Abstract
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 microM 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 beta-cells and suggest that it might play an important role in the regulation of insulin secretion.
Highlights
In this study we have provided evidence that MHC is a possible substrate for Ca2ϩ-dependent kinases and is both serine- and threonine-phosphorylated upon stimulation of the rat insulinoma cell line RINm5F
The maximal increase in threonine phosphorylation occurs slightly later or in parallel to (KCl) the peak rise in [Ca2ϩ]i, but its onset certainly precedes that of insulin secretion, which lags several minutes after nutrient delivery [7, 43]
Threonine phosphorylation was triggered by even small increases in [Ca2ϩ]i above 0.1 M and, because of the very low phosphothreonine content of MHC under basal conditions, showed a greater increase (6 –9-fold) than did serine phosphorylation (2-fold)
Summary
§ Recipient of an Australian Postgraduate Award. ʈ Supported by a Sternberg Research Fellowship. ** To whom correspondence should be addressed: Garvan Inst. of Medical Research, 384 Victoria St., Darlinghurst, NSW 2010, Australia. The importance of CaM kinase is better documented, and it has been widely reported to mediate phosphorylation of a 55-kDa islet cell protein in response to nutrient stimulation [12]. This protein has not yet been identified; tubulin, a cytoskeleton-associated protein, has been suggested as a possible candidate [15]. More recently there has been direct evidence to show that MAP2, a microtubule-associated protein, is a major substrate for CaM kinase II in the pancreatic -cell [16] This suggests that proteins associated with the cytoskeleton may be important kinase substrates linking the rise in [Ca2ϩ]i to insulin secretion. It functions primarily as a motor protein and is involved in a diverse range of cellular functions including cytokinesis and cellular movement and has been postulated to be involved in secretion
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