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

Although much is known about the oncogenic functions of chimeric Ewing sarcoma (EWS) fusion proteins that result from chromosomal translocations, the cellular role of the normal EWS protein is not well characterized. We have previously identified a WD domain–containing protein, serine-threonine kinase receptor-associated protein (STRAP), which inhibits transforming growth factor β (TGF-β) signaling through interaction with receptors and Smad7 and promotes growth and enhances tumorigenicity. Here, we report the interaction between STRAP and EWS using matrix-assisted laser desorption/ionization, time-of-flight and tandem mass spectrometry. Although STRAP is localized in both cytoplasm and nucleus, nuclear STRAP colocalizes and associates specifically with EWS in the nucleus through its NH₂ and COOH termini. We have found that normal EWS protein is up-regulated in human cancers, which correlates with the up-regulation of STRAP in 71% of colorectal cancers and 54% of lung cancers, suggesting a cooperative role of these two proteins in human cancers. TGF-β has no effect on STRAP and EWS interaction. However, EWS, like STRAP, attenuates TGF-β-dependent transcription. STRAP inhibits EWS-dependent p300-mediated transactivation of EWS target genes, such as ApoCIII and c-fos, in a TGF-β-independent manner. Interestingly, we have shown that STRAP blocks the interaction between EWS and p300, whereas the complex formation between STRAP and EWS is not affected by p300. These results suggest that STRAP inhibits the transactivation function of EWS by displacing p300 from the functional transcriptional complex. Thus, this study provides a novel TGF-β-independent function of STRAP and describes a mechanism by which STRAP regulates the function of oncogenic EWS protein. (Cancer Res 2006; 66(22): 10824-32)

WD40 domain–containing proteins seem to serve regulatory functions in various cellular processes, such as signal transduction, transcriptional regulation, RNA processing, vesicular trafficking, cytoskeletal assembly, and cell cycle progression (1, 2). The WD40 repeat structure seems to be a functional motif that facilitates defined protein-protein interactions, sometimes leading to multiprotein complexes, as shown for the β-subunit of heteromeric G proteins (3). Some of them consist only of WD40 repeats; others contain NH₂- or COOH-terminal extensions of various lengths (1). It is not clear whether the conserved core of each repeat binds to any common structure, although it has been shown that some F-box proteins contain WD40 repeats that allow them to bind to proteins with phosphorylated serine or sometimes threonine residues (4, 5). Several WD domain–containing proteins have been shown to be involved in human cancers (6–9).

In Ewing sarcoma (EWS) and several malignant tumors, the EWS gene displays a characteristic chromosomal translocation that results in fusion of the NH₂-terminal domain of the EWS protein to the COOH-terminal DNA-binding domain of transcription factors such as ETS family, activating transcription factor-1, Wilms' tumor 1, and nuclear orphan receptors (10). Specifically, EWS/Fli-1 translocation is exhibited by ∼80% of Ewing tumors. EWS protein is predicted to be an RNA-binding protein containing the transcriptional activation domain in the NH₂-terminal domain and the RNA recognition motif and three arginine-glycine-glycine repeats (RGG boxes 1-3) in its COOH-terminal domain. The transcriptional potency of the NH₂-terminal domain of EWS observed in its various tumorigenic fusion proteins suggests that EWS may function as a transcription factor. The EWS protein, primarily localized in the nucleus, has been found to associate with basal transcriptional machinery (11, 12) and functions as a transcriptional coactivator in a cell type– and promoter-specific manner. It requires CBP/p300 for hepatocyte nuclear factor 4 (HNF4)–mediated transcriptional activation (13, 14). Although EWS fusion proteins function as sequence-specific transcription factors, the role of native EWS protein and the regulatory mechanism controlling the coactivator function of EWS are largely unknown.

