Exposure on Cell Surface and Extensive Arginine Methylation of Ewing Sarcoma (EWS) Protein

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 matrixassisted 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.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 (1). The Nterminal 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 (2-7). 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. 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 intraabdominal desmoplasmic small round-cell tumor to WT-1, in myxoid liposarcoma to CHOP, and in myxoid chrondrosarcoma to CHN (8).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 (9 -11). The IQ d...

Oncogenic Serine-Threonine Kinase Receptor-Associated Protein Modulates the Function of Ewing Sarcoma Protein through a Novel Mechanism

“…S1D). This dimethylation on arginine residue (amino acid 615) in RGG motifs of EWS protein is consistent with previous report (24). Taken together, EWS protein was unambiguously identified as one of the STRAP binding proteins analyzed by MALDI-TOF MS.…”

“…Previous report suggests that EWS protein is exclusively in the nucleus (11). In contrast, other reports suggest both nuclear and cytoplasmic localization of EWS (24,25). To determine the subcellular localization of STRAP and EWS, and to examine whether or not they are colocalized, we did immunofluorescence analyses.…”

Fragmentation pathways of NG-methylated and unmodified arginine residues in peptides studied by ESI-MS/MS and MALDI-MS

“…For this reason, peptides containing modified arginines frequently give rise to relatively strong ion signals in MALDI spectra acquired from peptide mixtures, and the metastable fragment ion signals, which are less intense than the signals of intact DMA-containing peptides, are often well discernible even in spectra recorded from complex peptide mixtures. For example, human RNA-binding Ewing Sarcoma protein was shown, by using mass spectrometric peptide mass mapping and sequencing, to contain at least 29 dimethylated arginine residues [4]. We recorded MALDI mass spectra from trypsin and chymotrypsin digests of this protein and identified metastable peaks resulting from loss of dimethylamine, which indicated the presence of aDMA residues at 22 of these sites (unpublished data).…”

“…In general, trypsin is the enzyme of choice for generating peptide maps of known proteins, as well as for proteomics applications. In trypsin digests of proteins containing either aDMA and MMA [4] or sDMA and MMA [5], no peptides resulting from cleavages after modified arginines were identified. Synthetic peptides containing up to eight aDMA residues were 4 to 20 times more resistant to trypsin proteolysis than their non-methylated counterparts [10].…”

“…Type I substrates include many RNA binding and transporting proteins, transcription factors, nuclear matrix proteins and cytokines. Arginine methylation by Type I enzymes usually occurs in conserved glycine-and arginine-rich (GAR) sequences, typically in RG or RGG sequences [2,4]. Myelin basic protein and four spliceosomal proteins are the only substrates for Type II arginine methyltransferases reported in the literature [5,6].…”