Analysis of Ewing Sarcoma (EWS)-Binding Proteins: Interaction with

Analysis of Ewing Sarcoma (EWS)-Binding Proteins: Interaction with hnRNP M, U, and RNA-Helicases p68/72 within Protein-RNA Complexes Steffen Pahlich,† Lilian Quero,† Bernd Roschitzki,‡ Ruzanna P. Leemann-Zakaryan,† and Heinz Gehring*,† Department of Biochemistry, University of Zu ¨ rich, Winterthurerstrasse 190, 8057 Zu ¨ rich, Switzerland, and Functional Genomics Center Zu ¨ rich, Winterthurerstrasse 190, 8057 Zu ¨ rich, Switzerland Received March 10, 2009

The human Ewing Sarcoma (EWS) protein belongs to the TET family of RNA-binding proteins and consists of an N-terminal transcriptional activation domain (EAD) and a C-terminal RNA-binding domain (RBD), which is extensively methylated at arginine residues. This multifunctional protein acts in transcriptional co-activation, DNA-recombination, -pairing and -repair, in splicing, and mRNA transport. The role of arginine methylation in these processes as well as the time and place of methylation within cells is still unclear. In this study, we show that methylation of recombinant EWS protein in HEK cells occurs immediately after or even during translation. Pull-down experiments with recombinant EWS protein as bait, followed by mass spectrometric analysis identified more than 30 interacting proteins independent of whether the EWS protein was methylated or not. The EWS protein interacts via its RBD with RNase-sensitive protein complexes consisting of mainly heterogeneous nuclear ribonucleoproteins (hnRNPs) and RNA helicases. HnRNP M and U, the RNA-helicases p68 and p72, but also actin and tubulin were found to interact directly with the EWS protein. Co-precipitation experiments with recombinant proteins confirmed the interaction of the EWS protein with p68 via its RBD. Colocalization of the EWS protein and the RNA-helicases in the nucleus of HEK cells was visualized by expressing labeled EWS protein and p68 or p72. When co-expressed, the labeled proteins relocated from the nucleoplasm to nucleolar capping structures. As arginine methylation within the RBD of the EWS protein are neither needed for its subcellular localization nor for its protein-protein interaction, a role of EWS protein methylation in RNA-binding and affecting the activation/repression activity or even in the stabilization of the EWS protein seems very likely. Keywords: H-complex • arginine methylation • recombinant in vivo methylation • GST pull-down • mass spectrometry • nucleolar capping • fluorescence microscopy

Introduction The human Ewing sarcoma (EWS) protein belongs to the TET-family of RNA-binding proteins. It consists of an Nterminal transcriptional activation domain (EAD) and a Cterminal RNA-binding domain. The EAD interacts with calmodulin,1 splicing factor 1,2 protein kinases1,3 and RNA polymerase II,4 and functions as a transcriptional co-activator5-7 In Ewings sarcoma, a tumor-associated chromosomal translocation of the EWS gene results in the expression of fusion proteins of the EWS EAD with DNA-binding domains of several ETS transcription factors with transforming potential.8,9 The C-terminal RNAbinding domain (RBD) of the wild-type EWS protein, which represses the tumor-associated transcriptional activity of the EAD,10 consists of a RNA-recognition motif (RRM), a putative * To whom correspondence should be addressed. Heinz Gehring, Winterthurerstrasse 190, 8057 Zu ¨ rich, Switzerland, Tel. +41-44-635 5572, Fax: +41-44-635 5907, E-mail: [email protected]. † University of Zu ¨ rich. ‡ Functional Genomics Center Zu ¨ rich. 10.1021/pr900235t CCC: $40.75

 2009 American Chemical Society

C2C2 zinc finger, and three arginine-glycine rich domains (RGG boxes 1-3). Additional to the transcriptional activity of the EWS protein, a role in splicing and mRNA transport is proposed, as the EWS protein interacts with the splicing factors TASR-1 and -211,12 and YB-1.13 It was co-purified with hnRNPs A1 and C1/ C2,14 and was identified in a large-scale proteomic analysis of the human spliceosome.15 Recently, a role in DNA pairing16 and recombination was described, as EWS protein-deficient mice fail to develop functional B-lymphocytes and have defects in meiosis.17 SiRNA-silencing of EWSR1 in HeLa cells resulted in mitotic defects.18 The EWS protein is located mainly in the nucleus, directed by its C-terminal nuclear localization sequence19 via transportin.20 Minor fractions of the protein were also identified in the cytosol and at the cell membrane. Independent on its subcellular localization, the RGG boxes of the EWS protein were found to contain 30 asymmetrically dimethylated arginines.21 Arginine methylation is a eukaryotic post-translational modification catalyzed by a family of enzymes called protein arginine Journal of Proteome Research 2009, 8, 4455–4465 4455 Published on Web 08/12/2009

