Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of the EWS Protein

Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of... Hindawi Publishing Corporation Sarcoma Volume 2011, Article ID 218483, 6 pages doi:10.1155/2011/218483 Research Article Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of the EWS Protein 1, 2 1 1 Ruzanna P. Leemann-Zakaryan, Steffen Pahlich, Doris Grossenbacher, and Heinz Gehring Department of Biochemistry, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland Division of Experimental Pathology, Institute of Pathology, CHUV, Faculty of Biology and Medicine, University of Lausanne, Rue du Bugnon 25, 1005 Lausanne, Switzerland Correspondence should be addressed to Heinz Gehring, gehring@bioc.uzh.ch Received 2 September 2010; Revised 26 January 2011; Accepted 1 March 2011 Academic Editor: H. Kovar Copyright © 2011 Ruzanna P. Leemann-Zakaryan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ewing sarcoma (EWS) proto-oncoprotein, an RNA-binding protein, is involved in DNA recombination and repair, gene expression, RNA processing and transport, as well as cell signalling. Chimeric EWS oncoproteins generated by chromosomal translocations between EWSR1 and the genes of transcription factors cause malignant tumors. To understand the loss of function by these translocations, the role of the intact EWS protein has to be investigated. The predominantly nuclear localization of the EWS protein via a transportin-1-mediated mechanism is dependent on the recently identified C-NLS (also known as PY-NLS). Among other residues in the C-NLS, Y656 interacts with transportin-1 and is essential for its nuclear localization. Here, we show that Y656 is phosphorylated, which seems to be a critical factor for transportin-1-mediated nuclear import. If Y656 was mutated cytosolic aggregates of the EWS protein, colocalized with transportin-1, were observed, similar to those described with mutants of the closely related FUS/TLS protein that had amino acid substitutions in the PY-NLS causing familial amyothrophic lateral sclerosis. 1. Introduction homologous to that of the EWS protein (Figure 1(a)). Y440 of Sam68 corresponds to Y656 of the EWS protein. The The EWS protein is mainly located in the nucleus, accu- residues P and R at position -1 and -4 (from Y) correspond mulated in Cajal bodies and central regions of nucleoli, completely, and both proteins have positive charges at but it is also present in cytoplasm and associated with cell position -2 (H/R) and -8 (K/R) as well as a negative membrane [1, 2]. We have identified and characterized a charge at -3 (E/D). Phosphorylation and dephosphorylation nuclear localization and retention signal at the C-terminus regulate subcellular localization of numerous proteins [7]. of the EWS protein (C-NLS) (Figure 1(a)), which assures In the present study, we investigated and found that Y656 nuclear accumulation of the protein [3]. The EWS protein in the EWS protein occurs in a phosphorylated state and if has been shown to be a ligand of transportin-1, a mediator phosphorylation is abolished, it accumulates in the cytosol in nucleocytoplasmic protein transport, among many others, colocalized with transportin-1. including related RNA-binding proteins such as FUS/TLS, hnRNPA1, hnRNPM,and Sam68[4–6]. The C-NLS of 2. Results and Discussion the EWS protein has been classified as PY-NLS, a consensus sequence recognized by transportin-1 [5]. R648, R652, P655 Expression of the EWS-YFP fusion protein resulted exclu- and Y656 have been found to be essential residues in the C- sively in nuclear accumulation, with high concentration in NLS for the nuclear transport of the EWS protein [3]. nucleoplasmic speckles and a fraction in the subnuclear Brk (breast tumour kinases) phosphorylate tyrosine central region (Figure 1(b)), thereby interacting with par- residues present in the NLS of Sam68 [7], which is highly ticular proteins such as the RNA helicases p72 and 68 [8]. 2 Sarcoma SAM68 412 GQDDWNGTR PS L K A PPA R PV K GAYR EHPY GRY 443 EWS 639 PGK MDK GE HR QE RR DRPY 656 FUS 498 R GGGDR GGFGPGK MDSRG EHR QDRR ERPY 526 hnRNP A1 316 GNY NNQSSNFGPMK GNFGGR S S GPY GGGGQY FA K PR NQGGY 357 hnRNP M 38 GE GE R PA QNE K R K E K NIK R GGNR FE PY 64 (a) YFPEWS-YFPEWS (Y656A)-YFPEWS(Y656F)-YFP EWS(Y656D)-YFP (b) Figure 1: (a) Sequence alignment of PY-NLSs. The homologous regions of C-NLS of the EWS protein, NLS of Sam68 and FUS/TLS protein and M9 NLS of hnRNP A1 and hnRNP M, classified as PY-NLS are in yellow boxes. Phosphorylated Y656 of the EWS protein and Y440 of Sam68 are indicated (in red). Positions of identical residues in SAM68 and EWS C-NLS are indicated in bold and residues with identical charges are underlined. Known positions of the FUS/TLS mutations in ALS are in bold. (b) Subcellular localization of YFP, the C-terminally tagged EWS-YFP, EWS(Y656A)-YFP, EWS(Y656F)-YFP, and EWS(Y656D)-YFP (in green). Nuclei are shown by DAPI staining (in blue). Bars, 15 μm. YFP alone is diffusively distributed between the nucleic and C-terminal RNA-binding domain of the EWS protein con- cytoplasmic compartment. Single amino acid substitutions sists of the Zinc finger (Zf) motif followed by the arginine- of the C-terminal Y656 by alanine, phenylalanine, and glycine rich box 3 (RGG3) and C-NLS (Figure 2(a)). This aspartic acid revealed a drastic redistribution of the EWS fragment of the EWS protein (aa 525–656), hereafter called protein with cytoplasmic accumulations in the perinuclear Zf, contains Y656 as the only tyrosine residue and at the same region (Figure 1(b)). The resulting cytoplasmic aggregation time is large enough to avoid diffusive nuclear import of pattern demonstrates that none of these amino acids could the GFP-Zf fusion protein. Additionally, the construct GFP- successfully substitute the tyrosine residue. Phenylalanine Zf(Y656A), having alanine as the single amino acid substi- substitution does not fulfill the function of the Y656, tution for Y656, was produced as a negative control. GFP-Zf implying the importance of the hydroxyl group of tyrosine. shows the subcellular localization