PROTEIN N-ARGININE METHYLATION IN ADENOSINE DIALDEHYDE-TREATED LYMPHOBLASTOID-CELLS

Protein arginine methyltransferase was recently identified to be assoc iated with some proteins in signal transduction pathways. N-Arginine m ethylation in RNA binding proteins with arginine-and glycine-rich RGG motifs is known to be the major protein methylation in cells. Consider ing that arginine methylation might be involved in certain human disor ders, we used human lymphoblastoid cells that can be easily prepared f rom lymphocytes as a model system to study the methylation. Lymphoblas toid cells grown in the presence of 20 mu M indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) for 72 h appeared to accumulate high levels of hypomethylated proteins for the endogenous protein met hyltransferase or recombinant glutathion S-transferase-fused yeast arg inine methyltransferase (RMT1). Analysis of methyl-accepting polypepti des in AdOx-treated lymphoblastoid cells by SDS-PAGE and fluorography showed that many polypeptides between 29,000 and 90,000 Pa were methyl ated by the endogenous methyltransferase. A few polypeptides could be methylated to a higher extent upon the addition of yeast GST-RMT1 fusi on protein. A peptide (GGRGRGGGF) could compete for the majority of th e methyl-accepting protein substrates in the AdOx-treated lymphoblasto id cell extracts, whether or not exogenous yeast RMT1 was included in the reaction, When the arginine residues in the peptide were replaced by lysine, no competition was observed. The results indicated that the protein methyl accepters in lymphoblastoid cells share similar RGG mo tifs and that arginine residues should be the site of methylation. (C) 1998 Academic Press.