research articles methyltransferases (PRMTs). They catalyze the transfer of a methyl group from the donor S-adenosyl-L-methionine to the guanidino nitrogens of the arginine side chain. Methylated arginines are found mainly in RGG-motifs of RNA-binding proteinslikeheterogeneousnuclearribonucleoproteins(hnRNPs), RNA helicases, spliceosomal proteins, and histones. Methylated proteins function in transcriptional regulation, pre-m-RNA splicing, mRNA-transport, and translation, but also a role in signaling is proposed. So far, this modification was believed to be irreversible (for reviews, see refs 22 and 23). Recently JMJD6 has been indentified as the first arginine demethylase acting on histones H3 and H4.24 However, this activity is controversial because JMJD6 has been reported to catalyze lysine hydroxylation.55 For many proteins, like the EWS protein, the role of methylation, but also the time and place of methylation within the cell, is still not known. In this study, we could show that recombinant EWS protein is methylated immediately after translation or even co-translationally in the cytosol of HEK cells. To investigate the cellular function of methylation of EWS protein, we identified interaction partners of unmethylated and methylated recombinant EWS protein. Both, unmethylated and methylated EWS protein, were shown to interact with large, RNase-sensitive protein complexes including the RNA-helicases p68 and p72. Colocalization studies did not only confirm the interactions, but showed also a relocation of the EWS protein from the nucleoplasm to the nucleolar periphery, when co-expressing p68 or p72. The extensive arginine methylations within the RBD of the EWS protein, however, are neither needed for its subcellular localization19 nor for protein-protein interaction, as demonstrated in this study. Therefore, a role of EWS protein methylation in RNA-binding and thus in affecting the activation/ repression activity or even in stabilization of the EWS protein is proposed.

Materials and Methods Plasmids and Constructs. The cDNA of EWS was kindly provided by Dr. Olivier Delattre (Institut Curie, Pathologie Mole´culaire des Cancers, Paris Cedex). GST-p68 and GST-p72 expression vectors (pEBG2T) for expression in mammalian cells were a generous gift of Dr. Frances Fuller-Pace (Division of Pathology & Neuroscience, University of Dundee, U.K.), and His-tagged p68 expression vector (pET-30a) was kindly provided by Dr. Zhi-Ren Liu (Department of Animal and Dairy Sciences, Auburn University, AL). Expression of Recombinant Proteins. GST-EWS was expressed in Escherichia coli as described.25 Co-expression of GST-EWS (expression vector pGEX-6p-2) and His-PRMT1 (expression vector pET-28c) was done in E. coli XL-blue cells. His-tagged p68 was expressed in E. coli BL1-codon plus strain. For expression of His-EWS constructs,19 human embryonic kidney (HEK) 293 T cells were transiently transfected using the calcium phosphate precipitation method. Efficiency of transfection and subcellular localization of the different constructs was checked by fluorescence microscopy. Cell Culture and Preparation of Total Cell Lysates. Jurkat cells were cultivated in RPMI medium at 37 °C. Cells were harvested, washed twice in ice-cold PBS buffer, and lysed for 30 min on ice in lysis buffer A (50 mM Tris, pH 8.0; 200 mM NaCl; 1 mM PMSF; 1 mM DTT; 1% Triton-X 100; protease inhibitor mix (Roche)). The lysate was centrifuged for 30 min at 15 000g and the supernatant, designated as total Jurkat lysate, was used for pull-down experiments. 4456

