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• W2023679862 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. 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. cyclophilin Ewing sarcoma protein-arginineN-methyltransferase phosphate-buffered saline 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid matrix-assisted laser desorption ionization mass spectrometry tandem mass spectrometry polyacrylamide gel electrophoresis peripheral blood mononuclear cells erythroblastosis virus-transforming sequence heterogeneous nuclear ribonuclear protein While investigating a 90-kDa anti-cyclophilin (anti-CyP)1 immunoreactive band we noticed that anti-CyP antibodies recognized the RNA-binding Ewing sarcoma (EWS) protein and not a cyclophilin. The EWS gene is involved in tumor-related chromosomal translocations that associate part of EWS gene with various genes encoding transcription factors (1Delattre O. Zucman J. Melot T. Garau X.S. Zucker J.M. Lenoir G.M. Ambros P.F. Sheer D. Turc-Carel C. Triche T.J. et al.N. Engl. J. Med. 1994; 331: 294-299Crossref PubMed Scopus (906) Google Scholar). The N-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 (2Brown A.D. Lopez-Terrada D. Denny C. Lee K.A. Oncogene. 1995; 10: 1749-1756PubMed Google Scholar, 3Fujimura Y. Ohno T. Siddique H. Lee L. Rao V.N. Reddy E.S. Oncogene. 1996; 12: 159-167PubMed Google Scholar, 4May W.A. Lessnick S.L. Braun B.S. Klemsz M. Lewis B.C. Lunsford L.B. Hromas R. Denny C.T. Mol. Cell. Biol. 1993; 13: 7393-7398Crossref PubMed Scopus (442) Google Scholar, 5Ohno T. Rao V.N. Reddy E.S. Cancer Res. 1993; 53: 5859-5863PubMed Google Scholar, 6Prasad D.D. Ouchida M. Lee L. Rao V.N Reddy E.S. Oncogene. 1994; 9: 3717-3729PubMed Google Scholar, 7Zucman J. Melot T. Desmaze C. Ghysdael J. Plougastel B. Peter M. Zucker J.M. Triche T.J. Sheer D. Turc-Carel C. EMBO J. 1993; 12: 4481-4487Crossref PubMed Scopus (495) Google Scholar). 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. Often the ETS domain is derived from FLI-1 (Friend leukemia integration-1) and in rare cases from ERG (ETS-related gene), ETV-1 (ETS translocation variant-1), E1AF (E1A factor), or FEV (fifth Ewing variant). In malignant melanoma of soft parts, EWS is fused to ATF-1, in intra-abdominal desmoplasmic small round-cell tumor to WT-1, in myxoid liposarcoma to CHOP, and in myxoid chrondrosarcoma to CHN (8de Alava E. Gerald W.L. J. Clin. Oncol. 2000; 18: 204-213Crossref PubMed Google Scholar).The cellular role of wild-type EWS protein remains less clear. The EWS protein is a nuclear protein with unknown function containing a C-terminal RNA binding motif and a N-terminal activation domain (9Lessnick S.L. Braun B.S. Denny C.T. May W.A. Oncogene. 1995; 10: 423-431PubMed Google Scholar, 10Ohno T. Ouchida M. Lee L. Gatalica Z. Rao V.N. Reddy E.S. Oncogene. 1994; 9: 3087-3097PubMed Google Scholar, 11Ouchida M. Ohno T. Fujimura Y. Rao V.N. Reddy E.S. Oncogene. 1995; 11: 1049-1054PubMed Google Scholar). The IQ domain of the EWS protein is involved in calmodulin binding and protein kinase C phosphorylation (12Deloulme J.C. Prichard L. Delattre O. Storm D.R. J. Biol. Chem. 1997; 272: 27369-27377Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). EWS protein interacts with an SH3 domain of Bruton's tyrosine kinase and has been identified in B cells as a phosphotyrosine-containing protein (13Guinamard R. Fougereau M. Seckinger P. Scand. J. Immunol. 1997; 45: 587-595Crossref PubMed Scopus (61) Google Scholar). G-coupled receptor signaling and other stimuli of tyrosine kinase Pyk2 block the interaction between EWS protein and Pyk2. Partitioning of the EWS protein into a ribosome-associated fraction indicated that the role for EWS in gene expression includes an extranuclear action (14Felsch J.S. Lane W.S. Peralta E.G. Curr. Biol. 1999; 9: 485-488Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).In the present investigation we show the EWS protein is not only localized in the nucleus and cytosol but also on the surface of cells and that it is posttranslationally methylated at arginine residues. The identified ω-N G,N G-dimethylarginine residues of EWS protein let us modify a previously reported consensus sequence for asymmetric dimethylarginine formation in proteins.DISCUSSIONOur