Li C, Ai L S, Lin C H, Hsieh M, Li Y C, Li S Y
Institute of Medicine, Chung Shan Medical and Dental College, Taichung, Taiwan, Republic of China.
Arch Biochem Biophys. 1998 Mar 1;351(1):53-9. doi: 10.1006/abbi.1997.0526.
Protein arginine methyltransferase was recently identified to be associated with some proteins in signal transduction pathways. N-Arginine methylation in RNA binding proteins with arginine- and glycine-rich RGG motifs is known to be the major protein methylation in cells. Considering that arginine methylation might be involved in certain human disorders, we used human lymphoblastoid cells that can be easily prepared from lymphocytes as a model system to study the methylation. Lymphoblastoid cells grown in the presence of 20 microM indirect methyltransferase inhibitor adenosine dialdehyde (AdOx) for 72 h appeared to accumulate high levels of hypomethylated proteins for the endogenous protein methyltransferase or recombinant glutathion S-transferase-fused yeast arginine methyltransferase (RMT1). Analysis of methyl-accepting polypeptides in AdOx-treated lymphoblastoid cells by SDS-PAGE and fluorography showed that many polypeptides between 29,000 and 90,000 Da were methylated by the endogenous methyltransferase. A few polypeptides could be methylated to a higher extent upon the addition of yeast GST-RMT1 fusion protein. A peptide (GGRGRGGGF) could compete for the majority of the methyl-accepting protein substrates in the AdOx-treated lymphoblastoid 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 acceptors in lymphoblastoid cells share similar RGG motifs and that arginine residues should be the site of methylation.
蛋白精氨酸甲基转移酶最近被确定与信号转导途径中的一些蛋白质相关。已知在具有富含精氨酸和甘氨酸的RGG基序的RNA结合蛋白中，N-精氨酸甲基化是细胞中的主要蛋白质甲基化形式。考虑到精氨酸甲基化可能与某些人类疾病有关，我们使用可从淋巴细胞轻松制备的人淋巴母细胞作为模型系统来研究甲基化。在20微摩尔间接甲基转移酶抑制剂腺苷二醛（AdOx）存在下培养72小时的淋巴母细胞，似乎积累了高水平的内源性蛋白质甲基转移酶或重组谷胱甘肽S-转移酶融合酵母精氨酸甲基转移酶（RMT1）的低甲基化蛋白质。通过SDS-PAGE和荧光自显影分析AdOx处理的淋巴母细胞中的甲基接受多肽，结果表明，内源性甲基转移酶可使许多分子量在29,000至90,000道尔顿之间的多肽发生甲基化。添加酵母GST-RMT1融合蛋白后，少数多肽的甲基化程度可能更高。无论反应中是否包含外源性酵母RMT1，一种肽（GGRGRGGGF）都能竞争AdOx处理的淋巴母细胞提取物中大多数甲基接受蛋白底物。当该肽中的精氨酸残基被赖氨酸取代时，未观察到竞争现象。结果表明，淋巴母细胞中的蛋白质甲基受体具有相似的RGG基序，且精氨酸残基应为甲基化位点。