pattern of the full-length Thus, a possible phosphorylation of this amino acid residue EWS protein. However, its subnuclear partition is different in nuclear import function seems likely. However, not even from that of the full-length protein, as previously described an aspartic acid, which, due to its negative charge, is often [3](Figure 2(b)). GFP-Zf(Y656A) reveals the characteristic used as phosphomimetic of phosphorylated residues, could cytoplasmic distribution as is typical for Y656 mutations restore the nuclear localization of the protein. of the full-length EWS protein (Figure 2(b)). To show that To demonstrate a possible phosphorylation of Y656, the cytoplasmic accumulations of the GFP-Zf(Y656A) are GFP-Zf protein was constructed by fusion of His-GFP with not aggregation and precipitation of an insoluble mutant a part of EWS protein (aa 525–656). This part of the protein, but the result of specific inactivation of NLS Merged DAPI Sarcoma 3 EWS (aa525 656) - DAPI Transportin GFP-Zf (Y656A) Merged 6His GFP Zf RGG3 NLS (a) GFP-ZF(Y656A )- SV40NLS GFP-ZF(Y656A) GFP-Zf Figure 3: Colocalization of transportin-1 (in magenta) and GFP- Zf(Y656A) (in green). Nuclei are shown by DAPI staining (in blue). Bars, 15 μm. Representative examples of the subcellular localization are shown (all cells expressing the constructs are showing the indicated localization). GFP-Zf and GFP-Zf(Y656A) fusion proteins containing 6His-tag at the N-terminus of GFP were expressed in eukary- otic HEK 293(T) cells, extracted, subjected to SDS-PAGE, and analyzed on Western blots by using antiphosphotyrosine antibody. Phosphorylation was detected in GFP-Zf (band at ∼40 kDa) but not in GFP-Zf(Y656A) and not in the untransfected sample (Figure 2(c)). TheWestern blotswith the same samples but with anti-C-terminal EWS antibody, that recognized both fusion proteins, demonstrate similar expression level of both proteins (Figure 2(c)). The multiple protein bands detected by anti-C-terminal EWS antibody in the lysate of untransfected HEK 293(T) cells reflect (b) different degradation fragments of endogenous EWS protein (unpublished observations). Phosphorylation of a protein at Antiphosphotyrosine Ab Anti-C-terminal EWS Ab 55 kDa was also detected. This protein was identified by mass spectrometry as the nuclear RNA- and DNA-binding protein p54nrb. Whatever reasons might be responsible for binding 55 kDa to the resin (possibly endogenous histidine residues located 40 kDa in close proximity), p54nrb can serve here as an internal standard for equal loading. As nuclear import of the EWS protein is mediated by transportin-1 [5], we have visualized the subcellu- lar localization of transportin-1. In cells expressing GFP- Zf(Y656A), transportin-1 colocalizes with the mutant GFP- Zf(Y656A) in cytoplasmic accumulations, apart from its characteristic homogeneous nucleocytoplasmic distribution (Figure 3). Conceivably, the part of transportin-1 bound to GFP-Zf(Y656A) is spatially restricted to these cytoplasmic structures and cannot fulfill its functions in nucleocytoplas- (c) mic transport due to the missing phosphorylated tyrosine. Figure 2: (a) Schematic representation of the N-terminally tagged This finding indicates that phosphorylation is not required GFP-Zf fusion protein. 6His-GFP is fused to the EWS mutant for transportin-1 binding which is in accordance with protein (aa 525–656). (b) Representative examples of the subcellular previous data showing that unphosphorylated PY-NLS of localization (all cells expressing the construct are showing the EWS or the mutated peptides containing Y656A still bind indicated localization) of GFP-Zf, GFP-Zf(Y656A), and GFP- recombinant transportin-1 [5]. Why are these mutations Zf(Y656A)-SV40NLS (in green). Nuclei are shown by DAPI staining then causing loss of nuclear import function? Possibly, (in blue). Bars, 15 μm. (c) Phosphorylation analysis of GFP-Zf and binding in the absence of a phosphorylated C-NLS cannot GFP-Zf(Y656A) using antiphosphotyrosine antibodies. GFP-Zf and induce a conformational change in transportin-1, which GFP-Zf(Y656A) fusion protein bands are detectable at ∼40 kDa. might be essential for recruiting or binding to other partners The additional phosphorylated protein band detected at ∼55 kDa in the nuclear import process and leads, thus, to cytoplasmic was identified as p54nrb. accumulations of the transportin-1-EWS complex. Recently, mutations in FUS/TLS have been shown to be function, GFP-Zf(Y656A) was fused with the canonical SV40 responsible for familial amyotrophic lateral sclerosis (ALS), NLS. GFP-Zf(Y656A)-SV40NLS shows complete nuclear and mutants of the FUS/TLS protein accumulate in the localization similar to the GFP-Zf without any detectable cytoplasm of cortical neurons and lower motor neurons in cytoplasmic aggregates (Figure 2(b)). the brain of ALS patients [9, 10]. These results indicate that Merged DAPI Untransfected GFP-Z f GFP-Z f (Y656A) Untransfected GFP-Z f GFP-Z f (Y656A) 4 Sarcoma mutant FUS/TLS has a tendency to be insoluble compared to YFP fusions were constructed as described [3]. To produce the wild type. Remarkably, mutations are found in the C-NLS the EWS(Y656D)-YFP mutant, the Y656D reverse primer (PY-NLS) of FUS/TLS (Figure 1(a)), and the cytoplasmic was used. The vectors for expression of His-GFP-Zf and accumulation is similar with that of the EWS mutant GFP- His-GFP-Zf(Y656A) fusions were constructed as follows. Zf(Y656A). Our present data indicate that these cytoplasmic For construction of pcDNA3.1(−)B-Zf and pcDNA3.1(−)B- aggregations (or, alternatively, accumulations of protein Zf(Y656A) expression vectors, Zf and Zf(Y656A) fragments complexes) of FUS/TLS are formed, as in case of the EWS were amplified by traditional PCR using EWSR1 cDNA as mutants, due to a disturbed nuclear import leading to an template and Zf-XhoI-NotI-forward and EcoRI-stop-reverse increased cytoplasmic concentrations of these proteins. In or Y656A-stop-reverse primers 5 -TCTCTCGAGCGGCCG- the normal steady state, the predominant amount of these CGCCACCATGAATCCGGGTTGTGGAAACCAGAA-3 proteins is transported into the nucleus. Knowledge about and 5 -CCGAATTCTCAGTAGGGCCGATCTCTGCGC- the mechanisms of nuclear import, including the role of TCCTG-3 ,or5 -CCGAATTCTCAGGCGGGCCGATCT- tyrosine phosphorylation for the function of C-NLS, might CTGCGCTCCTG-3,respectively. ThePCR products have an impact particularly in better understanding the were treated with restriction enzymes XhoI and EcoRI pathogenesis of ALS in order to be able to develop a strategy and subcloned into pcDNA3.1(−)B/myc-His (Invitrogen) for its treatment. In addition, it is of interest to confirm the to generate in-frame fusions. Finally, the His-GFP-Zf presence of transportin-1 in these cytoplasmic aggregations and His-GFP-Zf(Y656A) vectors were constructed by and to further test the functionality and the role of this amplification of the His-GFP fragment from EWS-Myc- restricted protein in progression of the disease. 