data show that the anti-CyP immunoreactive protein located on the surface of T cells is not a cyclophilin but the EWS protein. The anti-CyP antibodies cross-react with the EWS protein as demonstrated by immunoprecipitation experiments. The cause of the cross-reactivity is not obvious. Global alignment of the EWS protein sequence either with human CyPA or CyPB using the program LALIGN revealed a low degree of identity (8.8%) in both cases, and some of the identity seems to be due to numerous glycines present in the proteins. The cross-reactivity led us, however, to the finding that the EWS protein is not only exposed on the cell surface of different cells but also that its arginine residues are extensively and asymmetrically dimethylated. These properties of the EWS protein shed a new light on the functionality of this unusual multidomain protein.The previously reported localization of EWS protein in the nucleus (20Bertolotti A. Melot T. Acker J. Vigneron M. Delattre O. Tora L. Mol. Cell. Biol. 1998; 18: 1489-1497Crossref PubMed Scopus (216) Google Scholar) and the cytosol (14Felsch J.S. Lane W.S. Peralta E.G. Curr. Biol. 1999; 9: 485-488Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), both of which we confirmed (data not shown), together with the present finding of the EWS protein to be accessible on the cell surface means that this protein shuttles between the nucleus, cytosol, and the cell surface. A similar behavior was reported for nucleolin. This major nucleolar protein shuttles between the cytosol and the nucleus and has also been detected on the cell surface of different cells. The C-terminal domain of nucleolin is, as in the EWS protein, rich in glycine residues and interspersed with dimethylarginines. It was suggested as a potential receptor in the human immunodeficiency virus binding processes by interaction with the V3 loop of gp120 (21Srivastava M. Pollard H.B. FASEB J. 1999; 13: 1911-1922Crossref PubMed Scopus (425) Google Scholar). So far we found cell-surface-exposed EWS protein in all investigated cells, i.e. Jurkat, H9, C816645 T cell lines, and PBM cells, but also NIH/3T3 fibroblasts (not shown). Remarkably, tumor cell lines showed a higher level of EWS protein expression on the cell surface (∼4-fold) than PBM cells and fibroblasts.Recognition Sequence of Asymmetric Methylation SitesArginine methylation is a post-translational modification found mainly in nuclear proteins that interact with RNA (22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). This modification is catalyzed by protein-arginine N-methyltransferases (PRMTs), utilizing S-adenosyl-l-methionine as the donor of methyl groups (23Lee H.W. Kim S. Paik W.K. Biochemistry. 1977; 16: 78-85Crossref PubMed Scopus (72) Google Scholar). Type I protein-arginineN-methyltransferases (EC 2.1.1.23) catalyze the formation of N G-monomethylarginine and asymmetric ω-N G,N G-dimethylarginine residues, whereas Type II enzymes catalyze the formation ofN G-monomethylarginine and symmetric ω-N G,N′G-dimethylarginine residues (22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). The Type III enzyme found in yeast catalyzes the monomethylation of the internal δ-guanidino nitrogen atom of arginine residues (24Zobel-Thropp P. Gary J.D. Clarke S. J. Biol. Chem. 1998; 273: 29283-29286Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Three Type I PRMTs from mammalian cells PRMT1 (25Lin W.J. Gary J.D. Yang M.C. Clarke S. Herschman H.R. J. Biol. Chem. 1996; 271: 15034-15044Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar), PRMT3 (26Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar), and coactivator-associated arginine methyltransferase 1 (CARM1) (27Chen D. Ma H. Hong H. Koh S.S. Huang S.M. Schurter B.T. Aswad D.W. Stallcup M.R. Science. 1999; 284: 2174-2177Crossref PubMed Scopus (991) Google Scholar) have been reported. PRMT1, a predominantly nuclear protein, methylates arginine residues of many proteins, among them RNA-binding proteins (22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar, 28Smith J.J. Rucknagel K.P. Schierhorn A. Tang J. Nemeth A. Linder M. Herschman H.R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). PRMT3 is a predominantly cytoplasmic protein whose activity overlaps with that of PRMT1 (26Tang J. Gary J.D. Clarke S. Herschman H.R. J. Biol. Chem. 1998; 273: 16935-16945Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). A number ofin vivo substrates for Type I PRMTs have been identified, including the heterogeneous nuclear ribonucleoprotein A1 (29Kim S. Merrill B.M. Rajpurohit R. Kumar A. Stone K.L. Papov V.V. Schneiders J.M. Szer W. Wilson S.H. Paik W.K. Williams K.R. Biochemistry. 