Increased methylation of endogenous 20-kDa protein in HIT beta-cell during insulin secretion.
Enzymatic methylation of endogenous proteins in clonal pancreatic beta-cell, HIT-T15, was investigated. When cell extract incubated with S-adenosyl-L-[methyl-3H]methionine was subjected to SDS-PAGE followed by fluorography, endogenous 20-kDa protein was highly [methyl-3H]-labeled. The increase of methylation was correlated with insulin secretion, when the cells were treated with secretagogue; at 5.5mM glucose, insulin secretion increased by 2.5-fold, while the 20-kDa methylation to about 3.2-fold. In the case of forskolin, another secretagogue, at 0.1mM, the methylation increased by approximately 4.5-fold. This increase of 20-kDa methylation was inhibited when the cells were treated with 3mM EGTA to inhibit insulin secretion by depleting extracellular calcium ion, indicating intercausal relation between methylation and insulin secretion. The [methyl-3H]-labeled amino acids were identified by thin layer chromatography as N(G)-methylated arginines. While arginyl residues in Gly-Arg-Gly sequence are known to be posttranslationally methylated, a synthetic nonapeptide, GGRGRGRGG, competed with the 20-kDa methylation; at 1 and 10 micro M nonapeptides, 62% and 78% of 20-kDa methylation were inhibited, respectively. Furthermore, Western immunoblot analysis of HIT cell extract against GGRGRGRGG antibodies strongly immunoreacted with the 20-kDa protein. These results suggested that methylation of the endogenous 20-kDa protein might play some role in insulin secretion.
Graduate School of Biomedical Sciences, Korea University College of Medicine, Seoul 136-705, Republic of Korea.
Yongchul
Y
Lim
Joon-Sup
JS
Shin
Woon Ki
WK
Paik
Sangduk
S
Kim
D020816:Amino Acid Motifs; D001120:Arginine / Q000032:analysis; D002460:Cell Line; D002855:Chromatography, Thin Layer; D004533:Egtazic Acid / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000187:drug effects / Q000378:metabolism*; D008745:Methylation; D008970:Molecular Weight; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
D007328:Insulin; D011506:Proteins; D004533:Egtazic Acid; D001120:Arginine; D005947:Glucose