6xHis/pEGFP-N2, a derivative of pEGFP-N2 plasmid (Clontech) using the NheI-His-forward and NotI-GFP- It is possible that different kinases are able to phospho- reverse primers 5 -TCTGCTAGCGCCACCATGGCCGTC- rylate the EWS protein individually in a cell cycle-dependent GACCATCATCATCATCATCAT-3 and 5 -TGCGTC- and a cell compartment-dependent manner, as it has been GCGGCCGCTCTTGTACAGCTCGTCCATGCCGAG-3 , found with Sam68 containing PY-NLS. Sam68 is phospho- respectively, and digestion with the appropriate restriction rylated in the nucleus by Brk (breast tumour kinases) and at enzymes, and by cloning the resulting product into the the cell membrane by Src kinases [7]. Remarkably, kinases pcDNA3.1(−)B-Zf and pcDNA3.1(−)B-Zf (Y656A) plas- of the Src-subfamily are localized in the cytoplasm and can mids. To obtain His-GFP-Zf(Y656A)-SV40NLS, His-GFP- be bound, due to N-terminal myristoylation, to the inner Zf(Y656A) vector was fused with an NLS from SV40 face of the plasma membrane [11], where the EWS protein large T antigen (PKKKRKV). The oligonucleotides 5 -AAT- has also been found [2, 12]. The members of the Brk family, TCCCAAAAAAGAAGAGAAAGGTCAGG-3 and 5 -AGC- related to the Src family, lack a myristoylation site, and TTCTGACCTTTCTCTTCTTTTTTGGG-3 were annealed, cytoplasmic and nuclear localization is more typical for these and the resulting fragment was digested with restriction kinases [13, 14]. Tyrosine kinases, known to interact with enzymes EcoRI-HindIII and was inserted into His-GFP- the EWS protein, are Pyk2 and Bruton tyrosine kinase, as Zf(Y656A). well as Lck, a member of Src kinases [15, 16]. Src kinases and Pyk2 recognize a similar tyrosine phosphorylation motif, which suggests that the latter might phosphorylate Y656 3.2. Cell Culture and Transfections. Human embryonic kid- of the EWS protein. Conceivably, tyrosine phosphorylation ney (HEK) 293 (T) cells, kindly provided by Professor P. might regulate, apart from its role in nucleocytoplasmic Sonderegger (Department of Biochemistry, University of transport, interactions with other proteins or with RNA, as Zurich, Switzerland), were grown in Dulbecco’s Modified was observed with the related Sam68 and QKI [7, 13, 17]. Eagle’s Medium (DMEM) (Sigma) supplemented with 10% Although phosphorylation of Y656 in the EWS protein fetal bovine serum (FBS) (Life technologies) and 1% (w/v) seems to be essential, it might not be the exclusive reg- each of penicillin and streptomycin (Life technologies) in ulating factor for nuclear localization, since other known a humidified 10% CO atmosphere at 37 C. For visual- or predicted mechanisms can be recruited to cooperatively ization analysis, HEK 293 T cells were cultured on glass or sequentially control nucleocytoplasmic distribution of a cover slips to 40% confluency and transiently transfected particular protein. SUMO-ylation, another reversible post- with mammalian expression constructs using the method translational modification of Sam68 [18] and of hnRNP M, of calcium phosphate precipitation. For protein visualiza- has been described to play a role in nucleocytoplasmic shut- tion, the cells were fixed 24 h after transfection with 4% tling of mRNA-binding protein complexes [19]. Remarkably, paraformaldehyde (PFA) for 5 min and stained with 4 ,6- the SUMO-ylation motif GKMD is predicted with high diamidino-2-phenylindol (DAPI) (Roche). Cover slips were probability also in the C-NLS region of the EWS protein mounted using Vectashiled medium (Vector) onto glass (http://us.expasy.org/, SUMOplot) and is a potential subject slides, and the cells were analyzed by fluorescence and confo- forfurtherinvestigation. cal microscopy. 3.3. Antibodies, Protein Purification, and Western Blotting. 3. Materials and Methods Primary rabbit anti-C-terminal EWS antibody SE 680, kindly 3.1. Expression Constructs. The eukaryotic vectors for expres- provided by Dr. O. Delattre (Institut Curie, Pathologie sion of the EWS-YFP, EWS(Y656A)-YFP, and EWS(Y656F)- Moleculair ´ e des Cancers, Paris Cedex), was used with Sarcoma 5 1 : 5000 dilution. Primary mouse anti-transportin-1 anti- [3] R. P. Zakaryan and H. Gehring, “Identification and character- ization of the nuclear localization/retention signal in the EWS body (TNPO1, ab10303, Abcam) was kindly provided Proto-oncoprotein,” Journal of Molecular Biology, vol. 363, no. by Professor I. Stamenkovic (Department of Experimen- 1, pp. 27–38, 2006. tal Pathology, Institute of Pathology, CHUV, Lausanne, [4] S. Guttinger ¨ , P. Muhlh ¨ ausser ¨ , R. Koller-Eichhorn, J. Bren- Switzerland). Primary rabbit antiphosphotyrosine antibody necke, and U. Kutay, “Transporting functions as importin and (Zymed) was used with 1 : 2000 dilution. The results were mediates nuclear import of HuR,” Proceedings of the National confirmed by using primary mouse antiphosphotyrosine Academy of Sciences of the United States of America, vol. 101, antibody (P-Tyr-100, Cell Signaling) with 1 : 2000 dilution no. 9, pp. 2918–2923, 2004. (data not shown). The secondary goat antirabbit and goat [5] B.J.Lee,A.E.Cansizoglu, K. E. Suel, ¨ T. H. Louis, Z. Zhang, antimouse antibodies, respectively, coupled to horseradish and Y. M. Chook, “Rules for nuclear