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INTRODUCTION |
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 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 (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
intra-abdominal 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 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).
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 (14).
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
-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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EXPERIMENTAL PROCEDURES |
Materials--
Stock cultures of Jurkat cells, a tumor T
lymphoma cell line, were kindly provided by Dr. J. Kemler-Carraneo
(Abteilung Klinische Immunologie, Universitätsspital
Zürich). Peripheral blood mononuclear (PBM) cells and human
cutaneous T lymphoma cell line (H9) were obtained from the National
Center for Retroviruses (University of Zürich). Purified
polyclonal antibodies raised against human CyPA and CyPB
(anti-CyP) were kindly provided by G. Woerly (Toxikologisches Institut der Universität Zürich, Schwerzenbach). Polyclonal antibody 677 against the N terminus of EWS protein (anti-EWS) was a
generous gift from Dr. Olivier Delattre (Institut Curie, Pathologie
Moléculaire des Cancers, Paris Cedex).
Cell Cultures--
Human Jurkat cells were grown in RPMI 1640 medium (Sigma) supplemented with 5% newborn calf serum (Life
Technologies), 15 mM HEPES, 2 mM
L-glutamine, 50 µM
-mercaptoethanol, and
1% (w/v) penicillin and streptomycin (Life Technologies) in a
humidified 5% CO2 atmosphere at 37 °C. PBM cells were
isolated by Ficoll-Hypaque gradient centrifugation. The cells were
washed with phosphate-buffered saline, pH 7.4 (PBS), and stimulated
with 2 µg/ml phytohemagglutinin in RPMI 1640 medium supplemented with
20% fetal calf serum (Life Technologies), 15 mM HEPES, 2 mM L-glutamine, 50 µM
-mercaptoethanol, and 1% (w/v) penicillin and streptomycin in a
humidified 5% CO2 atmosphere at 37 °C. Human H9 cell
line was grown in RPMI 1640 medium supplemented with 20% fetal calf
serum, 15 mM HEPES, 2 mM
L-glutamine, 50 µM
-mercaptoethanol, and
1% (w/v) penicillin and streptomycin in a humidified 5%
CO2 atmosphere at 37 °C.
Biotinylation of Cell Surface Proteins--
Cell-surface
biotinylation was performed as described (15) with some modification.
2 × 108 cells were washed three times with ice-cold
PBS, suspended in PBS (25 × 106 cells/ml), and
incubated with 0.5 mg/ml sulfosuccinimidobiotin (Calbiochem) for 1 h at 4 °C. The biotinylation procedure was stopped by replacing the
labeling solution with culture medium. After incubation for 10 min at
4 °C, cells were washed twice with PBS.
Biotinylated or non-labeled cells were solubilized (25 × 106 cells/ml) in lysis buffer containing 1% (w/v) Triton
X-100, PBS, protease inhibitor mixture (20 µg/ml pancreas extract, 5 µg/ml Pronase, 0.5 µg/ml thermolysin, 3 µg/ml chymotrypsin, 330 µg/ml papain; Roche Molecular Biochemicals), 2 mM EDTA,
0.5 mM phenylmethylsulfonyl fluoride, 0.5 mM
dithiothreitol for 40 min on ice with occasional vortexing. The cell
lysates were centrifuged at 480 × g for 5 min followed
by 15000 × g for 15 min. Obtained supernatants were then subjected to isoelectric focusing followed by avidin-agarose extraction or directly to avidin-agarose extraction and
immunoprecipitation experiments. The Bio-Rad protein assay kit was used
to determine protein concentrations.
Isolation of Membrane-enriched Fractions--
All steps were
performed at 4 °C. Biotinylated or nonlabeled cells were harvested
and washed three times with ice-cold PBS. Pelleted cells were suspended
(5 × 108 cells/ml) in homogenizing buffer containing
25 mM Tris-HCl, pH 7.6, 10% (v/v) glycerol, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM EDTA, and protease inhibitor mixture. Cells
were homogenized with a Dounce homogenizer (20 strokes), and the
suspension was centrifuged at 500 × g. The supernatant
was then centrifuged for 10 min at 15,000 × g, and a
membrane pellet obtained at 150000 × g for 1 h
was used for further purification. This supernatant was designated as
cytosolic fraction. Pellets were solubilized in 8 M urea,
2% (w/v) CHAPS, 15 mM dithiothreitol, 2% (v/v) Bio-Lyte ampholytes (Bio-Rad), pH 3-10, for 40 min at room temperature. Insoluble material was removed by centrifugation at 10,000 × g for 15 min, and the supernatant was designated as
membrane fraction.
