Threonine phosphorylations induced by RX-871024 and insulin secretagogues in betaTC6-F7 cells.
Treatment of the pancreatic beta-cell line betaTC6-F7 with an imidazoline compound, RX-871024, 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 ([Ca(2+)](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.
An
J
J
Endocrine Research, Lilly Research Laboratories, Indianapolis, Indiana 46285, USA.
Zhao
G
G
Churgay
L M
LM
Osborne
J J
JJ
Hale
J E
JE
Becker
G W
GW
Gold
G
G
Stramm
L E
LE
Shi
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D000959:Antihypertensive Agents / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002121:Calcium Channel Blockers / Q000494:pharmacology; D015220:Calcium Channels / Q000502:physiology; D002460:Cell Line; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003600:Cytosol / Q000737:chemistry / Q000201:enzymology; D003981:Diazoxide / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D007536:Isomerism; D008564:Membrane Potentials / Q000187:drug effects; D008565:Membrane Proteins / Q000032:analysis / Q000378:metabolism; D018995:Myosin Heavy Chains / Q000032:analysis / Q000737:chemistry; D009543:Nifedipine / Q000494:pharmacology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry; D013912:Threonine / Q000378:metabolism*; D014044:Tolbutamide / Q000494:pharmacology*
C101943:2-(2-imidazolin-2-yl)-1-phenyl-1H-indole; D000959:Antihypertensive Agents; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D007004:Hypoglycemic Agents; D007093:Imidazoles; D007211:Indoles; D007328:Insulin; D008565:Membrane Proteins; D013912:Threonine; D011189:Potassium Chloride; D014044:Tolbutamide; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D018995:Myosin Heavy Chains; D009543:Nifedipine; D003981:Diazoxide; D002118:Calcium