localization sequence peroxidase (Sigma) were used with 1 : 2000 dilution. His- recognition by karyopherinβ2,” Cell, vol. 126, no. 3, pp. 543– GFP-Zf and His-GFP-Zf(Y656A) protein purification by 558, 2006. His-SELECT Nickel affinty gel (Sigma) was performed [6] A. E. Cansizoglu, B. J. Lee, Z. I. C. Zhang, B. M. A. as described [2]. In all the purification buffers, sodium Fontoura, and Y. M. Chook, “Structure-based design of a orthovanadate was added as protein phosphatase inhibitor. pathway-specific nuclear import inhibitor,” Nature Structural Western blotting was performed as described [12]with and Molecular Biology, vol. 14, no. 5, pp. 452–454, 2007. some modifications. In case of phosphotyrosine detection, [7] K. E. Lukong, D. Larocque, A. L. Tyner, and S. Richard, 3% BSA instead of nonfat milk powder was used as blocking “Tyrosine phosphorylation of Sam68 by breast tumor kinase agent and in antibody solutions. regulates intranuclear localization and cell cycle progression,” The Journal of Biological Chemistry, vol. 280, no. 46, pp. 38639–38647, 2005. 3.4. Confocal Microscopy. Laser-scanning confocal fluores- [8] S. Pahlich, L. Quero, B. Roschitzki, R. P. Leemann-Zakaryan, cence microscopy was performed using a Leica SP2 AOBS and H. Gehring, “Analysis of Ewing sarcoma (EWS)-binding UV CLSM microscope and HCX PL APO lbd.BL 63.0x proteins: interaction with hnRNP M, U, and RNA-helicases NA 1.40 OIL UV objective. Images were acquired using p68/72 within protein-RNA complexes,” Journal of Proteome excitation wavelengths of 405 nm and 514 nm and the Research, vol. 8, no. 10, pp. 4455–4465, 2009. emission wavelengths of 470 nm and 528 nm for DAPI [9] T. J. Kwiatkowski, D. A. Bosco, A. L. LeClerc et al., “Mutations and YFP, respectively. Images were captured digitally using in the FUS/TLS gene on chromosome 16 cause familial Leica software and processed using Adobe Photoshop 8.0. amyotrophic lateral sclerosis,” Science, vol. 323, no. 5918, pp. The stacks of images were imported into Imaris (Bitplane) 1205–1208, 2009. software for the 3D rendering of the images. [10] C. Vance, B. Rogelj, T. Hortobag ´ yi et al., “Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6,” Science, vol. 323, no. 5918, pp. 1208–1211, Acknowledgments [11] M. T. Brown and J. A. Cooper, “Regulation, substrates and Current work was supported by Swiss National Science functions of src,” Biochimica et Biophysica Acta, vol. 1287, no. Foundation Grant no. 3100-066954. The authors are grateful 2-3, pp. 121–149, 1996. to Dr. O. Georgiev (Institute of Molecular Biology, University [12] L. L. Belyanskaya, P. M. Gehrig, and H. Gehring, “Exposure of Zurich, Switzerland) for providing facilities, reagents, and on cell surface and extensive arginine methylation of Ewing assistance. They would like to thank Professor I. Stamenkovic sarcoma (EWS) protein,” The Journal of Biological Chemistry, (Department of Experimental Pathology, Institute of Pathol- vol. 276, no. 22, pp. 18681–18687, 2001. ogy, CHUV, Lausanne, Switzerland) for providing facilities [13] J. J. Derry, S. Richard, H. V. Carvajal et al., “Sik (BRK) and the anti-transportin-1 antibody. They are grateful to phosphorylates sam68 in the nucleus and negatively regulates Dr. O. Delattre (Institut Curie, Pathologie Moleculair ´ e des Its RNA binding ability,” Molecular and Cellular Biology, vol. Cancers, Paris Cedex) for the human EWSR1 cDNA and the 20, no. 16, pp. 6114–6126, 2000. polyclonal antibody SE 680 against the C-terminus of the [14] M. S. Serfas and A. L. Tyner, “Brk, Srm, Frk, and Src42A form EWS protein. a distinct family of intracellular Src-Like tyrosine Kinases,” Oncology Research, vol. 13, no. 6–10, pp. 409–419, 2002. [15] J. S. Felsch,W.S.Lane, andE.G.Peralta,“Tyrosine kinase References Pyk2 mediates G-protein-coupled receptor regulation of the Ewing sarcoma RNA-binding protein EWS,” Current Biology, [1] R. P. Leemann-Zakaryan, S. Pahlich, M. J. Sedda, L. Quero, vol. 9, no. 9, pp. 485–488, 1999. D. Grossenbacher, and H. Gehring, “Dynamic subcellular [16] R. Guinamard, M. Fougereau, and P. Seckinger, “The SH3 localization of the Ewing sarcoma proto-oncoprotein and its association with and stabilization of microtubules,” Journal of domain of Bruton’s tyrosine kinase interacts with Vav, Sam68 and EWS,” Scandinavian Journal of Immunology, vol. 45, no. 6, Molecular Biology, vol. 386, no. 1, pp. 1–13, 2009. pp. 587–595, 1997. [2] S. Pahlich, R. P. Zakaryan, and H. Gehring, “Identification of proteins interacting with protein arginine methyltransferase [17] Y. Zhang, Z. Lu,L.I.Ku, Y. Chen,H.Wang, andY.Feng, 8: the Ewing sarcoma (EWS) protein binds independent of “Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism,” The EMBO Journal, its methylation state,” Proteins, vol. 72, no. 4, pp. 1125–1137, 2008. vol. 22, no. 8, pp. 1801–1810, 2003. 6 Sarcoma [18] I. Babic, E. Cherry, and D. J. Fujita, “SUMO modification of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis,” Oncogene, vol. 25, no. 36, pp. 4955–4964, 2006. [19] M. T. Vassileva and M. J. Matunis, “SUMO modification of heterogeneous nuclear ribonucleoproteins,” Molecular and Cellular Biology, vol. 24, no. 9, pp. 3623–3632, 2004. MEDIATORS of INFLAMMATION The Scientific Gastroenterology Journal of World Journal Research and Practice Diabetes Research Disease Markers Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Journal of Immunology Research Endocrinology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com BioMed PPAR Research Research International Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Obesity Evidence-Based Journal of Journal of Stem Cells Complementary and Ophthalmology International Alternative Medicine Oncology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Parkinson’s Disease Computational and Behavioural Mathematical Methods AIDS Oxidative Medicine and in Medicine Research and Treatment Cellular Longevity Neurology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Sarcoma Hindawi Publishing Corporation

Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of the EWS Protein

Loading next page...