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Pahlich et al. HEK cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% (v/v) fetal calf serum (FCS) in a humified 10% (v/v) CO2 atmosphere at 37 °C. The cells were cultured to 40% confluence on glass coverslips pretreated with poly-L-lysine (30 µg/mL) prior to transfection. Immunostaining and Confocal Microscopy. Twenty-four hours after transfection, HEK cells were washed with PBS, fixed with 4% paraformaldehyde, and permeabilized with 1% Triton X-100 for 15 min at room temperature (RT). The cells were washed three times with PBS and nonspecific binding sites were blocked with 10% FCS, 0.1% glycine in PBS, pH 7.4 for 1 h. After washing three times with PBS, cells were immunostained by using primary goat anti-GST antibody (Amersham, 1:2000) and secondary Cy3-coupled anti-goat antibody (Jackson Immuno Research, 1:400). DNA was stained with 4′,6-diamidino-2-phenylindol (DAPI) (Roche). Laser-scanning confocal fluorescence microscopy was performed at the Center for Microscopy and Image Analysis, University of Zurich, as described19 using excitation-emission wavelength of 405-470 nm for DAPI, 514-528 nm for YFP and 543-570 nm for Cy3. The pictures were merged and colocalized with Adobe Photoshop 8.0 and Imaris 5.7.0, respectively. GST Pull-Down. GST or GST-EWS (10 µg) was bound to glutathione-coupled agarose (Sigma) at RT (2 h). Afterward, the agarose was washed twice with 100 mM sodium borate and twice with 50 mM Tris, pH 8.0. Total Jurkat cell lysate (1 mg of total protein) was added in the presence of binding buffer (50 mM Tris, pH 8.0; 100 mM NaCl; 1 mM PMSF; 1 mM DTT; 0.1% Triton X-100; protease inhibitor mix (Roche)). If indicated, RNase A was added to a final concentration of 0.2 mg/mL. The sample was incubated for 1 h at RT and 1 h at 4 °C under constant shaking. The agarose was washed three times with binding buffer and bound proteins were specifically eluted with 20 mM glutathione/50 mM Tris, pH 8.0. The eluted proteins were separated using SDS-PAGE (10% acrylamide) and analyzed by mass spectrometry after in-gel digest or by Western blotting. Mass Spectrometry (MS). MS analyses were performed at the Functional Genomics Center Zu ¨ rich (FGCZ) and at the Protein Analysis Unit of the University of Zu ¨rich as described.25 Briefly, for identification of EWS-binding proteins from the GST pull-down, the whole gel lane was cut into slices and each slice was in-gel digested with trypsin. The tryptic peptides were pooled, desalted using C18-ZipTips (Millipore, Bedford, MA), and analyzed using nano-LC-MSMS using an LTQ-FT (Thermo Electron, Bremen, Germany). Peptides were separated on a nano-HPLC (Eksigent Technologies, Dublin CA) online prior to MS analysis on a homemade C18 reversed phase column (Magic 3 µm, 100 Å C18 AQ, Michrom, Auburn, CA) using an acetonitrile/water system at a flow rate of 200 nL/min. Tandem mass spectra were acquired in a data-dependent manner. Typically, 5 MS/MS were performed after each high accuracy spectral acquisition range survey. A custom database (38 246 entries) comprising the human portion (taxonomy ID: 9606) of the UniProt database (http://www.ebi.ac.uk) was interrogated using the Mascot search algorithm on an in-house server (FGCZ). For the search, three trypsin missed cleavages were allowed. The precursor ion tolerance and fragment ion tolerance were set to 2.5 ppm and 0.6 Da, respectively, and oxidized methionine and arginine methylation was set as variable modification. Only proteins, which were identified in at least two independent pull-down experiments, were counted as potential interaction partner of the EWS protein. Addition-

Interaction Partners of the EWS Protein

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Figure 1. Methylation state of nuclear and cytoplasmic EWS protein. (A) HEK cells were transiently transfected with His- and GFPtagged EWS wild-type protein (nuclear) or the GFP-EWS-Y/F mutant (cytoplasmic) and fluorescent proteins were analyzed by confocal microscopy using excitation-emission wavelengths of 405-470 nm and 488-509 nm for DAPI and GFP, respectively. Scale bars represent 5 µm. (B) Cells were lysed, and the His-tagged EWS constructs were affinity-purified using Ni-affinity agarose, and subjected to SDSPAGE followed by in-gel digestion and MALDI-TOF-MS analysis in the positive ion mode (M + H+). The spectrum shows the peptide covering the C-terminus of the EWS protein either without mutation (m/z 1275) or with the Y to F substitution (m/z 1259). The indicated peak at m/z 1276 was present in both samples and corresponds to the peptide RQDHPSSMGVY (534-544, see Supplemental Table 1 in Supporting Information). In the spectrum of the EWS wild-type, the peptide at m/z 1275 overlaps the one at m/z 1276. (C) Peptide coverage of the EWS and the EWS-Y/F mutant sequence after LC-MALDI-TOFTOF-MS analysis. Large parts of the sequence are covered by the tryptic (box) or chymotryptic (yellow) peptides. Methylated (R) and dimethylated (R) arginines are labeled.

ally, the MS/MS spectra of proteins, whose identification is based only on one or two assigned peptides, were checked manually for a correct peak assignment to exclude false positives. MALDI-TOF-MS analysis of the unmethylated and methylated His-EWS protein was performed on a Bruker-Daltonics Autoflex II MALDI mass spectrometer operated in the positive ion reflector mode using R-cyano-hydroxycinnamic acid (4 mg/ mL in 70% acetonitrile/0.1% TFA) as matrix and LC-MALDITOF/TOF-MS analysis was done as described.25

Results Methylation of the EWS Protein Occurs in the Cytosol. Until now, only methylated EWS protein could be found in eukaryotic cells, be it at the cell surface or in the nucleus ( see ref 21 and own observations). The methyltransferase PRMT1, which methylates the EWS protein efficiently in vitro,25 was described to be localized either predominantly in the cytosol or in the nucleus.26-30 The observed discrepancy might depend on the

different cell lines used and on different experimental setups, and reflect a dynamic subcellular distribution of PRMT1. Whether the EWS protein is methylated first in the cytoplasm and than transported to other cellular compartments or whether the methylation takes place in the nucleus, and fractions of the methylated protein might be exported and transferred to the cell membrane was still an open question. To determine where and when the methylation of the EWS protein occurs, HEK cells were transiently transfected with two different EWS constructs coding for EWS wild-type or a for EWS-Y/F mutant. In this mutant, the last residue, Y656, of the EWS protein was replaced with phenylalanine. This mutation in the C-terminal nuclear localization signal (C-NLS) of the EWS protein prevents a nuclear import of the protein.19 The subcellular localization of the constructs, both containing a His and GFP tag, was verified by fluorescence microscopy (Figure 1A) which confirmed nuclear localization with exclusion from the nucleoli of the tagged EWS protein and accumulation of the tagged EWS-Y/F mutant at the cytoplasmic side of the Journal of Proteome Research • Vol. 8, No. 10, 2009 4457