Isoelectric Focusing--
Isoelectric focusing using a Mini
Rotofor cell (Bio-Rad) was done as recommended by the supplier. After
cell-surface biotinylation, part of the detergent lysates (17 mg) or
solubilized membrane fraction (15 mg) were added to the Rotophor cell
containing 13 ml of 8 M urea, 2% (w/v) CHAPS, 15 mM dithiothreitol, 2% (v/v) Bio-Lyte ampholytes, pH 3-10,
after prefocusing for 1 h at 12 W. Focusing was carried out at 12 W for 5 h at 10 °C. The 20 fractions were harvested, and their
pH values were measured. After Western blot analysis, the fractions of
interest were pooled and concentrated with Centricon (YM-30;
Millipore), and biotinylated proteins were extracted with
avidin-agarose.
Avidin-Agarose Extraction--
Either the detergent lysate or
membrane fraction pooled after isoelectric focusing was incubated with
avidin-agarose (Sigma) overnight on a rotary device at 4 °C. The
affinity gel was then washed 3 times with 1% (w/v) Triton X-100, 0.2%
(w/v) SDS, 5 mM EDTA in PBS followed by a wash without SDS
and one with water. The captured biotinylated proteins were eluted from
the agarose with reducing 2-fold Laemmli buffer heated for 5 min at
95 °C.
In-gel Digestion and Mass Spectrometry--
Proteins were
separated by SDS-PAGE (7.5%, 0.75 mm thickness) and stained with 0.2%
(w/v) Coomassie Brilliant Blue R250 in 50% (v/v) ethanol and 10%
(v/v) acetic acid for 30 min. Protein bands of interest were excised
from the gel and in-gel-digested with trypsin (Promega) or chymotrypsin
(Roche Molecular Biochemicals) following the procedure of Shevchenko
et al. (16). Upon reduction of the disulfide bonds of the
protein with tris(2-carboxyethylphosphine) hydrochloride (Pierce),
cysteines were alkylated with iodoacetamide (Sigma). Digestions with
trypsin (400 ng) were carried out overnight at 37 °C in 100 mM ammonium bicarbonate buffer, pH 8.3, and 4 mM calcium chloride. Digestions with chymotrypsin (500 ng)
were performed overnight at 25 °C in the same buffer, pH 8.0. The
resulting peptides were extracted from the gel pieces and desalted
using pipette tips with C18 resins (Millipore).
Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)
mass spectra of the entire digests were recorded on a Bruker Biflex
instrument in the reflector mode employing pulsed ion extraction.
-Cyano-4-hydroxycinnamic acid (Fluka) was used as the matrix. The
MALDI mass spectra were mass calibrated using ion signals mainly from
autoproteolytic fragments of trypsin and chymotrypsin, respectively.
Electrospray ionization mass spectra (MS) and tandem mass spectra
(MS/MS) were acquired on an API III+ triple-quadrupole
instrument (PE-Sciex, Ontario, Canada) equipped with a nanoelectrospray
ion source (Protana, Odense, Denmark).
Immunoprecipitation--
The detergent lysates of cells were
preincubated with protein A-Sepharose (Amersham Pharmacia Biotech) for
2 h. Anti-CyP antiserum or anti-EWS antiserum were added to the
supernatants, and the mixture was incubated at 4 °C for at least
12 h, and then protein A-Sepharose was added for 2 h. The
agarose pellet was washed three times with 1% (w/v) Triton X-100, 2 mM EDTA, and 10 mM Tris-HCl, pH 7.4, and the
antigen was eluted from the gel with reducing 2-fold Laemmli buffer by
heating for 5 min at 95 °C.
Western Blotting and Protein Detection by Enhanced
Chemiluminescence--
Proteins separated on 7.5% SDS-PAGE were
transferred electrophoretically to a nitrocellulose membrane
(Schleicher and Schuell) as described (17). After transfer, the
membrane was blocked for 40 min with 5% (w/v) milk powder dissolved in
PBS and 0.25% (w/v) Tween 20 and incubated with the first antibodies
anti-CyP or anti-EWS, respectively, for 1 h at room temperature,
followed by incubation with goat anti-rabbit antibody coupled to
horseradish peroxidase (Sigma) for 1 h at room temperature. The
detection was performed by enhanced chemiluminescence (ECL, Amersham
Pharmacia Biotech).
