Identification of highly methylated arginine residues in an endogenous 20-kDa polypeptide in cancer cells

Enzymatic methylation of endogenous proteins in several cancer cell lines w as investigated to understand a possible relationship between protein-argin ine methylation and cellular proliferation. Cytosolic extracts prepared fro m several cancer cells (HeLa, HCT-48, A549, and HepG2) and incubated with S -adenosyl-L-[methyl-H-3]methionine revealed an intensely [methyl-H-3]-label ed 20-kDa polypeptide. On the other hand, cytosolic extracts prepared from normal colon cells did not show any methylation of the 20-kDa protein under identical conditions. To identify nature of the 20-kDa polypeptide, purifi ed histones were methylated with HCT-48 cytosolic extracts and analyzed by SDS-PAGE. However, none of the histones comigrated with the methylated 20-k Da polypeptide, indicating that it is unlikely to be any of the histone sub classes. The [methyl-H-3]group in the 20-kDa polypeptide was stable at pH 1 0-11 (37 degrees C for 30 min) and methylation was not stimulated by GTP ga mma S (4 mM), thus the reaction is neither carboxyl methylesterification on isoaspartyl residues, nor on C-terminal farnesylated cysteine. The present study together with the previous identification of N-G-methylated arginine residues in the HCT-48 cytosol fraction suggests that this novel endogenou s 20-kDa arginine-methylation is a cellular proliferation-related posttrans lational modification reaction.