Exposure on cell surface and extensive arginine methylation of ewing sarcoma (EWS) protein.
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.
Biochemisches Institut, Universität Zürich; Winterthurerstrasse 190, 8057 Zürich, Switzerland.
L L
LL
Belyanskaya
P M
PM
Gehrig
H
H
Gehring
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism*; D001360:Avidin / Q000378:metabolism; D019921:Biotinylation; D015153:Blotting, Western; D002462:Cell Membrane / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D021983:Cyclophilins / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005998:Glycine / Q000737:chemistry; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D007525:Isoelectric Focusing; D019169:Jurkat Cells; D008745:Methylation; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D011484:Protein-Arginine N-Methyltransferases / Q000378:metabolism; D012313:RNA / Q000378:metabolism; D034802:RNA-Binding Protein EWS; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012261:Ribonucleoproteins / Q000378:metabolism*; D012685:Sepharose / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D034441:Heterogeneous-Nuclear Ribonucleoproteins; D034802:RNA-Binding Protein EWS; D011993:Recombinant Fusion Proteins; D012261:Ribonucleoproteins; D001360:Avidin; D012313:RNA; D012685:Sepharose; D001120:Arginine; D011484:Protein-Arginine N-Methyltransferases; D021983:Cyclophilins; D005998:Glycine