Transforming growth factor β (TGF-β) represents an evolutionarily conserved family of secreted factors that mobilize a complex signaling network to control cell fate by regulating proliferation, differentiation, motility, adhesion, and apoptosis (15). TGF-β promotes the assembly of a cell-surface receptor complex composed of type I (TβRI) and type II (TβRII) receptor serine/threonine kinases. In response to TGF-β, receptor-regulated Smads (R-Smad), Smad2, and Smad3 form heteromeric complexes with common Smad, Smad4, and translocate to the nucleus where they modulate the transcription of TGF-β target genes. A distinct class of distantly related Smads, including Smad6 (16) and Smad7 (17), has been identified as inhibitors of these signaling pathways. In addition to Smads, several proteins interacting with TβRI and/or TβRII have been identified (18). Among them, serine-threonine kinase receptor-associated protein (STRAP) interacts with TGF-β receptors and Smad7 and negatively regulates TGF-β-induced gene expression and growth inhibition (6, 19). Our previous study suggests that STRAP is up-regulated in several cancers and functions as an oncogene (6). A recent study suggests that STRAP may be a predictive marker of 5-fluorouracil (5-FU)–based adjuvant chemotherapy benefit in colorectal cancer (7). The homozygous STRAP null allele embryos showed recessive embryonic lethality between embryonic days E 10.5 and E 12.5 and had defects in angiogenesis, cardiogenesis, somatogenesis, neural tube closure, and embryonic turning (20). These results suggest a broader role of STRAP in tumorigenesis and development. STRAP has also been shown to regulate the kinase activity of phosphoinositide-dependent kinase 1 through its physical interaction (21). A recent finding suggests how STRAP in survival motor neuron (SMN) protein complex plays an important role in pre-mRNA splicing (22). It is possible that STRAP may also function in a TGF-β-independent manner and may have cross-talk with other signaling pathways. Therefore, it is important to investigate how the function of STRAP is regulated through its interaction with other proteins or transcriptional coactivators.

To understand the function of STRAP, we have used proteomics analyses to search for intracellular signal mediators that interact with STRAP. We have shown that STRAP interacts with EWS protein in vitro and in vivo. STRAP binding with EWS is mostly confined in the nucleus. EWS is up-regulated in colorectal and lung cancers, which correlates with up-regulation of STRAP in colorectal (71%) and lung (54%) cancers, respectively. STRAP and EWS interaction resulted in the suppression of EWS-induced p300-mediated induction of the EWS target promoter activity. In addition, we have shown that STRAP displaces p300 from the EWS complex. Our results suggest a mechanism to explain how STRAP modulates EWS function in a TGF-β-independent manner.

Plasmids. pcDNA3-STRAP-Flag, pcDNA3-STRAP-hemagglutinin (HA), and the truncation mutant pcDNA3-STRAP(1-294)-Flag (CT1-Flag) have previously been described (19). Human Myc-EWS-WT and deletion mutants were a generous gift from Ralf Janknecht; pcDNA3-HA-HNF4, pHNF4x8-tk-Luc, and apolipoprotein CIII (ApoCIII)-Luc were kindly provided by Akiyoshi Fukamizu; and c-fos luciferase reporter plasmid, which contains a fragment of human c-fos promoter (nucleotide −404 to +41), was a gift from Tim Bowden.

Antibodies. Anti-EWS (sc-6532) polyclonal antibody, anti-p300 (sc-584) polyclonal antibody, anti-Myc (9E11) monoclonal antibody, anti-HA (Y-11) polyclonal antibody, anti-Rho-GDI, anti-Rho-A, and anti–poly(ADP-ribose) polymerase antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Flag M2 affinity gel (A2220) and anti-FLAG monoclonal antibody (M2) were from Sigma Biochemicals (St. Louis, MO).

Immunoprecipitation of STRAP. 293T cells were transfected with pcDNA3-STRAP-Flag, pcDNA3-CT1-Flag, and pcDNA3-Flag using FuGENE 6 (Roche Molecular Biochemicals, Indianapolis, IN). After 40 hours, cells were solubilized in lysis buffer as described (19). Cleared cell lysates were incubated with anti-Flag M2 affinity gel for 3 hours at 4°C. Immunoprecipitates were washed five times with lysis buffer and the immune complexes were eluted with either Flag peptide (40 μg/200 μL) or 4.0 mol/L urea in lysis buffer. The eluted proteins were precipitated with 10% trichloroacetic acid, washed with cold acetone, and then subjected to SDS-PAGE. The protein gels were stained with Colloidal Coomassie for 2 hours and destained with water for 24 hours.