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Figure 2. Methylation state of recombinant EWS protein co-expressed with His-PRMT1. GST-EWS protein, co-expressed with HisPRMT1 in E. coli, was affinity-purified with glutathione-coupled agarose, and the extracted EWS protein was subjected to SDS-PAGE and in-gel digested either with trypsin or chymotrypsin. The resulting peptides were analyzed by MALDI-TOF-MS in the positive ion mode. (A) Mass spectrum of the tryptic peptides. All peaks that could be assigned to the GST-EWS protein are labeled with a circle. Peptides with potential methylation sites (R) and the corresponding methylation state are enlarged. (B) Mass spectrum of the chymotryptic peptides. All peaks that could be assigned to the GST-EWS protein are labeled with a circle. Peptides with potential methylation sites (R) and the corresponding methylation state are enlarged.

nuclear envelope as described.19 The methylation state of the EWS constructs in the different subcellular compartments was determined by mass spectrometry after purifying the His-fusion proteins with Ni-affinity agarose and SDS-PAGE, followed by in-gel digestion of the dominant band at ∼116 kDa on a Coomassie-stained gel. The peptide RQERRDRPY (1275.7 Da) in the spectrum of the wild-type EWS construct covers the intact C-terminus and is absent in the spectrum of the His4458


EWS-Y/F mutant; instead the peptide RQERRDRPF (1259.7 Da) of the mutated C-terminus appeared, confirming the mutation of the EWS C-terminus (Figure 1B). In both cases, the tryptic and chymotryptic peptides cover large parts of the amino acid sequence of the constructs (Figure 1C). Methylation of the constructs was analyzed by LC-MALDI-TOFTOF MS. All identified peptides including their modifications can be found in Supplemental Table 1 in Supporting Information. From the 30


Figure 3. Pull-down with GST-EWS protein as bait. Total cell lysates from Jurkat cells were used for a pull-down experiment with recombinant GST-EWS protein in the absence (lane 1) or presence (lane 2) of RNase A, or recombinant GST (lane 3) as bait. Extracted proteins were subjected to SDS-PAGE and visualized by Coomassie blue staining of the gel.

arginines, which were found to be methylated in the EWS protein in vivo and in vitro,21,25 27 were asymmetrically dimethylated in the EWS wild-type construct, one arginine (R756) was found to be unmodified and the remaining two arginines (R569, R730) were not covered by the detected peptides. A similar methylation pattern was observed in the cytoplasmic His-EWS-Y/F mutant, 25 asymmetrically dimethylated arginines were detected, one arginine (R756) was found to be monomethylated, one arginine was unmodified (R730), and the remaining three arginines (R569, R838, R841) were not covered by the detected peptides. Completely methylated peptides were always the dominant signal in the mass spectra of the constructs, but also peptides in several methylation states were detected in both, nuclear and cytoplasmic mutated EWS protein, which is usually not observed in endogenous wildtype EWS protein (own observations). Incomplete methylation might be due to overexpression of the EWS constructs in the cells. Methylation of Recombinant EWS Protein by Co-Expression with PRMT1 in E. coli. As the purification of His-tagged EWS protein, expressed in HEK cells, yielded too low amounts for functional studies and multiple co-purifying proteins, we established in E. coli co-expression of human PRMT1 (Histagged) and of its substrate, the EWS protein (GST-tagged). The co-expressed GST-EWS protein was affinity-purified and its methylation state was controlled by MALDI-TOF MS analysis. All peptides in the spectrum, which contained potential arginine methylation sites, were found to be methylated (Figure 2). Complete methylation was dominant, although some incomplete methylated peptides were also present. The EWS protein co-expressed with PRMT1 we therefore designated here as methylated GST-EWS protein. GST Pull-down with Unmethylated and Methylated EWS Protein as Bait. To identify endogenous interaction partner of the unmethylated and methylated EWS protein, GST pull-