1997; 36: 5185-5192Crossref PubMed Scopus (107) Google Scholar), fibrillarin, nucleolin (22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar), high molecular weight fibroblast growth factor-2 (30Klein S. Carroll J.A. Chen Y. Henry M.F. Henry P.A. Ortonowski I.E. Pintucci G. Beavis R.C. Burgess W.H. Rifkin D.B. J. Biol. Chem. 2000; 275: 3150-3157Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar), and poly(A)-binding protein II (28Smith J.J. Rucknagel K.P. Schierhorn A. Tang J. Nemeth A. Linder M. Herschman H.R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).The sites of methylated or dimethylated arginine residues had been directly determined by Edman sequencing or by a combination of Edman sequencing and mass spectrometric approaches. In this study, the arginine dimethylation sites of the EWS protein from the cell surface of Jurkat and H9 cell lines were elucidated by mass spectrometric peptide mapping and sequencing. We found up to the present the EWS protein to be the most extensively methylated protein in vivo in higher eukaryotes. From sequences of known sites of asymmetric arginine methylation, a preferred recognition motif (G/F)GGRGG(G/F) had been proposed (Refs. 22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar and 29Kim S. Merrill B.M. Rajpurohit R. Kumar A. Stone K.L. Papov V.V. Schneiders J.M. Szer W. Wilson S.H. Paik W.K. Williams K.R. Biochemistry. 1997; 36: 5185-5192Crossref PubMed Scopus (107) Google Scholar; TableII). The shorter sequence GRG had also been suggested as a specificity determinant for arginine methylation by a Type I enzyme, although the Gly at position −1 could be replaced by a few amino acids other than glycine (31Hyun Y.L. Lew D.B. Park S.H. Kim C.W. Paik W.K. Kim S. Biochem. J. 2000; 348: 573-578Crossref PubMed Google Scholar). Only the Gly +1 C-terminal of the arginine was found to be completely conserved. In the present study, all 29 identified dimethylarginines are indeed followed by a glycine (Table II). Conversely, all arginines in Arg-Gly sequences mapped by mass spectrometry (29 out of a total of 30 Arg-Gly sequences) were at least partially methylated. The sequence Arg-Gly-Gly is present in 21 of the 29 methylation sites; however, only 11 (38%) methylated arginine residues were found in a Gly-Arg-Gly sequence. The prevalence of Gly at positions −2 and −3 in EWS protein is even higher than at position −1. Obvious deviations from the suggested consensus sequence are the small number of phenylalanines at position −3 and +3 and of glycines at position +3. Thus, the suggested recognition sequence for asymmetric arginine dimethylation is only partially consistent with the sequences identified in this study. Only the presence of the Gly +1 C-terminal of the arginine residue is a strict requirement for asymmetric dimethylation but not the Gly N-terminal of the arginine. The found differences may be attributed to an insufficiently defined consensus sequence based on only 20 in vivo arginine methylation sites or to different Type I PRMTs with similar specificity that are involved in methylation of the arginine residues in EWS protein. Arginine methylation sites very distinct from those found in EWS protein and the other sites listed in Table II were observed in poly(A)-binding protein II. In this protein, almost all of the asymmetrically dimethylated and monomethylated arginines were found in Arg-Xaa-Arg cluster (28Smith J.J. Rucknagel K.P. Schierhorn A. Tang J. Nemeth A. Linder M. Herschman H.R. Wahle E. J. Biol. Chem. 1999; 274: 13229-13234Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).Table IIComparison of the mapped sites of asymmetric dimethylation of EWS protein and other recently reported proteins with the proposed consensus sequence (22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar, 29Kim S. Merrill B.M. Rajpurohit R. Kumar A. Stone K.L. Papov V.V. Schneiders J.M. Szer W. Wilson S.H. Paik W.K. Williams K.R. Biochemistry. 