Belyanskaya L L, Gehrig P M, Gehring H
Biochemisches Institut, Universität Zürich; Winterthurerstrasse 190, 8057 Zürich, Switzerland.
J Biol Chem. 2001 Jun 1;276(22):18681-7. doi: 10.1074/jbc.M011446200. Epub 2001 Mar 9.
In contrast to the knowledge regarding the function of chimeric Ewing sarcoma (EWS) fusion proteins that arise from chromosomal translocation, the cellular function of the RNA binding EWS protein is poorly characterized. EWS protein had been found mainly in the nucleus. In this report we show that EWS protein is not only found in the nucleus and cytosol but also on cell surfaces. After cell-surface biotinylation, isoelectric focusing of membrane fraction, avidin-agarose extraction of biotinylated proteins, and SDS-polyacrylamide gel electrophoresis, EWS protein was identified by matrix-assisted laser desorption ionization and nanoelectrospray tandem mass spectrometry of in-gel-digested peptides. These analyses revealed that the protein, having repeated RGG motifs, is extensively asymmetrically dimethylated on arginine residues, the sites of which have been mapped by mass spectrometric methods. Out of a total of 30 Arg-Gly sequences, 29 arginines were found to be at least partially methylated. The Arg-Gly-Gly sequence was present in 21 of the 29 methylation sites, and in contrast to other methylated proteins, only 11 (38%) methylated arginine residues were found in the Gly-Arg-Gly sequence. The presence of Gly on the C-terminal side of the arginine residue seems to be a prerequisite for recognition by a protein-arginine N-methyltransferase (PRMT) catalyzing this asymmetric dimethylation reaction. One monomethylarginine and no symmetrically methylated arginine residue was found. The present findings imply that RNA-binding EWS protein shuttles from the nucleus to the cell surface in a methylated form, the role of which is discussed.
与源于染色体易位的嵌合性尤因肉瘤（EWS）融合蛋白功能的相关知识相比，RNA结合EWS蛋白的细胞功能却鲜为人知。EWS蛋白主要存在于细胞核中。在本报告中，我们发现EWS蛋白不仅存在于细胞核和细胞质中，还存在于细胞表面。通过细胞表面生物素化、膜组分的等电聚焦、生物素化蛋白的抗生物素蛋白-琼脂糖提取以及SDS-聚丙烯酰胺凝胶电泳后，通过对胶内消化肽段进行基质辅助激光解吸电离和纳升电喷雾串联质谱鉴定出EWS蛋白。这些分析表明，该蛋白具有重复的RGG基序，在精氨酸残基上广泛存在不对称二甲基化，其位点已通过质谱方法确定。在总共30个Arg-Gly序列中，发现29个精氨酸至少部分被甲基化。29个甲基化位点中有21个存在Arg-Gly-Gly序列，与其他甲基化蛋白不同的是，在Gly-Arg-Gly序列中仅发现11个（38%）甲基化精氨酸残基。精氨酸残基C端侧存在甘氨酸似乎是催化这种不对称二甲基化反应的蛋白精氨酸N-甲基转移酶（PRMT）识别的先决条件。未发现单甲基精氨酸，也未发现对称甲基化的精氨酸残基。目前的研究结果表明，RNA结合EWS蛋白以甲基化形式从细胞核穿梭至细胞表面，并对其作用进行了讨论。