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RESULTS |
Partial Purification of a 90-kDa Cell-surface
Protein--
Cell-surface proteins of Jurkat and PBM cells were
labeled under physiological conditions with the impermeable
biotinylation reagent. Labeled surface proteins were extracted with
avidin-coupled agarose and separated by SDS-PAGE. Western blot analysis
with anti-CyP antibodies showed a strong signal of a protein band with a molecular mass of about 90 kDa (Fig.
1A). To obtain a pure protein in amounts high enough for identification, a membrane fraction of
Jurkat and PBM cells was prepared by differential centrifugation after
cell-surface biotinylation. The solubilized proteins were further
purified by isoelectric focusing under denaturing conditions. The
anti-CyP immunoreactive protein was found in a pH range from 7.2 to
9.3. These fractions were pooled, and the biotinylated proteins
extracted with avidin-agarose and subjected to SDS-PAGE. The
Coomassie-stained band (Fig. 1B) containing the
immunoreactive 90-kDa protein of interest (Fig. 1C) was
clearly separated from other proteins and was sufficiently pure to be
subjected for an identification procedure.

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Fig. 1.
Detection of a cell-surface anti-cyclophilin
reactive protein. PBM and Jurkat cells were biotinylated with
sulfosuccinimidobiotin. Equal amounts of cell proteins (see
"Experimental Procedures") were added to avidin-agarose gel, and
biotinylated proteins extracted. After elution with Laemmli buffer,
aliquots were separated on 7.5% SDS-PAGE, transferred to
nitrocellulose, and analyzed by Western blot using anti-CyP antibodies
(A). The pellets of the membrane fractions of Jurkat cells
or PBM cells (PBMC) were solubilized (see "Experimental
Procedures") and loaded for isoelectric focusing into a Rotophor
cell. After focusing, fractions containing the anti-CyP-immunoreactive
protein were pooled, and biotinylated proteins were extracted with
avidin-agarose, separated on 7.5% SDS-PAGE, and stained with Coomassie
Blue (B) or transferred and immunoblotted with anti-CyP
antibodies (C). The molecular masses of marker proteins are
indicated (in kDa).
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Identification of the 90-kDa Band--
The 90-kDa
Coomassie-stained gel band containing the protein of interest was
excised and in-gel-digested with trypsin, and the resulting peptides
were analyzed by MALDI-MS (see "Experimental Procedures"). A
protein sequence data base search (Mascot Search) performed with
the obtained peptide masses (Table I) did
not provide an unequivocal result. RNA-binding protein EWS (1) was
retrieved with the highest score; however, some of the most intense
signals of the MALDI mass spectrum could not be assigned to this
protein. To confirm the identification of the protein, nanoelectrospray
MS/MS sequencing of selected peptides was performed. Four MS/MS spectra
were in complete agreement with the predicted fragmentation pattern of
peptides 269-292, 411-424, 425-439, and 472-486 from RNA-binding
EWS protein and, thus, unambiguously verified the identification of
this protein (Fig. 2, Table I).
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Table I
Summary of mass spectrometric data of proteolytic fragments from the
in-gel-digested protein (see Fig. 1B)
Arginines found to be dimethylated in the corresponding peptides are in
bold and underlined, and the only monomethylated arginine at position
471 is in italics.
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Fig. 2.
Amino acid sequence of the EWS protein.
The C-terminal arginine- and glycine-rich domain of RNA-binding protein
EWS contains 30 Arg-Gly sequences. Completely dimethylated arginine
residues are in bold and underlined, and
arginines found to be either dimethylated or non-methylated are only in
bold. Arginine 471 (shown in bold and in
italics) is either dimethylated or non-methylated and also
monomethylated to a small extent. No proteolytic peptide containing
arginine 330, which represents a potential site of arginine
methylation, had been identified. Regions of protein covered by peptide
fragments from MS data (Table I) are highlighted.