1997; 36: 5185-5192Crossref PubMed Scopus (107) Google Scholar)Gly/Phe −3Gly −2Gly −1Arg 0Gly 1Gly 2Gly/Phe 3%EWS protein (29 sites)45 /32-aAsp, 3; Leu, 3; Met, 3; Asn, 7; Pro, 7; Arg, 28;522-bAsp, 3; Glu, 3; Phe, 3; Met, 14; Pro, 7; Arg, 10; Ser, 3;382-cAsp, 21; Phe, 7; Leu, 3; Met, 7; Asn, 3; Pro, 7; Arg, 7; Ser, 7;100100722-dMet, 7; Asn, 3; Pro, 3; Arg, 10; Ser, 3;14 /142-eAsp, 10; Leu, 7; Met, 7; Pro, 24; Arg, 24.Review (20 sites) (from Ref.22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar)35 /3575801001007535 /30hnRNP A1 (3 sites) (from Ref. 29Kim S. Merrill B.M. Rajpurohit R. Kumar A. Stone K.L. Papov V.V. Schneiders J.M. Szer W. Wilson S.H. Paik W.K. Williams K.R. Biochemistry. 1997; 36: 5185-5192Crossref PubMed Scopus (107) Google Scholar)33 /333310010010010033 /33High molecular weight fibroblast growth factor-2 (8 sites) (from Ref.30Klein S. Carroll J.A. Chen Y. Henry M.F. Henry P.A. Ortonowski I.E. Pintucci G. Beavis R.C. Burgess W.H. Rifkin D.B. J. Biol. Chem. 2000; 275: 3150-3157Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar)50 /0388810088050 /0Total (60 sites)42 /155761100986527 /18a–e Other amino acid residues found in these positions (%):2-a Asp, 3; Leu, 3; Met, 3; Asn, 7; Pro, 7; Arg, 28;2-b Asp, 3; Glu, 3; Phe, 3; Met, 14; Pro, 7; Arg, 10; Ser, 3;2-c Asp, 21; Phe, 7; Leu, 3; Met, 7; Asn, 3; Pro, 7; Arg, 7; Ser, 7;2-d Met, 7; Asn, 3; Pro, 3; Arg, 10; Ser, 3;2-e Asp, 10; Leu, 7; Met, 7; Pro, 24; Arg, 24. Open table in a new tab Role of Methylation of EWS ProteinEWS protein belongs to a large family of RNA-binding proteins that includes heterogeneous nuclear ribonucleoproteins, mRNA poly(A)-binding proteins (32Weighardt F. Biamonti G. Riva S. Bioessays. 1996; 18: 747-756Crossref PubMed Scopus (180) Google Scholar), and alternative splicing factors (33Krainer A.R. Mayeda A. Kozak D. Binns G. Cell. 1991; 66: 383-394Abstract Full Text PDF PubMed Scopus (411) Google Scholar, 34Zamore P.D. Patton J.G. Green M.R. Nature. 1992; 355: 609-714Crossref PubMed Scopus (457) Google Scholar). The methylation state of RNA-binding proteins (Table II) is likely to have an important effect on their function. One type of methylated RNA binding domain consists of RGG boxes, defined as closely repeated RGG tripeptides interrupted by other amino acids. The RNA binding of EWS protein is localized in the C-terminal domain, which contains RGG boxes (Ref. 10Ohno T. Ouchida M. Lee L. Gatalica Z. Rao V.N. Reddy E.S. Oncogene. 1994; 9: 3087-3097PubMed Google Scholar; Fig. 2, residues 288–656). Considering the RNA binding properties of these proteins, arginine methylation in the RNA binding domain may regulate the RNA-EWS protein interaction most likely by preventing hydrogen bonding, by short range ionic interactions, and/or by introducing steric constraints and therefore diminishes otherwise strong interactions between unmodified arginine residues and RNA (35Calnan B.J. Tidor B. Biancalana S. Hudson D. Frankel A.D. Science. 1991; 252: 1167-1171Crossref PubMed Scopus (562) Google Scholar). The methylated form of one of the well studied shuttling heterogeneous nuclear ribonucleoproteins, A1, showed decreased affinity for single-stranded DNA compared with the unmethylated form (36Rajpurohit R. Paik W.K Kim S. Biochem. J. 1994; 304: 903-909Crossref PubMed Scopus (60) Google Scholar).The changes in the physico-chemical properties, e.g.increased hydrophobicity, loss of hydrogen bonding, and ionic interactions resulting from arginine methylation of nuclear as well as of cell-surface-localized EWS protein, might favor crossing the various cellular membranes or its embedding in the plasma membrane. Arginine methylation was found to facilitate export of certain heterogeneous nuclear ribonucleoproteins out of the nucleus, especially of proteins that bind mRNA (37Shen E.C. Henry M.F. Weiss V.H. Valentini S.R. Silver P.A. Lee M.S. Genes Dev. 1998; 12: 679-691Crossref PubMed Scopus (250) Google Scholar). These observations suggest that methylation of EWS protein may play a role in its nucleocytoplasmic shuttling and that it acts as a carrier for export of RNA constituents to the cytosol due