the Ca²⁺/calmodulin-dependent kinase family. The 200-kDa protein was identified as myosin heavy chain by immunoprecipitation with a polyclonal nonmuscle myosin antibody. Phosphopeptide mapping indicated that the site of phosphorylation on myosin heavy chain was the same for both KCl- and glyceraldehyde-stimulated cells. Phosphoamino acid analysis confirmed a low basal phosphothreonine content of myosin heavy chain, which increased 6-fold in response to KCl. A lesser (2-fold) increase in serine phosphorylation was also detected using this technique. Although myosin IIA and 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 results identify myosin heavy chain as a novel kinase substrate in pancreatic -cells and suggest that it might play an important role in the regulation of insulin secretion.

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