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Assignment of Arginine Methylation Sites--
The tandem mass
spectrum shown in Fig. 3 appeared to
match peptide 615-632 from this protein, but the observed mass was 28 Da higher than the mass calculated from the amino acid sequence. Detailed analysis of the spectrum indicated that this peptide most
likely contains a dimethylated arginine residue located at position 615 (Table I). Another MS/MS spectrum was found to be consistent with
peptide 465-486 containing a dimethylarginine residue at position
471.

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Fig. 3.
Tandem mass spectrum of the tryptic peptide
615-632 of EWS protein. Nanoelectrospray tandem mass spectrum of
the triply charged ion at m/z 593.5 corresponding
to tryptic peptide RGGPGGPPGPLMEQMGGR (R,
dimethylarginine). The singly and doubly charged fragment ions are
labeled according to the Roepstorff-Biemann nomenclature (38).
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Re-examination of the MALDI mass spectrum of the tryptic digest
revealed the presence of eight additional dimethylarginines located in the C-terminal arginine- and glycine-rich domain of the
protein (Fig. 2). Arginines 464, 471, 490, and 503 appear to be
dimethylated to a large extent but not completely. Trypsin does not
cleave after methylated arginines (18, 19). Analyses of tryptic
peptides indicated cleavages at these four arginines that were found to
be modified in other peptides. In addition, peptides containing the
non-methylated arginines 471 and 503 were identified. A small fraction
of arginine 471 (
15%) also occurs in the monomethylated form
(Table I, peptide 465-486).
To analyze the sites of arginine methylation more completely,
RNA-binding protein EWS was in-gel-cleaved with chymotrypsin. MALDI-MS
of the entire chymotryptic digest allowed the identification of 19 additional dimethylarginines (Table I and Fig. 2). Mainly peptides
resulting from specific cleavage after the known chymotryptic cleavage
sites phenylalanine, tyrosine, methionine, and leucine were
assigned; however, arginine 292 and 614 were also found to be
susceptible to chymotryptic cleavage. Only uniquely assignable mass
peaks were reported. The identification of all peptides listed in Table
I was confirmed by the excellent agreement between the observed
molecular masses with those predicted from the known sequences (within
0.006%). The assignment of three dimethylarginine-containing peptides
and of peptide 233-247 was ascertained by nanoelectrospray MS/MS
analyses as noted in Table I.
A qualitative amino acid analysis (data not shown) of an EWS protein
hydrolysate revealed the presence of asymmetric dimethylarginine residues, whereas symmetric dimethylarginine, if at all present, or
monomethylarginine residues were below the detection limit, although
monomethylarginine is present in small amounts according to the MS
data. To check the methylation state of EWS protein in the nucleus, the
protein was isolated from the nucleus as described (12), in-gel
digested with either trypsin or chymotrypsin, and subjected to MALDI-MS
analysis (data not shown). The peptide map of nuclear EWS protein was
found to be comparable with the peptide map of EWS protein isolated
from the plasma membrane, indicating a similar dimethylation pattern of
arginine residues of the nuclear EWS protein.
Cross-reactivity of EWS Protein with Anti-CyP Antibodies--
The
detection of an immunoreactive band with anti-CyP antibodies at the
same position in SDS-PAGE as the EWS protein indicated either
cross-reactivity of the antibodies or cross-contamination of the
proteins. Thus immunoprecipitations with anti-CyP antibodies and
anti-EWS antibody were performed from cell lysates followed by Western
analysis with anti-CyP antibodies (Fig.
4A) as well as with anti-EWS
antibody (Fig. 4B). In any case a stronger signal was
obtained in the Western with anti-EWS antibody even if
immunoprecipitation was done with anti-CyP antibodies, indicating
cross-reactivity of both antibodies with EWS protein. No signals were
observed if (when) goat anti-rabbit antibody alone or rabbit antibodies against several other proteins was used.

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Fig. 4.
Cross-reactivity of EWS protein with
anti-cyclophilin antibodies. After preincubating the detergent
lysates of Jurkat cells with protein A-Sepharose, immunoprecipitation
with the anti-CyP antibodies (lane 1) or with the anti-EWS
antibody (lane 2) was performed. Immunoprecipitated proteins
were subjected to Western blot analysis using the anti-CyP antibodies
(A) or the anti-EWS antibody (B). For
quantification of the bands, the ECL films were scanned, and
intensities of the bands were calculated by using Multi-Analyst
software (Bio-Rad). The large band at 55 kDa (lanes 1 and
2) corresponds to the heavy chain of the antibodies.