to its RNA binding properties.Asymmetric methylation might also play a role in signal transduction. Stimulation of pre-B cells with lipopolysaccharide resulted in increased methylation of membrane proteins (22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). EWS protein, which is as the present results show extensively methylated on arginine residues, was found to interact with cellular kinases, suggesting its participation in cell signaling and proliferation. Under conditions leading to Bruton's tyrosine kinase activation, EWS protein was phosphorylated on tyrosines in mitotically arrested B cells (13Guinamard R. Fougereau M. Seckinger P. Scand. J. Immunol. 1997; 45: 587-595Crossref PubMed Scopus (61) Google Scholar) and interacted with Pyk2 in an activation-dependent manner (14Felsch J.S. Lane W.S. Peralta E.G. Curr. Biol. 1999; 9: 485-488Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar).The presence of asymmetrically arginine-dimethylated EWS protein as well as of nucleolin on the cell surface raises the question as to the significance of this localization. The present findings,i.e. the shuttling properties, together with the discussed properties of the EWS protein allow one to speculate that EWS protein or putative splicing products thereof can function as binding proteins or receptors for various ligands on the cell surface, e.g.nucleic acids, be it alone or in a complex with other proteins and, thus, might mediate, in an alternative regulation mechanism, between extracellular and nuclear events. While investigating a 90-kDa anti-cyclophilin (anti-CyP)1 immunoreactive band we noticed that anti-CyP antibodies recognized the RNA-binding Ewing sarcoma (EWS) protein and not a cyclophilin. The EWS gene is involved in tumor-related chromosomal translocations that associate part of EWS gene with various genes encoding transcription factors (1Delattre O. Zucman J. Melot T. Garau X.S. Zucker J.M. Lenoir G.M. Ambros P.F. Sheer D. Turc-Carel C. Triche T.J. et al.N. Engl. J. Med. 1994; 331: 294-299Crossref PubMed Scopus (906) Google Scholar). The N-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 (2Brown A.D. Lopez-Terrada D. Denny C. Lee K.A. Oncogene. 1995; 10: 1749-1756PubMed Google Scholar, 3Fujimura Y. Ohno T. Siddique H. Lee L. Rao V.N. Reddy E.S. Oncogene. 1996; 12: 159-167PubMed Google Scholar, 4May W.A. Lessnick S.L. Braun B.S. Klemsz M. Lewis B.C. Lunsford L.B. Hromas R. Denny C.T. Mol. Cell. Biol. 1993; 13: 7393-7398Crossref PubMed Scopus (442) Google Scholar, 5Ohno T. Rao V.N. Reddy E.S. Cancer Res. 1993; 53: 5859-5863PubMed Google Scholar, 6Prasad D.D. Ouchida M. Lee L. Rao V.N Reddy E.S. Oncogene. 1994; 9: 3717-3729PubMed Google Scholar, 7Zucman J. Melot T. Desmaze C. Ghysdael J. Plougastel B. Peter M. Zucker J.M. Triche T.J. Sheer D. Turc-Carel C. EMBO J. 1993; 12: 4481-4487Crossref PubMed Scopus (495) Google Scholar). 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. Often the ETS domain is derived from FLI-1 (Friend leukemia integration-1) and in rare cases from ERG (ETS-related gene), ETV-1 (ETS translocation variant-1), E1AF (E1A factor), or FEV (fifth Ewing variant). In malignant melanoma of soft parts, EWS is fused to ATF-1, in intra-abdominal desmoplasmic small round-cell tumor to WT-1, in myxoid liposarcoma to CHOP, and in myxoid chrondrosarcoma to CHN (8de Alava E. Gerald W.L. J. Clin. Oncol. 2000; 18: 204-213Crossref PubMed Google Scholar). The cellular role of wild-type EWS protein remains less clear. The EWS protein is a nuclear protein with unknown function containing a C-terminal RNA binding motif and a N-terminal activation domain (9Lessnick S.L. Braun B.S. Denny C.T. May W.A. Oncogene. 1995; 10: 423-431PubMed Google Scholar, 10Ohno T. Ouchida M. Lee L. Gatalica Z. Rao V.N. Reddy E.S. Oncogene. 1994; 9: 3087-3097PubMed Google Scholar, 11Ouchida M. Ohno T. Fujimura Y. Rao V.N. Reddy E.S. Oncogene. 1995; 11: 1049-1054PubMed Google Scholar). The IQ domain of the EWS protein is involved in calmodulin binding and protein kinase C phosphorylation (12Deloulme J.C. Prichard L. Delattre O. Storm D.R. J. Biol. Chem. 1997; 272: 27369-27377Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). EWS protein interacts with an SH3 domain of Bruton's tyrosine kinase and has been identified in B cells as a phosphotyrosine-containing protein (13Guinamard R. Fougereau M. Seckinger P. Scand. J. Immunol. 