Protein identification by mass spectrometry and database interrogation. Protein bands of interest were excised from the gel and processed for mass spectrometry (MS). Excised proteins were equilibrated within the gel slices in 100 mmol/L NH₄HCO₃, then reduced with DTT (3 mmol/L in 100 mmol/L NH₄HCO₃, 37°C for 15 minutes), and alkylated with iodoacetamide (6 mmol/L in 100 mmol/L NH₄HCO₃, 15 minutes). Gel slices were dehydrated with acetonitrile and then rehydrated with 15 μL of 25 mmol/L NH₄HCO₃ containing 0.01 μg/μL modified trypsin (Promega, Madison, WI) for 2-hour digestion at 37°C. Peptides were extracted with 60% acetonitrile, 0.1% trifluoroacetic acid, then dried and reconstituted in 10 μL of 0.1% trifluoroacetic acid. Peptides were desalted and then concentrated. Aliquots (0.4 μL) were applied to target plates and overlaid with 0.4 μL of α-cyano-4-hydroxycinnamic acid matrix. Matrix-assisted laser desorption/ionization, time-of-flight mass spectrometry (MALDI-TOF MS) was carried out using a Voyager DE-STR mass spectrometer (Applied Biosystems, Foster City, CA) operated in reflectron mode. The mass spectrum was calibrated to within 50 ppm using trypsin autolytic peptides present in the sample (m/z 842.50, 1,045.56, and 2,211.10 Da). Ions ([M+H]) corresponding to peptide masses were entered into the MASCOT database search algorithm,³

which compared the data against proteins present in the Swiss-Prot and NCBInr databases (allowing for complete carbamidomethylation of cysteine and up to one missed cleavage). MS/MS was carried out using a Qstar hybrid quadrupole/time-of-flight mass spectrometer (Applied Biosystems) equipped with a nanoelectrospray ion source.

Immunoprecipitation and immunoblot analyses. 293T cells were transfected with expression constructs and then were solubilized in lysis buffer as described before (19). Cell lysates were incubated with anti-Flag antibody or anti-EWS antibody and the immunocomplexes were analyzed by immunoblotting with anti-EWS antibody or anti-Flag antibody. Cell lysates prepared from VMRC-LCD, A549, and STRAP^+/+ cell lines were used for STRAP and EWS endogenous coimmunoprecipitation experiments. Cell lysates were also prepared from human colon and lung cancer tissues for Western blot analysis of STRAP and EWS protein expression. For subcellular localizations of endogenous STRAP and EWS, nuclear and cytoplasmic protein extracts were prepared from VMRC-LCD, ACC-LC-176, A549, 293T, and STRAP^+/+ cells according to the protocol of the manufacturer (NE-PER, Pierce Biotechnology, Inc., Rockford, IL) and then analyzed by Western blotting as indicated in figure legends. In brief, 20 μg of each cytoplasmic and nuclear extract were analyzed by Western blotting with anti-EWS and anti-STRAP antibodies. Complete fractionation of cytoplasmic and nuclear proteins was verified by Western blotting with antibodies against Rho-GDI or Rho-A and poly(ADP-ribose) polymerase, respectively.

Reverse transcription-PCR analyses. 293T cells were transiently cotransfected with Myc-EWS, p300, and STRAP-Flag expression plasmids, either alone or combination as indicated. Total RNA was isolated from transfected cells using TRIzol reagent according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Two micrograms of total RNA were reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Promega). The reaction was conducted at 42°C for 1 hour. cDNA mixture, 2.5 μL, was subjected to PCR reaction of 27 cycles of denaturation for 60 seconds at 95°C, annealing for 60 seconds at 55°C, and elongation for 60 seconds at 72°C. The specific primers for c-fos and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are as follows: c-fos 5′-primer, 5′-ATGGCTGCAGCCAAATGCCGCAACC, and 3′-primer, 5′-CAGTCAGATCAAGGGAAGCCACAGAC; GAPDH 5′-primer, 5′-ACCACAGTCCATGCCATCAC, and 3′-primer, 5′-TCCACCACCCTGTTGCTGTA.

Immunofluorescence staining. NIH 3T3 cells were plated and transfected with expression constructs STRAP-HA or Myc-EWS either alone or together. Cells were fixed, permeabilized, and then used for immunofluorescent staining as previously described (6). Primary antibodies, anti-HA and anti-Myc, were used and then detected with Cy3 goat anti-rabbit antibody and/or FITC-labeled goat anti-mouse antibody. STRAP^+/+ and STRAP^−/− cells were transiently transfected with Myc-EWS expression construct using Lipofectamine plus reagent (Invitrogen). Transfected cells were processed for immunofluorescence analyses with anti-Myc antibody and then detected with antimouse Cy3-conjugated secondary antibody. Stained cells were mounted with Vectashield H-1000 with 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA) and visualized by either fluorescence microscopy or confocal microscopy as mentioned.

Transcriptional response assays. NIH 3T3 cells were transiently transfected with various constructs as indicated. In each experiment, equal amounts of total DNA were transfected. Twenty hours after transfection, cells were incubated without serum for c-fos promoter and with serum (7%) for all other promoters for 20 hours as indicated. Luciferase and β-galactosidase activities were determined. Luciferase activity was normalized to β-galactosidase activity and the relative luciferase activity was presented.