Molecular mass standards (kDa) are indicated.
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Excision of the immunoprecipitated 90-kDa Coomassie-stained bands,
in-gel digestion with trypsin, and MALDI-MS analysis of tryptic
peptides confirmed the presence of methylated RNA-binding protein EWS
in anti-CyP and anti-EWS-precipitated samples.
Occurrence of Cell Surface-localized EWS Protein in Different Cell
Lines--
To ascertain that EWS protein is indeed present on the cell
surface, Jurkat, PBM, but also H9 cells were surface-labeled with the
impermeable biotinylation reagent, and biotinylated proteins were
extracted with avidin-coupled agarose. The Western blot, now performed
with anti-EWS antibody, revealed the presence of EWS protein on all
three cell types (Fig. 5A).
Densitometry showed an identical high content of EWS protein on the
surface of the tumor cell lines Jurkat and H9, whereas in PBM cells the
content of EWS protein was drastically lower. On purifying the
cell-surface biotinylated proteins by isoelectric focusing of
membrane-enriched fractions as described in Fig. 1B, a
strong signal was obtained again in the Western blot analysis with
anti-EWS antibody (Fig. 5B), showing that EWS protein is
indeed present on the cell surface. To verify that the 90-kDa protein
represents a surface-exposed molecule and not an unspecifically
extracted protein, proteins from nonbiotinylated cells were extracted
and analyzed by the same procedure. Signals in the anti-CyP or anti-EWS
Western blot analysis were not found (not shown).

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Fig. 5.
The presence and variation of EWS protein on
cell surfaces. Jurkat, H9, and PBM cells (PBMC) were
biotinylated with sulfosuccinimidobiotin. Equal amounts of cell
proteins were extracted as described in Fig. 1A. Aliquots
were separated on 7.5% SDS-PAGE, transferred to nitrocellulose, and
analyzed by Western blot using anti-EWS antibody (A).
Samples obtained by cell surface biotinylation, membrane fractionation,
and isoelectric focusing followed by avidin-agarose extraction (see
Fig. 1B) were separated on 7.5% SDS-PAGE and subjected to
Western blot analysis with anti-EWS antibody (B). Molecular
mass standards (kDa) are indicated.
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 |
DISCUSSION |
Our 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 (20)
and the cytosol (14), 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 (21). 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 Sites--
Arginine
methylation is a post-translational modification found mainly in
nuclear proteins that interact with RNA (22). This modification is
catalyzed by protein-arginine N-methyltransferases (PRMTs),
utilizing S-adenosyl-L-methionine as the
donor of methyl groups (23). Type I protein-arginine
N-methyltransferases (EC 2.1.1.23) catalyze the
formation of NG-monomethylarginine and
asymmetric
-NG,NG-dimethylarginine
residues, whereas Type II enzymes catalyze the formation of
NG-monomethylarginine and symmetric
-NG,N'G-dimethylarginine
residues (22). The Type III enzyme found in yeast catalyzes the
monomethylation of the internal
-guanidino nitrogen atom of arginine
residues (24). Three Type I PRMTs from mammalian cells PRMT1 (25),
PRMT3 (26), and coactivator-associated arginine methyltransferase 1 (CARM1) (27) have been reported. PRMT1, a predominantly nuclear
protein, methylates arginine residues of many proteins, among them
RNA-binding proteins (22, 28). PRMT3 is a predominantly cytoplasmic
protein whose activity overlaps with that of PRMT1 (26). A number of
in vivo substrates for Type I PRMTs have been identified,
including the heterogeneous nuclear ribonucleoprotein A1 (29),
fibrillarin, nucleolin (22), high molecular weight fibroblast growth
factor-2 (30), and poly(A)-binding protein II (28).
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. 22 and 29; Table
II). 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 (31). 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 (28).
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Table II
Comparison of the mapped sites of asymmetric dimethylation of EWS
protein and other recently reported proteins with the proposed
consensus sequence (22, 29)
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Role of Methylation of EWS Protein--
EWS protein belongs to a
large family of RNA-binding proteins that includes heterogeneous
nuclear ribonucleoproteins, mRNA poly(A)-binding proteins (32), and
alternative splicing factors (33, 34). 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. 10; 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 (35). 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 (36).
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 (37). 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 (22). 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 (13)
and interacted with Pyk2 in an activation-dependent manner
(14).
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.