1997; 45: 587-595Crossref PubMed Scopus (61) Google Scholar). G-coupled receptor signaling and other stimuli of tyrosine kinase Pyk2 block the interaction between EWS protein and Pyk2. Partitioning of the EWS protein into a ribosome-associated fraction indicated that the role for EWS in gene expression includes an extranuclear action (14Felsch J.S. Lane W.S. Peralta E.G. Curr. Biol. 1999; 9: 485-488Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). In the present investigation we show the EWS protein is not only localized in the nucleus and cytosol but also on the surface of cells and that it is posttranslationally methylated at arginine residues. The identified ω-N G,N G-dimethylarginine residues of EWS protein let us modify a previously reported consensus sequence for asymmetric dimethylarginine formation in proteins. DISCUSSIONOur data show that the anti-CyP immunoreactive protein located on the surface of T cells is not a cyclophilin but the EWS protein. The anti-CyP antibodies cross-react with the EWS protein as demonstrated by immunoprecipitation experiments. The cause of the cross-reactivity is not obvious. Global alignment of the EWS protein sequence either with human CyPA or CyPB using the program LALIGN revealed a low degree of identity (8.8%) in both cases, and some of the identity seems to be due to numerous glycines present in the proteins. The cross-reactivity led us, however, to the finding that the EWS protein is not only exposed on the cell surface of different cells but also that its arginine residues are extensively and asymmetrically dimethylated. These properties of the EWS protein shed a new light on the functionality of this unusual multidomain protein.The previously reported localization of EWS protein in the nucleus (20Bertolotti A. Melot T. Acker J. Vigneron M. Delattre O. Tora L. Mol. Cell. Biol. 1998; 18: 1489-1497Crossref PubMed Scopus (216) Google Scholar) and the cytosol (14Felsch J.S. Lane W.S. Peralta E.G. Curr. Biol. 1999; 9: 485-488Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), both of which we confirmed (data not shown), together with the present finding of the EWS protein to be accessible on the cell surface means that this protein shuttles between the nucleus, cytosol, and the cell surface. A similar behavior was reported for nucleolin. This major nucleolar protein shuttles between the cytosol and the nucleus and has also been detected on the cell surface of different cells. The C-terminal domain of nucleolin is, as in the EWS protein, rich in glycine residues and interspersed with dimethylarginines. It was suggested as a potential receptor in the human immunodeficiency virus binding processes by interaction with the V3 loop of gp120 (21Srivastava M. Pollard H.B. FASEB J. 1999; 13: 1911-1922Crossref PubMed Scopus (425) Google Scholar). So far we found cell-surface-exposed EWS protein in all investigated cells, i.e. Jurkat, H9, C816645 T cell lines, and PBM cells, but also NIH/3T3 fibroblasts (not shown). Remarkably, tumor cell lines showed a higher level of EWS protein expression on the cell surface (∼4-fold) than PBM cells and fibroblasts.Recognition Sequence of Asymmetric Methylation SitesArginine methylation is a post-translational modification found mainly in nuclear proteins that interact with RNA (22Gary J.D Clarke S. Prog. Nucleic Acid Res. Mol. Biol. 1998; 61: 65-131Crossref PubMed Google Scholar). This modification is catalyzed by protein-arginine N-methyltransferases (PRMTs), utilizing S-adenosyl-l-methionine as the donor of methyl groups (23Lee H.W. Kim S. Paik W.K. Biochemistry. 1977; 16: 78-85Crossref PubMed Scopus (72) Google Scholar). Type I protein-arginineN-methyltransferases (EC 2.1.1.23) catalyze the formation of N G-monomethylarginine and asymmetric ω-N G,N G-dimethylarginine residues, whereas Type II enzymes catalyze the formation ofN G-monomethylarginin" @default.
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• W2023679862 title "Exposure on Cell Surface and Extensive Arginine Methylation of Ewing Sarcoma (EWS) Protein" @default.
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