Enzymatic methylation of endogenous proteins in clonal pancreatic P-cell, HIT-T15, was investigated. When cell extract incubated with S-adenosyl-L-[methyl-H-3]methionine was subjected to SDS-PAGE followed by fluorography, endogenous 20-kDa protein was highly [methyl-H-3]-labeled. The increase of methylation was correlated with insulin secretion, when the cells were treated with secretagogue; at 5.5 mM 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 0.1 mM, the methylation increased by approximately 4.5-fold. This increase of 20-kDa methylation was inhibited when the cells were treated with 3 mM EGTA to inhibit insulin secretion by depleting extracellular calcium ion, indicating intercausal relation between methylation and insulin secretion. The [methyl-H-3]-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 posttranlationally methylated, a synthetic nonapeptide, GGRGRGRGG, competed with the 20-kDa methylation; at 1 and 10 muM 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. (C) 2003 Elsevier Science (USA). All rights reserved.