research articles down experiments were performed. First, recombinant unmethylated GST-EWS protein, expressed in E. coli, was bound to glutathione-coupled agarose and total cell lysate from Jurkat cells was added. The bound proteins were specifically eluted with glutathione and subjected to SDS-PAGE followed by Coomassie-blue staining of the gel (Figure 3, lane 1). Beside the GST-EWS protein band at ∼116 kDa, several copurifying protein bands can be seen which are not present when GST was used as bait (lane 3). The dominant bands at ∼30 kDa represent GST-fragments of the GST-EWS fusion protein. To identify the co-purified proteins, the whole gel lane was cut into slices, each slice was in-gel digested, and the resulting peptides were analyzed by LC-MSMS as described in Materials and Methods. More than 30 different proteins were identified, among them mainly RNA-binding proteins like hnRNPs, RNA-helicases, or TLS/FUS and TAFII68 which belong together with the EWS protein to the TET family of proteins (Table 1). But also the methyltransferase PRMT1 and the structural proteins actin, tubulin, and vimentin were found. A methylation of the GST-EWS protein by endogenous PRMT1 during the pull-down can be excluded as no methylated peptides of the GST-EWS could be identified by mass spectrometry (data not shown). All identified proteins and their Mascot scores are listed in Supplemental Table 2 in Supporting Information. Among the identified proteins, the hnRNPs A0, A1, D0, G, and H, but also TLS/FUS and TAFII68 were found to contain methylated arginine residues (Table 2). The identified methylation sites of hnRNP H (R232) and TAFII68 (R475, R483, R528, R535, R570) have not been described to our knowledge, whereas the other identified methylation sites have been reported.31 HnRNPs are known to form protein-RNA complexes in the nucleus. To test, whether the identified proteins bind directly to the EWS protein or whether RNA-protein complexes are co-purified, cell lysates were treated with RNase A during the pull-down experiments. Subjecting the co-purified proteins to SDS-PAGE showed that several protein bands were absent (Figure 3, lane 2). MS analysis after in-gel digestion revealed that almost all RNA-binding proteins, except hnRNP M, hnRNP U, and the RNA-helicases p68 and p72, were absent after the treatment with RNase (Table 1, Supplemental Table 2 in Supporting Information). To investigate the influence of arginine methylation on EWS protein-protein interactions, the pull-down experiments in the presence and absence of RNase A were performed using as bait recombinant methylated EWS protein which was obtained by co-expressing PRMT1 in E. coli. Similar RNA-binding proteins as in the pull-down experiments with unmethylated EWS protein were extracted and identified in the presence or absence of RNase A (Table 1, Supplemental Table 2 in Supporting Information). The EWS protein consists of an N-terminal EAD and a C-terminal RBD. To determine which domain is interacting with the RNA-protein complex, pull-down experiments with individual EWS domains that were expressed in E. coli were performed in the presence and absence of RNase A. The same set of hnRNPs and RNA-helicases were identified with the RBD as bait, whereas none of them bound to the EAD or the GST control. Remarkably, complete methylation of the RBD had again no impact on the interacting proteins. Interaction of the EWS Protein with RNA Helicase p68. In the pull-down experiments, the RNA helicase p68 was found to interact with the RBD of the EWS protein independent of Journal of Proteome Research • Vol. 8, No. 10, 2009 4459

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Table 1. List of Proteins Identified in the Indicated Pull-Down Experiments

a

Proteins identified in at least two independent experiments are labeled in bold (X), proteins identified in only one experiment are labeled in roman (X).

Table 2. Methylated Peptides of Proteins Identified in the EWS Pull-Down Experimentsa accession number

protein

m/z

Q13151

hnRNP A0

1968,85

SNSGPYRGGYGGGGGYGGSSF

1 dimethylation (R291)

P09651

hnRNP A1

1422,67 2058,92 1340,64

QEMASASSSQRGR SGSGNFGGGRGGGFGGNDNFGR GGNFSGRGGFGGSR

1 dimethylation (R193) 1 dimethylation (R205) 1 dimethylation (R224)

Q14103

hnRNP D0

1334,65

RGGHQNSYKPY


P38159

hnRNP G

770,47

APVSRGR


P31943

hnRNP H

2901,24

RGAYGGGYGGYDDYNGYNDGYGFGSDR


P35637

TLS/FUS

1762,85

GGRGRGGSGGGGGGGGGGYNR

2 dimethylations (R216, R218)

Q92804

TAF(II)68

2189,91 1428,62 1843,85 1285,59

GGGYGGDRGGGYGGDRGGYGGDR GGGYGGDRGGYGGDR SRGGYGGDRGGGSGYGGDR GGGYGGDRGGYGGK

2 1 2 1

a

peptide sequence

dimethylations (R475, R483) dimethylation (R475) dimethylations (R528, R535) dimethylation (R570)

new

new new new new

The methylation sites of hnRNP H and TAFII68 are not described so far.

its methylation state or RNase A treatment. To confirm this interaction, pull-down experiments with affinity-purified recombinant proteins were performed. Purified recombinant GST-RBD was incubated with purified recombinant His-tagged 4460

modification


p68 and bound p68 was detected by Western blotting using anti-His antibodies (Figure 4). Binding of p68 was observed with both, unmethylated and methylated RBD as bait, whereas no p68 could be detected in the GST control. These results confirm


Figure 4. Purified His-p68 was used for a pull-down with GSTRBD as bait. Recombinant GST-RBD, co-expressed with HisPRMT1 (methylated RBD) or not (unmethylated RBD) and prebound to glutathione-coupled agarose, was incubated with purified His-p68. After extensive washing, eluted GST-RBD and bound proteins were subjected to SDS-PAGE. Bound recombinant Hisp68 was detected by Western blotting using a His-tag-specific antibody. Similar amounts of GST-RBD were loaded on the SDSPAGE, whereas that of the GST control was even higher, as indicated by ponceau staining.

