Abstract
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.
Highlights
Terminal transcriptional activation domain of Ewing sarcoma (EWS) is fused to C-terminal DNA binding domains of corresponding partners
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
In the present investigation we show the EWS protein is localized in the nucleus and cytosol and on the surface of cells and that it is posttranslationally methylated at arginine residues
Summary
CyP, cyclophilin; EWS, Ewing sarcoma; PRMT, protein-arginine N-methyltransferase; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PAGE, polyacrylamide gel electrophoresis; PBM, peripheral blood mononuclear cells; ETS, erythroblastosis virus-transforming sequence; hnRNP, heterogeneous nuclear ribonuclear protein. Terminal transcriptional activation domain of EWS is fused to C-terminal DNA binding domains of corresponding partners. The translocation produces chimeric EWS proteins with transforming potential [2,3,4,5,6,7]. The EWS gene of Ewing sarcoma and primitive neuroectodermal tumor is translocated to one of different members of the ETS (erythroblastosis virus-transforming sequence) family that contains the highly conserved DNA binding ETS domain. The EWS protein is a nuclear protein with unknown function containing a C-terminal RNA binding motif and a N-terminal activation domain (9 –11). The IQ domain of the EWS protein is involved in calmodulin binding and protein kinase C phosphorylation [12]. EWS protein interacts with an SH3 domain of Bruton’s tyrosine kinase and has been identified in B cells as a phosphotyrosine-containing protein [13]. The identified -NG,NG-dimethylarginine residues of EWS protein let us modify a previously reported consensus sequence for asymmetric dimethylarginine formation in proteins
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