 
/lp/hindawi-publishing-corporation/tyrosine-phosphorylation-in-the-c-terminal-nuclear-localization-and-zPlNoffs0g

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Hindawi Publishing Corporation
Copyright
Copyright © 2011 Ruzanna P. Leemann-Zakaryan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ISSN
1357-714X
eISSN
1369-1643
DOI
10.1155/2011/218483
Publisher site
See Article on Publisher Site

Abstract

Hindawi Publishing Corporation Sarcoma Volume 2011, Article ID 218483, 6 pages doi:10.1155/2011/218483 Research Article Tyrosine Phosphorylation in the C-Terminal Nuclear Localization and Retention Signal (C-NLS) of the EWS Protein 1, 2 1 1 Ruzanna P. Leemann-Zakaryan, Steffen Pahlich, Doris Grossenbacher, and Heinz Gehring Department of Biochemistry, University of Zurich, Winterthurerstraße 190, 8057 Zurich, Switzerland Division of Experimental Pathology, Institute of Pathology, CHUV, Faculty of Biology and Medicine, University of Lausanne, Rue du Bugnon 25, 1005 Lausanne, Switzerland Correspondence should be addressed to Heinz Gehring, gehring@bioc.uzh.ch Received 2 September 2010; Revised 26 January 2011; Accepted 1 March 2011 Academic Editor: H. Kovar Copyright © 2011 Ruzanna P. Leemann-Zakaryan et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Ewing sarcoma (EWS) proto-oncoprotein, an RNA-binding protein, is involved in DNA recombination and repair, gene expression, RNA processing and transport, as well as cell signalling. Chimeric EWS oncoproteins generated by chromosomal translocations between EWSR1 and the genes of transcription factors cause malignant tumors. To understand the loss of function by these translocations, the role of the intact EWS protein has to be investigated. The predominantly nuclear localization of the EWS protein via a transportin-1-mediated mechanism is dependent on the recently identified C-NLS (also known as PY-NLS). Among other residues in the C-NLS, Y656 interacts with transportin-1 and is essential for its nuclear localization. Here, we show that Y656 is phosphorylated, which seems to be a critical factor for transportin-1-mediated nuclear import. If Y656 was mutated cytosolic aggregates of the EWS protein, colocalized with transportin-1, were observed, similar to those described with mutants of the closely related FUS/TLS protein that had amino acid substitutions in the PY-NLS causing familial amyothrophic lateral sclerosis. 1. Introduction homologous to that of the EWS protein (Figure 1(a)). Y440 of Sam68 corresponds to Y656 of the EWS protein. The The EWS protein is mainly located in the nucleus, accu- residues P and R at position -1 and -4 (from Y) correspond mulated in Cajal bodies and central regions of nucleoli, completely, and both proteins have positive charges at but it is also present in cytoplasm and associated with cell position -2 (H/R) and -8 (K/R) as well as a negative membrane [1, 2]. We have identified and characterized a charge at -3 (E/D). Phosphorylation and dephosphorylation nuclear localization and retention signal at the C-terminus regulate subcellular localization of numerous proteins [7]. of the EWS protein (C-NLS) (Figure 1(a)), which assures In the present study, we investigated and found that Y656 nuclear accumulation of the protein [3]. The EWS protein in the EWS protein occurs in a phosphorylated state and if has been shown to be a ligand of transportin-1, a mediator phosphorylation is abolished, it accumulates in the cytosol in nucleocytoplasmic protein transport, among many others, colocalized with transportin-1. including related RNA-binding proteins such as FUS/TLS, hnRNPA1, hnRNPM,and Sam68[4–6]. The C-NLS of 2. Results and Discussion the EWS protein has been classified as PY-NLS, a consensus sequence recognized by transportin-1 [5]. R648, R652, P655 Expression of the EWS-YFP fusion protein resulted exclu- and Y656 have been found to be essential residues in the C- sively in nuclear accumulation, with high concentration in NLS for the nuclear transport of the EWS protein [3]. nucleoplasmic speckles and a fraction in the subnuclear Brk (breast tumour kinases) phosphorylate tyrosine central region (Figure 1(b)), thereby interacting with par- residues present in the NLS of Sam68 [7], which is highly ticular proteins such as the RNA helicases p72 and 68 [8]. 2 Sarcoma SAM68 412 GQDDWNGTR PS L K A PPA R PV K GAYR EHPY GRY 443 EWS 639 PGK MDK GE HR QE RR DRPY 656 FUS 498 R GGGDR GGFGPGK MDSRG EHR QDRR ERPY 526 hnRNP A1 316 GNY NNQSSNFGPMK GNFGGR S S GPY GGGGQY FA K PR NQGGY 357 hnRNP M 38 GE GE R PA QNE K R K E K NIK R GGNR FE PY 64 (a) YFPEWS-YFPEWS (Y656A)-YFPEWS(Y656F)-YFP EWS(Y656D)-YFP (b) Figure 1: (a) Sequence alignment of PY-NLSs. The homologous regions of C-NLS of the EWS protein, NLS of Sam68 and FUS/TLS protein and M9 NLS of hnRNP A1 and hnRNP M, classified as PY-NLS are in yellow boxes. Phosphorylated Y656 of the EWS protein and Y440 of Sam68 are indicated (in red). Positions of identical residues in SAM68 and EWS C-NLS are indicated in bold and residues with identical charges are underlined. Known positions of the FUS/TLS mutations in ALS are in bold. (b) Subcellular localization of YFP, the C-terminally tagged EWS-YFP, EWS(Y656A)-YFP, EWS(Y656F)-YFP, and EWS(Y656D)-YFP (in green). Nuclei are shown by DAPI staining (in blue). Bars, 15 μm. YFP alone is diffusively distributed between the nucleic and C-terminal RNA-binding domain of the EWS protein con- cytoplasmic compartment. Single amino acid substitutions sists of the Zinc finger (Zf) motif followed by the arginine- of the C-terminal Y656 by alanine, phenylalanine, and glycine rich box 3 (RGG3) and C-NLS (Figure 2(a)). This aspartic acid revealed a drastic redistribution of the EWS fragment of the EWS protein (aa 525–656), hereafter called protein with cytoplasmic accumulations in the perinuclear Zf, contains Y656 as the only tyrosine residue and at the same region (Figure 1(b)). The resulting cytoplasmic aggregation time is large enough to avoid diffusive nuclear import of pattern demonstrates that none of these amino acids could the GFP-Zf fusion protein. Additionally, the construct GFP- successfully substitute the tyrosine residue. Phenylalanine Zf(Y656A), having alanine as the single amino acid substi- substitution does