the direct interaction between the RBD of the EWS protein and the RNA-helicase p68. Colocalization of the EWS Protein and RNA Helicases p68 and p72. To investigate, whether the EWS protein and the RNA helicases p68 and p72 colocalize in vivo, HEK 293 (T) cells were co-transfected with YFP-fused EWS protein and with GSTp68 or GST-p72. The YFP-EWS protein alone shows the expected nuclear localization with exclusion from the nucleoli (Figure 5).19 As no methyltransferase inhibitors were used, the EWS protein is methylated completely. The GST-tagged RNAhelicases, detected by immunostaining using anti-GST antibodies, are similar to the EWS protein, also located in the nucleus with exclusion from the nucleoli. Minor fractions are found in the cytosol (Figure 5). As endogenous GST of untransfected cells was hardly detectable in the nucleus (Figure 5), the nuclear GST-signals must be due to the presence of GSTRNA-helicases. Upon co-expression of YFP-EWS and GST-p68 or GST-p72, large parts of the EWS protein and the RNA helicases colocalize in the nucleus. Interestingly, the EWS protein, as well as p68 and p72 even change its subnuclear distribution from nucleoplasm to the nucleolar periphery (Figure 5). EWS-YFP is expressed at physiological concentration ranges as indicated by Western blots which showed only a one to 2-fold higher expression of EWS-YFP than the endogenous EWS protein.32

Discussion In the present study, the time and place of EWS protein methylation in the cell and the influence of this posttranslational modification on EWS protein-protein interactions were investigated. PRMTs are present in cytosol and nucleus, and therefore, it is not clear where and when in the cell proteins, in particular the most extensive methylated EWS protein, become methylated. The present results show that recombinant EWS protein gets extensively methylated immediately after or even during translation in HEK cells as no unmethylated EWS protein could be detected, be it in

research articles the nucleus or even if EWS stays in the cytosol. This indicates to a static rather than a dynamic or regulatory function of most of the methylation sites of the EWS protein. Whether some few specific sites might be methylated in the nucleus is out of the scope of the present investigation. Recombinant, in E. coli expressed, unmethylated and methylated EWS protein was used in this study to investigate whether EWS protein-protein interactions are dependent on this post-translational modification. The methylated EWS protein was obtained by co-expressing EWS protein with PRMT1 in E. coli both under the control of the same inducible promoters. This simple procedure resulted in more or less complete methylation of the EWS protein demonstrating efficient methylation and that PRMT1 has not to be controlled by its own strong E. coli promoter to ensure its presence in the cell prior to the expression of the substrate as reported previously.33 More than 30 proteins, which were pulled down by the unmethylated EWS protein, could be unequivocally identified by mass spectrometric analyses. Among them were mainly heterogeneous nuclear ribonucleoproteins (hnRNPs), but also RNA-helicases and structural proteins like actin, tubulin, and vimentin. Some of the identified proteins were co-purified in an RNase A-sensitive complex. Several methylated proteins were found among the identified proteins, and for hnRNP H and TAFII68, we identified the methylation sites which, to our knowledge, have not been described yet. Methylation of the EWS protein, however, had no influence on its interaction properties with this RNase A-sensitive complex as it did not change the pattern of the co-purified proteins. An influence of EWS protein methylation on its interaction to low-affinity binding partners cannot be excluded. HnRNPs are highly abundant nuclear RNA-binding proteins which function in transcriptional regulation, mRNA splicing and processing, mRNA transport, and translation.34,35 Characteristic structural features of hnRNPs are RNArecognition motifs (RRM), RGG-boxes containing asymmetrically dimethylated arginines, or KH-domains. In the nucleus, hnRNPs have been identified to form RNA-dependent complexes, most likely with (pre)-mRNA, consisting of ∼20 major hnRNP-components with varying composition and stoichiometry (H-complex). Although a function in splicing of several hnRNPs was proposed, they are no key elements of the spliceosome.36 The H-complex rather seems to bind free RNA or cover intron sequences of the pre-mRNA to guide them to degradation within the nucleus.37 Here we showed that the EWS protein, be it methylated or not, pulled down an hnRNP-RNA complex. HnRNPs M and U seem to be direct binding partners of the EWS protein, as their interaction is mediated even after digestion of RNA with RNase A. The responsible domain in the EWS protein for binding is the C-terminal RBD of the EWS protein, which contains a RNArecognition motif (RRM) and RGG-boxes with dimethylated arginines, both structural features characteristic for hnRNPs. RBD binds to the H-complex independent if it is methylated or not. As methylation of the hnRNPs is also not necessary for assembling the H-complex,38 and as the EWS-RBD and hnRNPs have structural features in common, similar interactions of the EWS-RBD in the H-complex seem likely. Transcriptional activation domains like the N-terminal EAD of the EWS protein are, however, absent in hnRNPs. Therefore, the EWS protein might target hnRNP-complexes to their place of action in the cell. Considering the fact that no core Journal of Proteome Research • Vol. 8, No. 10, 2009 4461