Identification of STRAP binding proteins. STRAP is a ubiquitously expressed WD domain–containing protein, which has been shown to be up-regulated in human cancers (6) and to be a strong predictive marker of 5-FU-based adjuvant chemotherapy benefit in colorectal cancer (7). Moreover, STRAP interacts with TGF-β receptors and negatively regulates the expression of TGF-β target genes (23). To understand the mechanism of function of STRAP, we used proteomics studies to identify STRAP binding partners. Coimmunoprecipitation assays in combination with MS have recently been shown to be a powerful technique for identifying components of protein complexes. Flag-tagged STRAP (full-length), Flag-tagged CT1 (COOH-terminal deletion mutant of STRAP), or Flag vector was transfected into 293T cells. Cell lysates were subjected to immunoprecipitation with antibodies to Flag epitope. Immune complexes were then eluted with a Flag peptide or urea. STRAP-bound protein bands were visualized with Colloidal Coomassie staining and were subjected to MS after trypsin digestion (Fig. 1A). A protein sequence data base search (Mascot Search) was done with the obtained peptides for proteins in bands 1 and 2 (Table 1; bands 1 and 2 denoted in Fig. 1B). The EWS protein and a SMN-interacting protein (Gemin2) were unambiguously identified from the peptide mass map of protein band 1 (Supplementary Fig. S1A) and band 2, respectively, by using MALDI-TOF mass spectrum (Table 1). Ions labeled with an asterisk (Supplementary Fig. S1A) indicate peptide masses [M+H] that match predicted peptide masses for EWS to within 50 ppm using trypsin autolytic peptide ions for internal calibration.

Despite this unambiguous statistically significant protein identification, nano-electrospray-tandem mass spectrometry (ESI-MS/MS) was done to confirm the EWS amino acid sequences of select peptides (highlighted in Table 1) that were found in the MALDI-TOF peptide mass maps. The fragmentation patterns of peptide ions at m/z 725.8 (doubly charged ion that corresponds to m/z 1,450.68 in the MALDI-TOF mass spectrum; Supplementary Fig. S1B) and 562.3 (triply charged ion that corresponds to m/z 1,684.80 in the mass spectrum; Supplementary Fig. S1C) were consistent with the amino acid sequences predicted for these peptides from the EWS protein. Nano-ESI-MS/MS on the corresponding triply charged 604.30 ion was consistent with a predicted EWS-derived peptide containing dimethyl arginine (Supplementary Fig. S1D). This dimethylation on arginine residue (amino acid 615) in RGG motifs of EWS protein is consistent with previous report (24). Taken together, EWS protein was unambiguously identified as one of the STRAP binding proteins analyzed by MALDI-TOF MS.

STRAP interacts with EWS. To verify the binding between STRAP and EWS and to map the binding region on STRAP, we generated several NH₂- and COOH-terminal truncated constructs of STRAP by progressively deleting one or two WD domains. However, the truncated STRAP proteins were not stable except CT1-Flag, which has all WD domains intact but lacks 57 COOH-terminal amino acids (Supplementary Fig. S2A). This is consistent with the fact that all WD domains are usually required for the stability of these proteins. STRAP-Flag and CT1-Flag constructs were transfected in 293T cells and cell lysates were subjected to anti-Flag immunoprecipitation followed by immunoblotting with anti-EWS antibody. Endogenous EWS was detected in the immune complex of STRAP (Fig. 2A). In a reciprocal experiment, we observed that STRAP coimmunoprecipitated with EWS, showing the association of STRAP with EWS. The binding between STRAP and EWS was not affected by COOH-terminal 57-amino-acid deletion. To determine the in vivo interaction between STRAP and EWS, STRAP was immunoprecipitated from whole-cell lysates of two lung adenocarcinoma cell lines, A549 and VMRC-LCD, with a polyclonal antibody and EWS was detected in the immune complex (Supplementary Fig. S2B). To determine whether TGF-β has any effect on the interaction between STRAP and EWS, a similar experiment was done after treating A549 cells with TGF-β for different time points. However, we did not observe any effect of TGF-β on this binding (Supplementary Fig. S2C). To confirm the specificity of STRAP and EWS binding, we used STRAP^+/+ and STRAP^−/− mouse embryonic fibroblasts that express good levels of EWS protein (Fig. 2B,, top). STRAP was immunoprecipitated from both cell lysates and the immune complexes were analyzed by Western blot with anti-EWS monoclonal antibody and vice versa. We detected the interaction between STRAP and EWS in STRAP^+/+ cells, and STRAP^−/− cells that do not have STRAP expression lack the interaction (Fig. 2B). These results indicate a specific interaction between STRAP and EWS. To determine the domains in EWS required for binding with STRAP, STRAP-HA plasmid was cotransfected with Myc-tagged EWS and deletion constructs EWS_2-502, EWS_2-374, EWS_2-246, EWS_246-656, and EWS_374-656. Cell lysates were subjected to anti-HA antibody immunoprecipitation, and each immunoprecipitate was then probed with antibodies to Myc. In reciprocal experiments, we detected STRAP in immunocomplexes obtained with an antibody against Myc epitope. Although we observed difference in binding efficiency, we did not find complete abrogation of binding with any EWS deletion constructs, suggesting that the association of STRAP takes place at the NH₂ and COOH termini of EWS protein (Fig. 2C). These results suggest that STRAP associates with EWS in vivo and that a functional link between these two proteins may exist.