not fulfill the function of the Y656, tution for Y656, was produced as a negative control. GFP-Zf implying the importance of the hydroxyl group of tyrosine. shows the subcellular localization pattern of the full-length Thus, a possible phosphorylation of this amino acid residue EWS protein. However, its subnuclear partition is different in nuclear import function seems likely. However, not even from that of the full-length protein, as previously described an aspartic acid, which, due to its negative charge, is often [3](Figure 2(b)). GFP-Zf(Y656A) reveals the characteristic used as phosphomimetic of phosphorylated residues, could cytoplasmic distribution as is typical for Y656 mutations restore the nuclear localization of the protein. of the full-length EWS protein (Figure 2(b)). To show that To demonstrate a possible phosphorylation of Y656, the cytoplasmic accumulations of the GFP-Zf(Y656A) are GFP-Zf protein was constructed by fusion of His-GFP with not aggregation and precipitation of an insoluble mutant a part of EWS protein (aa 525–656). This part of the protein, but the result of specific inactivation of NLS Merged DAPI Sarcoma 3 EWS (aa525 656) - DAPI Transportin GFP-Zf (Y656A) Merged 6His GFP Zf RGG3 NLS (a) GFP-ZF(Y656A )- SV40NLS GFP-ZF(Y656A) GFP-Zf Figure 3: Colocalization of transportin-1 (in magenta) and GFP- Zf(Y656A) (in green). Nuclei are shown by DAPI staining (in blue). Bars, 15 μm. Representative examples of the subcellular localization are shown (all cells expressing the constructs are showing the indicated localization). GFP-Zf and GFP-Zf(Y656A) fusion proteins containing 6His-tag at the N-terminus of GFP were expressed in eukary- otic HEK 293(T) cells, extracted, subjected to SDS-PAGE, and analyzed on Western blots by using antiphosphotyrosine antibody. Phosphorylation was detected in GFP-Zf (band at ∼40 kDa) but not in GFP-Zf(Y656A) and not in the untransfected sample (Figure 2(c)). TheWestern blotswith the same samples but with anti-C-terminal EWS antibody, that recognized both fusion proteins, demonstrate similar expression level of both proteins (Figure 2(c)). The multiple protein bands detected by anti-C-terminal EWS antibody in the lysate of untransfected HEK 293(T) cells reflect (b) different degradation fragments of endogenous EWS protein (unpublished observations). Phosphorylation of a protein at Antiphosphotyrosine Ab Anti-C-terminal EWS Ab 55 kDa was also detected. This protein was identified by mass spectrometry as the nuclear RNA- and DNA-binding protein p54nrb. Whatever reasons might be responsible for binding 55 kDa to the resin (possibly endogenous histidine residues located 40 kDa in close proximity), p54nrb can serve here as an internal standard for equal loading. As nuclear import of the EWS protein is mediated by transportin-1 [5], we have visualized the subcellu- lar localization of transportin-1. In cells expressing GFP- Zf(Y656A), transportin-1 colocalizes with the mutant GFP- Zf(Y656A) in cytoplasmic accumulations, apart from its characteristic homogeneous nucleocytoplasmic distribution (Figure 3). Conceivably, the part of transportin-1 bound to GFP-Zf(Y656A) is spatially restricted to these cytoplasmic structures and cannot fulfill its functions in nucleocytoplas- (c) mic transport due to the missing phosphorylated tyrosine. Figure 2: (a) Schematic representation of the N-terminally tagged This finding indicates that phosphorylation is not required GFP-Zf fusion protein. 6His-GFP is fused to the EWS mutant for transportin-1 binding which is in accordance with protein (aa 525–656). (b) Representative examples of the subcellular previous data showing that unphosphorylated PY-NLS of localization (all cells expressing the construct are showing the EWS or the mutated peptides containing Y656A still bind indicated localization) of GFP-Zf, GFP-Zf(Y656A), and GFP- recombinant transportin-1 [5]. Why are these mutations Zf(Y656A)-SV40NLS (in green). Nuclei are shown by DAPI staining then causing loss of nuclear import function? Possibly, (in blue). Bars, 15 μm. (c) Phosphorylation analysis of GFP-Zf and binding in the absence of a phosphorylated C-NLS cannot GFP-Zf(Y656A) using antiphosphotyrosine antibodies. GFP-Zf and induce a conformational change in transportin-1, which GFP-Zf(Y656A) fusion protein bands are detectable at ∼40 kDa. might be essential for recruiting or binding to other partners The additional phosphorylated protein band detected at ∼55 kDa in the nuclear import process and leads, thus, to cytoplasmic was identified as p54nrb. accumulations of the transportin-1-EWS complex. Recently, mutations in FUS/TLS have been shown to be function, GFP-Zf(Y656A) was fused with the canonical SV40 responsible for familial amyotrophic lateral sclerosis (ALS), NLS. GFP-Zf(Y656A)-SV40NLS shows complete nuclear and mutants of the FUS/TLS protein accumulate in the localization similar to the GFP-Zf without any detectable cytoplasm of cortical neurons and lower motor neurons in cytoplasmic aggregates (Figure 2(b)). the brain of ALS patients [9, 10]. These results indicate that Merged DAPI Untransfected GFP-Z f GFP-Z f (Y656A) Untransfected GFP-Z f GFP-Z f (Y656A) 4 Sarcoma mutant FUS/TLS has a tendency to be insoluble compared to YFP fusions were constructed as described [3]. To produce the wild type. Remarkably, mutations are found in the C-NLS the EWS(Y656D)-YFP mutant, the Y656D reverse primer (PY-NLS) of FUS/TLS (Figure 1(a)), and the cytoplasmic was used. The vectors for expression of His-GFP-Zf and accumulation is similar with that of the EWS mutant GFP- His-GFP-Zf(Y656A) fusions were constructed as follows. Zf(Y656A). Our present data indicate that these cytoplasmic For construction of pcDNA3.1(−)B-Zf and pcDNA3.1(−)B- aggregations (or, alternatively, accumulations of protein Zf(Y656A) expression vectors, Zf and Zf(Y656A) fragments complexes) of FUS/TLS are formed, as in case of the EWS were amplified by traditional PCR using EWSR1 cDNA as mutants, due to a disturbed nuclear import leading to an template and Zf-XhoI-NotI-forward and EcoRI-stop-reverse increased cytoplasmic concentrations of these proteins. In or Y656A-stop-reverse primers 5 -TCTCTCGAGCGGCCG- the normal steady state, the predominant amount of these CGCCACCATGAATCCGGGTTGTGGAAACCAGAA-3 proteins is transported into the nucleus. Knowledge about and 5 -CCGAATTCTCAGTAGGGCCGATCTCTGCGC- the mechanisms of nuclear import, including the role of TCCTG-3 ,or5 -CCGAATTCTCAGGCGGGCCGATCT- tyrosine phosphorylation for the function of C-NLS, might CTGCGCTCCTG-3,respectively. ThePCR products have an impact particularly in better understanding the were treated with restriction enzymes XhoI and EcoRI pathogenesis of ALS in order to be able to develop a strategy and subcloned into pcDNA3.1(−)B/myc-His (Invitrogen) for its treatment. In addition, it is of interest to confirm the to generate in-frame fusions. Finally, the His-GFP-Zf presence of transportin-1 in these cytoplasmic aggregations and His-GFP-Zf(Y656A) vectors were constructed by and to further test the functionality and the role of this amplification of the His-GFP fragment from EWS-Myc- restricted protein in progression of the disease. 