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Figure 5. Colocalization of YFP-EWS and p68-GST or p72-GST. HEK cells were transiently transfected with either YFP-EWS, p68-GST, or p72-GST individually, or co-transfected with YFP-EWS and p68, or YFP-EWS and p72-GST, respectively. P68-GST and p72-GST were visualized by immunofluorescence using a mouse anti-GST primary antibody and indocarbocyanine (Cy3)-coupled goat antirabbit secondary antibodies. The fluorescent proteins were analyzed by confocal microscopy using excitation-emission wavelengths of 405-470, 488-509, and 550-570 nm for DAPI, YFP, and Cy3, respectively. The pictures were merged using the programs Adobe Photoshop and Imaris. Scale bars represent 15 µm.

proteins of the spliceosome were identified in this study, and that the EAD of the EWS protein is able to bind RNA polymerase II, it might function as scaffold protein in targeting hnRNPs and other RNA binding proteins to premRNA already during transcription rather than playing a direct role in splicing. Another direct interaction partner of the EWS protein, identified in this study, is the RNA-helicase p68 (DDX5). P68 and its homologue p72 (DDX17) are ATP-dependent RNA helicases located in the nucleus and, like the EWS protein, excluded from the nucleoli.39 They were identified as part of the human spliceosome in a large-scale proteomic analysis15 4462


and a function as a promoter-specific transcriptional coactivator/repressor was reported.40,41 Recently, a role in ribosome biogenesis and cell proliferation was observed.42 In this study, we could show that the EWS protein interacts directly with p68 via the RBD, independent of its methylation state. In HEK cells, we observed colocalization of the EWS protein with both p68 and p72. Interestingly, the subnuclear distribution of the EWS protein and p68 or p72 changed from nucleoplasm, when expressed alone, to an accumulation in the periphery of the nucleoli, when co-expressed. Several nuceloplasmic RNAbinding proteins like PSF, p54, p68, TLS/FUS, and the EWS protein were reported to relocate to such nucleolar caps under

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Interaction Partners of the EWS Protein 43

transcriptional arrest, and a proteomic study of the nucleolar dynamics revealed an upregulation of p68, among others, in the nucleolus when transcription was inhibited.44 In that study, ∼700 different proteins were identified as components of the nucleoli, but only one-third are involved in rRNA biogenesis, an important function of the nucleolus. Therefore, an additional role of the nucleoli in gene expression was proposed. Nucleoli might serve as a place, where cellular regulators and their activity are sequestered45 or even degraded, as proposed for the transcription factor c-myc.46 The determination of the exact roles of the EWS protein and p68 and p72 at the nucleolar periphery, however, needs further investigations. Actin, tubulin, and vimentin were also found to interact with the EWS protein. These proteins are highly abundant in the cell and might be extracted unspecifically in such pull-down experiments. However, they were not found in any GST control pull-down indicating a functional relevance of these interactions. It was shown that the EWS protein is present in RNAtransporting granules in dendrites, which transport their cargo via the microtubules,47 and in quiescent serum-starved cells, the EWS protein forms a net-like structure in the cytoplasm and is associated with microtubules.32 Actin, beside its cytoplasmic localization, was shown to be present as monomer also in the nucleus.48 The role of its interaction with the EWS protein needs to be addressed, but similar to the interaction of actin with hnRNP U in the nucleus, which is necessary for productive transcription of the RNA polymerase II,49 a functional relevance seems feasible. PRMT1, the main arginine methyltransferase in human cells, was also identified in the present study to bind to the unmethylated EWS protein in the presence and absence of RNase. As PRMT1 efficiently binds to and methylates the EWS protein in vitro and in vivo,25,50 its identification in this pulldown experiments confirms the specificity of the used method. Additionally, PRMT1 was found here to interact also with the methylated EWS protein. Even if all arginines are methylated, the methyltransferase PRMT1 seems to retain a certain affinity toward the EWS protein indicating also a role of PRMT1 as mediator than just being a methyltransferase. We could recently show that also PRMT8, a membrane-associated methyltransferase with high sequence similarity to PRMT1, interacts with both, methylated and unmethylated RGG3-box of the EWS protein.51 Binding partners of the EWS protein, which have been previously reported to interact with the EAD of the EWS protein, are the RNA polymerase II,4,52 CBP,52 calmodulin,1 the splicing factor 1,2 or protein kinases.1,3 They were not found in the present study, be it with the full-length EWS protein or the EAD only as bait. The EAD was predicted to be completely unstructured, a typical feature of human tumor-related transcription factors.53 Such intrinsically disordered proteins can mediate a stable secondary structure only upon ligand binding, as shown when CBP binds to ACTR,54 or after post-translational modifications such as phosphorylation. Recombinant EWS protein or EAD might lack such ligands or modifications and therefore also a proper conformation for pulling down known and unknown interaction partners. In summary, several unknown direct and indirect interaction partners of the EWS protein were identified in this study leading to new insights into the roles of the multifunctional EWS protein. However, its extensive arginine methylation did neither affect the binding of proteins and protein-RNA complexes to the RBD of the EWS protein nor the transport of the EWS

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protein to the nucleus. As no unmethylated EWS protein is found in cells, it seems that the methylation of the EWS protein might be needed either for RNA-binding properties and activation/repression activity, or for its stability rather than being a regulatory element of EWS protein function. Abbreviations: GST, Glutathione-S-transferase; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; GFP, green fluorescent protein; YFP, yellow fluorescent protein; R-CCA, R-cyano-4-hydroxycinnamic acid; RT, room temperature; ETS domain, erythroblast transformation specific domain.