Subcellular localization of STRAP and EWS. Previous report suggests that EWS protein is exclusively in the nucleus (11). In contrast, other reports suggest both nuclear and cytoplasmic localization of EWS (24, 25). To determine the subcellular localization of STRAP and EWS, and to examine whether or not they are colocalized, we did immunofluorescence analyses. NIH 3T3 cells were cotransfected with STRAP-HA and Myc-EWS plasmids and then processed for immunofluorescence analyses with anti-Myc or anti-HA antibody. Antimouse FITC and antirabbit Cy3 secondary antibodies were used to detect EWS and STRAP, respectively. We observed STRAP staining in both cytoplasm and nucleus as reported before (ref. 6; Fig. 3A,, b and g), whereas EWS was found to be mostly in the nucleus (a and f). The overlay images showed that the nuclear STRAP colocalizes with EWS (c). To verify whether STRAP has any effect on localization of EWS, we did immunofluorescence analyses using STRAP^+/+ and STRAP^−/− MEFs that were transiently transfected with Myc-EWS expression construct. Myc-EWS was detected with antimouse Cy3-conjugated secondary antibody in transfected cells and visualized by confocal microscopy. EWS is localized in the nucleus of STRAP^+/+ or STRAP^−/− MEFs, and this nuclear localization is not affected by STRAP (Fig. 3B,, top). We then confirmed the subcellular localization of endogenous EWS by Western blotting using cytoplasmic and nuclear lysates from STRAP^+/+ and STRAP^−/− MEFs. Consistent with the confocal data, EWS is mostly localized in nuclear fractions of STRAP^+/+ and STRAP^−/− MEFs (Fig. 3B,, bottom). To further confirm the subcellular localization of STRAP and EWS proteins and to determine whether there is any difference in expression of these proteins in normal and tumor cell lines, we analyzed the cytoplasmic and nuclear fractions of 293T and STRAP^+/+ MEFs and human lung tumor cell lines, including VMRC-LCD, ACC-LC-176, and A549, by Western blot analyses for STRAP and EWS proteins. STRAP protein was equally detected in cytoplasmic and nuclear fractions in 293T and STRAP^+/+ MEFs. In contrast, nuclear lysates of tumor cells showed less expression of STRAP in the nucleus (Fig. 3C). However, EWS was detected mostly in nuclear fractions with little expression in the cytoplasm of VMRC-LCD and ACC-LC-176 cells (Fig. 3C). Complete separation of cytoplasmic and nuclear proteins was tested by Western blot analyses for Rho-GDI and poly(ADP-ribose) polymerase, respectively. These results suggest that STRAP is expressed in both nuclear and cytoplasmic fractions and EWS is expressed mostly in the nucleus. To determine the subcellular compartment where the interaction between these two proteins takes place, we used cytoplasmic and nuclear fractions from A549, VMRC-LCD, 293T, and STRAP^+/+ cell lines to immunoprecipitate STRAP. The immune complex was used for immunoblotting with anti-EWS antibody (Fig. 3D). We observed that EWS was coprecipitated by STRAP from nuclear lysates. Together, these data indicate that STRAP and EWS colocalize and interact in the nucleus.