6xHis/pEGFP-N2, a derivative of pEGFP-N2 plasmid (Clontech) using the NheI-His-forward and NotI-GFP- It is possible that different kinases are able to phospho- reverse primers 5 -TCTGCTAGCGCCACCATGGCCGTC- rylate the EWS protein individually in a cell cycle-dependent GACCATCATCATCATCATCAT-3 and 5 -TGCGTC- and a cell compartment-dependent manner, as it has been GCGGCCGCTCTTGTACAGCTCGTCCATGCCGAG-3 , found with Sam68 containing PY-NLS. Sam68 is phospho- respectively, and digestion with the appropriate restriction rylated in the nucleus by Brk (breast tumour kinases) and at enzymes, and by cloning the resulting product into the the cell membrane by Src kinases [7]. Remarkably, kinases pcDNA3.1(−)B-Zf and pcDNA3.1(−)B-Zf (Y656A) plas- of the Src-subfamily are localized in the cytoplasm and can mids. To obtain His-GFP-Zf(Y656A)-SV40NLS, His-GFP- be bound, due to N-terminal myristoylation, to the inner Zf(Y656A) vector was fused with an NLS from SV40 face of the plasma membrane [11], where the EWS protein large T antigen (PKKKRKV). The oligonucleotides 5 -AAT- has also been found [2, 12]. The members of the Brk family, TCCCAAAAAAGAAGAGAAAGGTCAGG-3 and 5 -AGC- related to the Src family, lack a myristoylation site, and TTCTGACCTTTCTCTTCTTTTTTGGG-3 were annealed, cytoplasmic and nuclear localization is more typical for these and the resulting fragment was digested with restriction kinases [13, 14]. Tyrosine kinases, known to interact with enzymes EcoRI-HindIII and was inserted into His-GFP- the EWS protein, are Pyk2 and Bruton tyrosine kinase, as Zf(Y656A). well as Lck, a member of Src kinases [15, 16]. Src kinases and Pyk2 recognize a similar tyrosine phosphorylation motif, which suggests that the latter might phosphorylate Y656 3.2. Cell Culture and Transfections. Human embryonic kid- of the EWS protein. Conceivably, tyrosine phosphorylation ney (HEK) 293 (T) cells, kindly provided by Professor P. might regulate, apart from its role in nucleocytoplasmic Sonderegger (Department of Biochemistry, University of transport, interactions with other proteins or with RNA, as Zurich, Switzerland), were grown in Dulbecco’s Modified was observed with the related Sam68 and QKI [7, 13, 17]. Eagle’s Medium (DMEM) (Sigma) supplemented with 10% Although phosphorylation of Y656 in the EWS protein fetal bovine serum (FBS) (Life technologies) and 1% (w/v) seems to be essential, it might not be the exclusive reg- each of penicillin and streptomycin (Life technologies) in ulating factor for nuclear localization, since other known a humidified 10% CO atmosphere at 37 C. For visual- or predicted mechanisms can be recruited to cooperatively ization analysis, HEK 293 T cells were cultured on glass or sequentially control nucleocytoplasmic distribution of a cover slips to 40% confluency and transiently transfected particular protein. SUMO-ylation, another reversible post- with mammalian expression constructs using the method translational modification of Sam68 [18] and of hnRNP M, of calcium phosphate precipitation. For protein visualiza- has been described to play a role in nucleocytoplasmic shut- tion, the cells were fixed 24 h after transfection with 4% tling of mRNA-binding protein complexes [19]. Remarkably, paraformaldehyde (PFA) for 5 min and stained with 4 ,6- the SUMO-ylation motif GKMD is predicted with high diamidino-2-phenylindol (DAPI) (Roche). Cover slips were probability also in the C-NLS region of the EWS protein mounted using Vectashiled medium (Vector) onto glass (http://us.expasy.org/, SUMOplot) and is a potential subject slides, and the cells were analyzed by fluorescence and confo- forfurtherinvestigation. cal microscopy. 3.3. Antibodies, Protein Purification, and Western Blotting. 3. Materials and Methods Primary rabbit anti-C-terminal EWS antibody SE 680, kindly 3.1. Expression Constructs. The eukaryotic vectors for expres- provided by Dr. O. Delattre (Institut Curie, Pathologie sion of the EWS-YFP, EWS(Y656A)-YFP, and EWS(Y656F)- Moleculair ´ e des Cancers, Paris Cedex), was used with Sarcoma 5 1 : 5000 dilution. Primary mouse anti-transportin-1 anti- [3] R. P. Zakaryan and H. Gehring, “Identification and character- ization of the nuclear localization/retention signal in the EWS body (TNPO1, ab10303, Abcam) was kindly provided Proto-oncoprotein,” Journal of Molecular Biology, vol. 363, no. by Professor I. Stamenkovic (Department of Experimen- 1, pp. 27–38, 2006. tal Pathology, Institute of Pathology, CHUV, Lausanne, [4] S. Guttinger ¨ , P. Muhlh ¨ ausser ¨ , R. Koller-Eichhorn, J. Bren- Switzerland). Primary rabbit antiphosphotyrosine antibody necke, and U. Kutay, “Transporting functions as importin and (Zymed) was used with 1 : 2000 dilution. The results were mediates nuclear import of HuR,” Proceedings of the National confirmed by using primary mouse antiphosphotyrosine Academy of Sciences of the United States of America, vol. 101, antibody (P-Tyr-100, Cell Signaling) with 1 : 2000 dilution no. 9, pp. 2918–2923, 2004. (data not shown). The secondary goat antirabbit and goat [5] B.J.Lee,A.E.Cansizoglu, K. E. Suel, ¨ T. H. Louis, Z. Zhang, antimouse antibodies, respectively, coupled to horseradish and Y. M. Chook, “Rules for nuclear localization sequence peroxidase (Sigma) were used with 1 : 2000 dilution. His- recognition by karyopherinβ2,” Cell, vol. 126, no. 3, pp. 543– GFP-Zf and His-GFP-Zf(Y656A) protein purification by 558, 2006. His-SELECT Nickel affinty gel (Sigma) was performed [6] A. E. Cansizoglu, B. J. Lee, Z. I. C. Zhang, B. M. A. as described [2]. In all the purification buffers, sodium Fontoura, and Y. M. Chook, “Structure-based design of a orthovanadate was added as protein phosphatase inhibitor. pathway-specific nuclear import inhibitor,” Nature Structural Western blotting was performed as described [12]with and Molecular Biology, vol. 14, no. 5, pp. 452–454, 2007. some modifications. In case of phosphotyrosine detection, [7] K. E. Lukong, D. Larocque, A. L. Tyner, and S. Richard, 3% BSA instead of nonfat milk powder was used as blocking “Tyrosine phosphorylation of Sam68 by breast tumor kinase agent and in antibody solutions. regulates intranuclear localization and cell cycle progression,” The Journal of Biological Chemistry, vol. 280, no. 46, pp. 38639–38647, 2005. 