Acknowledgment. We are grateful to Doris Grossenbacher for her support in the production and purification of the recombinant proteins. We would like to thank Dr. Peter Gehrig and Dr. Bertran Gerrits from the FGCZ for their contributions in recording and interpreting the MS data, and Dr. Philipp Christen for helpful discussions. This work was supported by the Olga Mayenfisch Stiftung, Hartmann Mu ¨ ller-Stiftung fu ¨r medizinische Forschung, and the Julius Mu ¨ ller Stiftung zur Unterstu ¨ tzung der Krebsforschung. Supporting Information Available: Supplemental Table 1, identified peptides of the His-EWS and His-EWS-Y/F constructs. The His-EWS constructs were purified with Niaffinity agarose, in-gel digested with trypsin or chymotrypsin and the resulting peptides were desalted using C18 ZipTips and subjected to LC-MALDI-TOFTOF-MS analysis. The presence of the ion at m/z 46 confirms arginine dimethylation within a peptide. Supplemental Table 2, proteins identified in GST pulldown experiments. To identify the interaction partner, bound proteins were specifically eluted with glutathione, subjected to SDS-PAGE, the whole gel lane was cut into slices, each slice was in-gel digested, and the resulting peptides were analyzed by LC-MSMS as described in Materials and Methods. Only proteins, which were not found in the GST controls, are listed. The Mascot scores of each identification are indicated. The MS/ MS spectra of proteins, whose identification is based only on one or two assigned peptides, were checked manually for a correct peak assignment to exclude false positives. This material is available free of charge via the Internet at http://pubs.acs.org. References (1) Deloulme, J. C.; Prichard, L.; Delattre, O.; Storm, D. R. The prooncoprotein EWS binds calmodulin and is phosphorylated by protein kinase C through an IQ domain. J. Biol. Chem. 1997, 272 (43), 27369–27377. (2) Zhang, D.; Paley, A. J.; Childs, G. The transcriptional repressor ZFM1 interacts with and modulates the ability of EWS to activate transcription. J. Biol. Chem. 1998, 273 (29), 18086–18091. (3) Felsch, J. S.; Lane, W. S.; Peralta, E. G. Tyrosine kinase Pyk2 mediates G-protein-coupled receptor regulation of the Ewing sarcoma RNA-binding protein EWS. Curr. Biol. 1999, 9 (9), 485– 488. (4) Bertolotti, A.; Melot, T.; Acker, J.; Vigneron, M.; Delattre, O.; Tora, L. EWS, but not EWS-FLI-1, is associated with both TFIID and RNA polymerase II: interactions between two members of the TET family, EWS and hTAFII68, and subunits of TFIID and RNA polymerase II complexes. Mol. Cell. Biol. 1998, 18 (3), 1489–1497. (5) Thomas, G. R.; Faulkes, D. J.; Gascoyne, D.; Latchman, D. S. EWS differentially activates transcription of the Brn-3a long and short isoform mRNAs from distinct promoters. Biochem. Biophys. Res. Commun. 2004, 318 (4), 1045–1051. (6) Lee, J.; Rhee, B. K.; Bae, G. Y.; Han, Y. M.; Kim, J. Stimulation of Oct-4 activity by Ewing’s sarcoma protein. Stem Cells 2005, 23 (6), 738–751.

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research articles

Interaction Partners of the EWS Protein (54) Demarest, S. J.; Martinez-Yamout, M.; Chung, J.; Chen, H.; Xu, W.; Dyson, H. J.; Evans, R. M.; Wright, P. E. Mutual synergistic folding in recruitment of CBP/p300 by p160 nuclear receptor coactivators. Nature 2002, 415 (6871), 549–553. (55) Webby, C. J.; Wolf, A.; Gromak, N.; Dreger, M.; Kramer, H.; Kessler, B.; Nielsen, M. L.; Schmitz, C.; Butler, D. S.; Yates, J. R., III;

Delahunty, C. M.; Hahn, P.; Lengeling, A.; Mann, M.; Proudfoot, N. J.; Schofield, C. J.; Bottger, A. Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing. Science 2009, 325 (5936), 90–93.

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Journal of Proteome Research • Vol. 8, No. 10, 2009 4465

Analysis of Ewing Sarcoma (EWS)-Binding Proteins: Interaction with

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