Coexpression of STRAP and EWS in human cancers. Expression pattern of EWS protein in human tumors is not known. In addition, up-regulation of STRAP in colorectal and lung cancers (6) and interaction between STRAP and EWS prompted us to investigate whether or not STRAP and EWS are co-upregulated in human tumors. We analyzed colon tumor and patient-matched adjacent normal colon tissue samples by Western blot analyses using anti-STRAP or anti-EWS antibody. STRAP was found to be up-regulated in 14 of 20 (70%; marked by solid triangle) and EWS was up-regulated in 11 of 20 (55%; marked by line) colon tumor samples (Fig. 4A). Interestingly, 10 of 14 samples (marked by asterisk), where STRAP was up-regulated, showed up-regulation of EWS protein expression (71% correlation), suggesting a good correlation of up-regulation of these two proteins. In addition, we observed co-upregulation of STRAP and EWS in higher-stage colon tumors (9 of 10) and the level of EWS was higher in all three liver metastases (Supplementary Table S1A). We further investigated the possibility of coexpression of both STRAP and EWS in lung cancer. We observed up-regulation of STRAP in 11 of 16 (68%) tumors including squamous, adenocarcinoma, and large-cell carcinoma (Fig. 4B). However, Western blot analyses with anti-EWS antibody showed up-regulation of EWS in 7 of 16 tumors (44%; marked by line). We found that EWS is coexpressed in 6 of 11 (54%) tumors that overexpressed STRAP. However, we did not find any correlation between up-regulation of these proteins and stage, grade, histologic type, or smoking status in lung tumors. Therefore, these results suggest that EWS is up-regulated in colorectal and lung tumors, which correlates with elevated level of STRAP.

STRAP inhibits the coactivator function of EWS. EWS was shown to act as a transcriptional activator in a cell type– and promoter-specific manner. As EWS does not have any DNA binding domain, it may be recruited to the target promoters by protein-protein interaction with DNA binding transcription factors and act as a coactivator. EWS has a potent NH₂-terminal transcriptional activation domain (EAD) that is important for oncogenic fusion to various members of the DNA binding transcription factors in EWS. Recent reports indicate that EWS is dependent on CBP/p300 to function as a transcriptional coactivator (12). Furthermore, EWS acts as a coactivator for HNF4-mediated transcriptional activation (13). In an attempt to determine the functional role of STRAP in EWS-activated transcriptional responses, we used HNF4-specific reporter, pHNF4x8-tk-Luc (containing eight HNF4 binding sites), in transcriptional assays. NIH 3T3 cells were transfected with the reporter alone or in combination with expression vectors such as HNF4, STRAP, and EWS (Fig. 5A). Expression of HNF4 strongly induced the reporter activity as expected, whereas STRAP or EWS alone had no effect on the reporter. Coexpression of EWS and p300 with HNF4 further induced transcription. EWS-dependent transcriptional activation was totally abolished by STRAP, suggesting its inhibitory role in EWS- and HNF4-induced transactivation. Previous report suggests that EWS enhances ApoCIII promoter activity in a HNF4-dependent manner (13). To investigate the role of STRAP in EWS-mediated transcriptional activation of endogenous promoter, similar experiments were carried out using ApoCIII-Luc promoter reporter. EWS induced the promoter activity strongly in the presence of HNF4. STRAP had no effect on HNF4-dependent transactivation, but it reduced the promoter activity induced by EWS (Fig. 5B). The promoter of the human c-fos proto-oncogene has been reported to be stimulated by EWS, which is dependent on CBP/p300 but not on HNF4 (12). c-fos promoter was activated by EWS in a dose-dependent manner, and STRAP suppressed EWS-mediated activation of this promoter (Fig. 5C). To confirm this effect of STRAP, we tested the level of c-fos mRNA and protein after transfecting 293T cells with expression vectors for EWS, p300, and/or STRAP. Total RNAs from transfected cells were isolated and used for semiquantitative reverse transcription-PCR (RT-PCR) analyses. EWS modestly induced the c-fos mRNA. p300 strongly induced EWS-mediated c-fos mRNA expression, which was significantly reduced by STRAP (Fig. 5D,, left). Cell lysates were prepared from transfected cells and subjected to immunoblotting with anti-c-fos antibody (Fig. 5D,, right). EWS induced the expression of c-fos, which was further enhanced by p300. Expression of STRAP decreased the level of c-fos induced by EWS and p300 (Fig. 5D , right). These data show that STRAP inhibits EWS-activated transcription of c-fos gene. TGF-β had no effect on this STRAP-mediated inhibition of EWS coactivator function (data not shown). To examine whether EWS has any effect on TGF-β signaling, we did reporter assays using TGF-β responsive reporters, including (CAGA)₉ MLP-Luc, p3TP-Lux, and p21^Cip1-Luc. TGF-β-induced reporter activities were inhibited by EWS, suggesting a role for EWS in the negative regulation of TGF-β-induced transcription (Supplementary Fig. S3A-C). Taken together, these results suggest that STRAP inhibits transactivation function of EWS in both a HNF4-dependent and a HNF4-independent manner.