3.4. Confocal Microscopy. Laser-scanning confocal fluores- [8] S. Pahlich, L. Quero, B. Roschitzki, R. P. Leemann-Zakaryan, cence microscopy was performed using a Leica SP2 AOBS and H. Gehring, “Analysis of Ewing sarcoma (EWS)-binding UV CLSM microscope and HCX PL APO lbd.BL 63.0x proteins: interaction with hnRNP M, U, and RNA-helicases NA 1.40 OIL UV objective. Images were acquired using p68/72 within protein-RNA complexes,” Journal of Proteome excitation wavelengths of 405 nm and 514 nm and the Research, vol. 8, no. 10, pp. 4455–4465, 2009. emission wavelengths of 470 nm and 528 nm for DAPI [9] T. J. Kwiatkowski, D. A. Bosco, A. L. LeClerc et al., “Mutations and YFP, respectively. Images were captured digitally using in the FUS/TLS gene on chromosome 16 cause familial Leica software and processed using Adobe Photoshop 8.0. amyotrophic lateral sclerosis,” Science, vol. 323, no. 5918, pp. The stacks of images were imported into Imaris (Bitplane) 1205–1208, 2009. software for the 3D rendering of the images. [10] C. Vance, B. Rogelj, T. Hortobag ´ yi et al., “Mutations in FUS, an RNA processing protein, cause familial amyotrophic lateral sclerosis type 6,” Science, vol. 323, no. 5918, pp. 1208–1211, Acknowledgments [11] M. T. Brown and J. A. Cooper, “Regulation, substrates and Current work was supported by Swiss National Science functions of src,” Biochimica et Biophysica Acta, vol. 1287, no. Foundation Grant no. 3100-066954. The authors are grateful 2-3, pp. 121–149, 1996. to Dr. O. Georgiev (Institute of Molecular Biology, University [12] L. L. Belyanskaya, P. M. Gehrig, and H. Gehring, “Exposure of Zurich, Switzerland) for providing facilities, reagents, and on cell surface and extensive arginine methylation of Ewing assistance. They would like to thank Professor I. Stamenkovic sarcoma (EWS) protein,” The Journal of Biological Chemistry, (Department of Experimental Pathology, Institute of Pathol- vol. 276, no. 22, pp. 18681–18687, 2001. ogy, CHUV, Lausanne, Switzerland) for providing facilities [13] J. J. Derry, S. Richard, H. V. Carvajal et al., “Sik (BRK) and the anti-transportin-1 antibody. They are grateful to phosphorylates sam68 in the nucleus and negatively regulates Dr. O. Delattre (Institut Curie, Pathologie Moleculair ´ e des Its RNA binding ability,” Molecular and Cellular Biology, vol. Cancers, Paris Cedex) for the human EWSR1 cDNA and the 20, no. 16, pp. 6114–6126, 2000. polyclonal antibody SE 680 against the C-terminus of the [14] M. S. Serfas and A. L. Tyner, “Brk, Srm, Frk, and Src42A form EWS protein. a distinct family of intracellular Src-Like tyrosine Kinases,” Oncology Research, vol. 13, no. 6–10, pp. 409–419, 2002. [15] J. S. Felsch,W.S.Lane, andE.G.Peralta,“Tyrosine kinase References Pyk2 mediates G-protein-coupled receptor regulation of the Ewing sarcoma RNA-binding protein EWS,” Current Biology, [1] R. P. Leemann-Zakaryan, S. Pahlich, M. J. Sedda, L. Quero, vol. 9, no. 9, pp. 485–488, 1999. D. Grossenbacher, and H. Gehring, “Dynamic subcellular [16] R. Guinamard, M. Fougereau, and P. Seckinger, “The SH3 localization of the Ewing sarcoma proto-oncoprotein and its association with and stabilization of microtubules,” Journal of domain of Bruton’s tyrosine kinase interacts with Vav, Sam68 and EWS,” Scandinavian Journal of Immunology, vol. 45, no. 6, Molecular Biology, vol. 386, no. 1, pp. 1–13, 2009. pp. 587–595, 1997. [2] S. Pahlich, R. P. Zakaryan, and H. Gehring, “Identification of proteins interacting with protein arginine methyltransferase [17] Y. Zhang, Z. Lu,L.I.Ku, Y. Chen,H.Wang, andY.Feng, 8: the Ewing sarcoma (EWS) protein binds independent of “Tyrosine phosphorylation of QKI mediates developmental signals to regulate mRNA metabolism,” The EMBO Journal, its methylation state,” Proteins, vol. 72, no. 4, pp. 1125–1137, 2008. vol. 22, no. 8, pp. 1801–1810, 2003. 6 Sarcoma [18] I. Babic, E. Cherry, and D. J. Fujita, “SUMO modification of Sam68 enhances its ability to repress cyclin D1 expression and inhibits its ability to induce apoptosis,” Oncogene, vol. 25, no. 36, pp. 4955–4964, 2006. [19] M. T. Vassileva and M. J. Matunis, “SUMO modification of heterogeneous nuclear ribonucleoproteins,” Molecular and Cellular Biology, vol. 24, no. 9, pp. 3623–3632, 2004. MEDIATORS of INFLAMMATION The Scientific Gastroenterology Journal of World Journal Research and Practice Diabetes Research Disease Markers Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 International Journal of Journal of Immunology Research Endocrinology Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Submit your manuscripts at http://www.hindawi.com BioMed PPAR Research Research International Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Journal of Obesity Evidence-Based Journal of Journal of Stem Cells Complementary and Ophthalmology International Alternative Medicine Oncology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 Parkinson’s Disease Computational and Behavioural Mathematical Methods AIDS Oxidative Medicine and in Medicine Research and Treatment Cellular Longevity Neurology Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation Hindawi Publishing Corporation http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014 http://www.hindawi.com Volume 2014

Journal

SarcomaHindawi Publishing Corporation

Published: May 2, 2011

References