TY - JOUR
T1 - Threonine phosphorylations induced by RX-871024 and insulin secretagogues in βTC6-F7 cells
AU - An, Jie
AU - Zhao, Genshi
AU - Churgay, Lisa M.
AU - Osborne, John J.
AU - Hale, John E.
AU - Becker, Gerald W.
AU - Gold, Gerald
AU - Stramm, Lawrence E.
AU - Shi, Yuguang
PY - 1999/11
Y1 - 1999/11
N2 - Treatment of the pancreatic β-cell line βTC6-F7 with an imidazoline compound, RX871024, KCl, or tolbutamide resulted in increased threonine phosphorylation of a 220-kDa protein (p220) concurrent with enhanced insulin secretion, which can be partially antagonized by diazoxide, an ATP-sensitive potassium (K(ATP)) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free calcium concentration ([Ca2+](i)), membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated by protein kinase A, protein kinase C, or insulin exocytosis. Analysis of subcellular fractions indicated that p220 is a hydrophilic protein localized exclusively in the cytosol. Subsequently, p220 was purified to homogeneity, sequenced, and identified as nonmuscle myosin heavy chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle MHC-A by KCl treatment also resulted in increased phosphorylation of a 40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A. Our results suggest that both nonmuscle MHC-A and the 40-kDa protein may play roles in regulating signal transduction, leading to insulin secretion.
AB - Treatment of the pancreatic β-cell line βTC6-F7 with an imidazoline compound, RX871024, KCl, or tolbutamide resulted in increased threonine phosphorylation of a 220-kDa protein (p220) concurrent with enhanced insulin secretion, which can be partially antagonized by diazoxide, an ATP-sensitive potassium (K(ATP)) channel activator. Although phosphorylation of p220 was regulated by cytoplasmic free calcium concentration ([Ca2+](i)), membrane depolarization alone was not sufficient to induce phosphorylation. Phosphorylation of p220 also was not directly mediated by protein kinase A, protein kinase C, or insulin exocytosis. Analysis of subcellular fractions indicated that p220 is a hydrophilic protein localized exclusively in the cytosol. Subsequently, p220 was purified to homogeneity, sequenced, and identified as nonmuscle myosin heavy chain-A (MHC-A). Stimulation of threonine phosphorylation of nonmuscle MHC-A by KCl treatment also resulted in increased phosphorylation of a 40-kDa protein, which was coimmunoprecipitated by antibody to MHC-A. Our results suggest that both nonmuscle MHC-A and the 40-kDa protein may play roles in regulating signal transduction, leading to insulin secretion.
KW - Free calcium concentration
KW - Imidazoline
KW - Nonmuscle myosin
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U2 - 10.1152/ajpendo.1999.277.5.e862
DO - 10.1152/ajpendo.1999.277.5.e862
M3 - Article
C2 - 10567013
AN - SCOPUS:33747251565
SN - 0193-1849
VL - 277
SP - E862-E869
JO - American Journal of Physiology - Endocrinology and Metabolism
JF - American Journal of Physiology - Endocrinology and Metabolism
IS - 5 40-5
ER -