STRAP expression abrogates the interaction between EWS and p300. Previous reports suggest that the association between EWS and p300 is required for EWS-dependent transactivation (12, 13). In an attempt to understand the mechanism by which STRAP inhibits EWS-induced transcription, we first determined whether STRAP can interfere with the binding between EWS and p300. 293T cells were cotransfected with p300, Myc-tagged EWS, and increasing doses of Flag-tagged STRAP expression construct. Cell lysates were subjected to immunoprecipitation with anti-p300 antibody and the immunoprecipitates were analyzed by immunoblotting with an anti-Myc antibody (Fig. 6A). We observed that EWS was coprecipitated with p300 as expected. However, the coprecipitation of EWS was significantly reduced by coexpression of STRAP. This result suggests that STRAP blocks the interaction between EWS and p300 by direct binding with EWS protein. It is possible that STRAP binding with EWS is stronger in affinity than the binding between EWS and p300. To confirm this, we analyzed whether p300 expression has any effect on STRAP and EWS interaction. 293T cells were cotransfected with HA-STRAP, Myc-EWS, and increasing amounts of p300 expression plasmids, and cell lysates were subjected to anti-HA immunoprecipitation followed by immunoblotting with anti-Myc antibodies (Fig. 6B). We observed efficient coprecipitation of EWS with STRAP, and increasing expression of p300 has no effect on the interaction between STRAP and EWS. Reciprocal experiment showed coprecipitation of STRAP with EWS (second from top) and this interaction was not affected by p300. These results show that the binding between STRAP and EWS is stronger than that between EWS and p300, and STRAP may inhibit EWS-mediated transactivation by dissociating the complex between EWS and p300.

STRAP, a novel WD40 domain–containing protein, interacts with both TβRI and TβRII and negatively regulates TGF-β-induced gene expression (23). STRAP also associates with Smad7 and prevents Smad2 and Smad3 activation by the receptor complex (19). STRAP is up-regulated in 60% of colon cancers, 78% of lung carcinomas (6), and 46% breast cancers (26) and has been shown to be a strong predictive marker of 5-FU-based adjuvant chemotherapy benefit in colorectal cancer (7). It activates the mitogen-activated protein kinase/extracellular signal–regulated kinase pathway and down-regulates the cyclin-dependent kinase inhibitor p21^Cip1, which leads to pRb phosphorylation in a TGF-β-independent manner (6). Therefore, we decided to identify STRAP binding proteins that may provide an insight into the TGF-β-dependent and/or TGF-β-independent role of STRAP in human cancers. In this study, we have identified the oncoprotein EWS as STRAP-interacting protein by using proteomics studies. Interaction between these two oncoproteins opens up the avenue to explore their functions.

Much of the knowledge about the function of EWS is derived from studies of oncogenic fusion proteins that are involved in several malignancies and that arise from chromosomal fusions with multiple partners (27). Gene rearrangement after translocation is associated with more than 85% of Ewing tumors that involve EWS. However, the cellular function of the normal EWS protein is not well characterized. In this report, we show that EWS interacts with STRAP in the nucleus and this is important for STRAP-induced inhibition of EWS transactivation function, thus implicating a nuclear function of STRAP. Thus, this study provides a mechanism by which the physiologic role of EWS is regulated in the nucleus. Although STRAP is localized in both cytoplasm and nucleus, our previous studies indicate the cytoplasmic function of STRAP (6, 19). In this study, we investigate STRAP-associated proteins by sensitive and accurate MALDI-TOF and MS/MS (Supplementary Fig. S1A). The powerful nature of this technique in identifying proteins is shown by sequencing of the fragment peptides (Supplementary Fig. S1B and C), including one that is dimethylated on arginine residue of the peptide sequence (amino acid 615-632; Supplementary Fig. S1D). We have confirmed this specific binding between STRAP and EWS in vivo in several cell lines including STRAP null and wild-type MEFs (Fig. 2). However, TGF-β has no effect on the association between STRAP and EWS (Supplementary Fig. S2C) and on STRAP-mediated inhibition of EWS function. Although there are some controversies about the localization of EWS, our data show that EWS is localized mostly in the nucleus, and this is in agreement with previous studies (12, 28). It is known that methylation status of EWS influences nuclear-cytoplasmic shuttling (29) and our mass spectral data support the methylation of EWS protein (Supplementary Fig. S1D). Recent studies show EWS interaction with cytoplasmic proteins, including the tyrosine kinase Pyk2 (25), Btk (30), and calmodulin (28), suggesting that EWS may also play a significant role in signal transduction. Although TGF-β has no effect on STRAP and EWS functional interaction, our preliminary data suggest that EWS, like STRAP, inhibits TGF-β-dependent transcription (Supplementary Fig. S3A-C). However, we do not see any synergy between STRAP and EWS in the inhibition of TGF-β-dependent transcription, suggesting that the effect of these two proteins on TGF-β signaling may be through different mechanisms.

As STRAP binds with EWS through both the NH₂ and COOH termini and EWS-fusion proteins share the same NH₂-terminal EAD domain, it is possible that STRAP may also modulate the function of fusion proteins in EWS through binding. EWS does not have any DNA-binding domain. It may be recruited to target promoters by protein-protein interaction with transcription factors for its coactivator function. The physical association of EWS with CBP/P300 seems to be required for HNF4-induced transcriptional activation. STRAP inhibits this EWS-mediated induction of HNF4 target genes (Fig. 5A and B). STRAP also inhibits EWS (Fig. 5C and D) and the p300-dependent and HNF4-independent transactivation of c-fos (12). In an attempt to determine the mechanism, we have observed that STRAP dissociates the interaction between EWS and p300, whereas p300 has no effect on the interaction between EWS and STRAP (Fig. 6). Although there is controversy about the region of EWS protein required for interaction with CBP/p300, STRAP can displace p300 from the complex with EWS, as it binds through both the NH₂ and COOH termini. This is in agreement with a previous study suggesting that adenoviral E1A oncoprotein abolishes c-fos activation by EWS through the interaction with CBP/p300 (12). However, in this case, STRAP binds EWS with stronger affinity and p300 has no effect on this interaction.

We have identified another STRAP-interacting protein, Gemin2, by using MALDI-TOF-MS (Table 1). The SMN protein, a product of the spinal muscular atrophy disease-related gene, forms a large macromolecular complex with Gemin2-Gemin8, which is involved in a number of cellular processes including pre-mRNA splicing, transcription, apoptosis, ribosomal assembly, and nuclear-cytoplasmic transportation (31). Our finding, showing the binding between STRAP and Gemin2, is consistent with a previous study (31) and suggests a function of STRAP in several other cellular pathways related to RNA splicing and metabolism. In addition, SMN interacts and colocalizes with EWS (32), suggesting that a multiprotein complex containing STRAP, EWS, and SMN plays an important role in small nuclear ribonucleoprotein biogenesis.

Although EWS fusion proteins are known to be expressed in more than 85% of Ewing family tumors, nothing is known about the expression and function of EWS normal protein in human tumors. To our knowledge, for the first time, we show that EWS is up-regulated in 55% of colorectal tumors and 44% of lung tumors. STRAP is also up-regulated in 70% of colorectal cancers and 68% of lung cancers. Interestingly, up-regulation of STRAP correlates with that of EWS in 71% of colon tumors and 54% of lung tumors based on STRAP. Protein tyrosine kinases stimulate the NH₂-terminal transactivation domain of EWS fusion protein (33). Here we show that EWS could be regulated in its transcriptional properties through mitogenic signaling of oncogenic STRAP in human cancers. Future studies will investigate whether or not STRAP is involved in activation of EWS in human tumors through phosphorylation. Taken together, functional interaction, colocalization, and increased level of coexpression in human tumors suggest that EWS in cooperation with STRAP could potentially be involved in tumor progression and thus provide a distinct function of normal EWS protein.

Grant support: R01 CA95195 and CA113519, a Career Development Award from SPORE in lung cancer (5P50CA90949), a Clinical Innovator Award from Flight Attendant Medical Research Institute (P.K. Datta), and institutional support from Vanderbilt University through the Academic Venture Capital Fund for funding for proteomics services.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Ralf Janknecht (Department of Biochemistry and Molecular Biology, Mayo Clinic College of Medicine, Rochester, MN), Akiyoshi Fukamizu (Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Tsukuba, Ibaraki, Japan), and Tim Bowden (Department of Molecular and Cellular Biology, University of Arizona, Tucson, AZ) for providing us with constructs, and Pierre Massion (Division of Allergy, Pulmonary, and Critical Care Medicine, Vanderbilt-Ingram Comprehensive Cancer Center, Vanderbilt University Medical Center, Nashville, TN) and M. Key Washington (Department of Pathology, Vanderbilt University Medical Center, Nashville, TN) for providing us with human tumor tissues.