Proteomic Analysis of a PDEF Ets Transcription Factor-Interacting

Feb 9, 2009 - The prostate derived Ets transcription factor (PDEF) is reported to be a breast ... E26 leukemia virus.1 The human Ets transcription fac...
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Proteomic Analysis of a PDEF Ets Transcription Factor-Interacting Protein Complex Je-Yoel Cho,*,† Minjung Lee,† Jung-Mo Ahn,† Eun-Sung Park,† Ji-Hoon Cho,† Seung-Jin Lee,† Byung-Gyu Kim,† Sun-Hee Heo,† Hye-Jeong Park,† Luiz F. Zerbini,§ Daehee Hwang,† and Towia A. Libermann§ Department of Biochemistry, School of Dentistry, Kyungpook National University and ProtAn, Daegu 700-422, Korea, School of Interdisciplinary Bioscience and Bioengineering, Pohang University of Science and Technology, Pohang, Kyungpook, 790-784, Korea, and BIDMC Genomics Center, Harvard Medical School, Boston, Massachusetts 02115 Received August 29, 2008

Ets proteins are a family of transcription factors that share an 85 amino acid conserved DNA binding domain, the ETS domain. The 27 known human Ets transcription factors control multiple biological processes, including cellular proliferation, differentiation, apoptosis, angiogenesis, transformation, and invasion. Overexpression of some Ets genes has been linked to numerous malignancies, including breast cancers. The prostate derived Ets transcription factor (PDEF) is reported to be a breast and prostate tumor-associated Ets factor. To understand the roles of PDEF in breast cancers, we transiently overexpressed PDEF in MDA-MB-231 human breast cancer cells by adenoviral-mediated gene delivery. PDEF binding protein complexes were isolated by immunoprecipitation and PDEF-interacting proteins were analyzed by LC-MS/MS. After subtracting the proteins binding nonspecifically to antibody-bead complexes, we identified 286 proteins in the PDEF-associated protein complex. By comparison to published protein-protein interactions, we selected 121 proteins for further analysis. PDEF interactors distribute not only in the nucleus, but also in the cytoplasm, as well as other subcellular compartments. Our data reveals that PDEF interacts with a variety of proteins involved in cell cycle, DNA repair, cytoskeleton organization, mRNA processing, tRNA biosynthesis, protein folding, and cell signaling. Furthermore, the EGFR1- (Erbb1) and Erbb2- (HER2) related proteins erbin, an ERBB2 interacting protein, catenin δ-1 (which interacts with Erbin), and EGFR (a HER2-homology receptor) were associated with PDEF. These findings indicate that PDEF may be regulated by Erbb2 or EGFR-activated signaling pathways in breast cancer cells. Further analysis of these proteins will identify the roles of PDEFinteracting proteins in breast tumorigenesis. Keywords: Breast cancer • Prostate derived ETS transcription factor • LC-MS/MS

1. Introduction Transcription factor protein-protein interactions are involved in every step of cell signaling, including receiving signals, selection of target genes, regulation of DNA binding, regulation of transcriptional activity, and protein turnover. Ets (E26 Transformation-Specific) was first identified as one of two viral oncogenes (Ets and Myb) transduced by the avian E26 leukemia virus.1 The human Ets transcription factor family consists of 27 multifunctional transcription factors that share a common DNA-binding domain, known as the Ets domain, which binds to a consensus 5′-GGA(A/T)-3′ sequence within the promoters of target genes. Ets transcription factor family * To whom correspondence should be addressed. Dept. of Biochemistry, School of Dentistry, Kyungpook National University, 101 Dong In-Dong, Jung-Gu, Daegu, Korea 700-422. Tel., 82-53-420-4997; fax, 82-53-421-1417; e-mail, [email protected]. † Kyungpook National University. † Pohang University of Science and Technology. § Harvard Medical School. 10.1021/pr800683b CCC: $40.75

 2009 American Chemical Society

members also have a second conserved domain. This domain is the pointed domain which is found in 11 of 27 human Ets genes and functions in protein-protein interactions and oligomerization.2 Ets proteins are downstream nuclear targets of signal transduction cascades and Ets factors regulate a number of cellular processes including growth, apoptosis, development, differentiation, hematopoiesis, vasculogenesis, and angiogenesis.3 Furthermore, increased or decreased expression of many ETS-domain transcription factors has been linked to cancer. Also, it is known that fusions of certain Ets genes with other regulatory gene elements result in the overexpression of the oncogenic transcription factors in cancers, which includes frequent rearrangements of ERG, ETV1 and ETV4 ETS factors in prostate cancers.4-6 Generally, Ets family members are nuclear targets of signal transduction and function in concert with other proteins.7 The prostate-derived Ets transcription factor (PDEF) is a member of the Ets family and contains an Ets domain and a pointed domain. Unlike the majority of Ets factors, PDEF is Journal of Proteome Research 2009, 8, 1327–1337 1327 Published on Web 02/09/2009

research articles expressed exclusively in tissues and cell lines from prostate and breast. In addition, PDEF preferentially binds to GGAT on DNA, unlike other epithelial-specific Ets (ESE) factors.8 PDEF is involved in the transcriptional activation of prostate-specific antigen (PSA), a diagnostic indicator for prostate cancer. PDEF also interacts with the androgen receptor and NKX-3.1, a suspected prostate tumor suppressor gene that encodes a homeodomain transcription factor.9 The transcriptional activity of PDEF is modulated by other factors/partners. PDEF is also a breast tumor-associated molecule. However, conflicting results have been reported for the expression and role of PDEF in breast tumors. High levels of PDEF mRNA and protein were identified in breast cancers compared to normal breast tissue.10,11 However, some reports have shown that expression of PDEF in breast cancers led to the reductions in invasion, migration, and growth of cancer cells, suggesting that PDEF may act as a suppressor of metastasis.12,13 Identifying the proteins interacting with PDEF will elucidate the role of PDEF in breast cancer cells. In this study, in order to examine the proteins that interact with PDEF in the breast cancer cells, Pdef was transiently overexpressed in MDA-MB231 human breast cancer cells, which do not express PDEF, by adenoviral-mediated gene delivery. PDEF-interacting proteins were then analyzed by LC-MS/MS. A total of 286 proteins were identified. Following a protein-protein network analysis, 121 proteins were found to be either directly or indirectly associated with PDEF.

2. Materials and Methods 2.1. Cell Culture. The human breast cancer cell lines MDAMB-231 and MDA-MB-453 were maintained as subconfluent monolayer cultures in Dulbecco’s Modified Eagle’s Medium (DMEM; Hyclone, South Logan, UT) supplemented with 10% fetal bovine serum (FBS; Logan, UT) and antibiotic-antimycotic liquid (Gibco Grand Island, NY) at 37 °C under 5% CO2. 2.2. RT-PCR Analysis. RT-PCR was performed as previously reported.14 Briefly, total cellular RNAs were isolated from the MDA-MB-231 cells infected with β-gal or PDEF and MDA-MB453 using TRIZOL Reagent (Invitrogen, Grand Island, NY) following the manufacturer’s instructions. All samples were treated with DNase I for 30 min at 37 °C. The PCR primers for PDEF detection were as follows: forward primer, 5′-GAAAGAGCGGACTTCACCTG-3′, and reverse primer, 5′-TTCTCCTT GTTGAGCCACCT-3′. 2.3. Immunoprecipitation (IP). MDA-MB-231 cells were cultured to 70% confluence in a 150 mm dish. For infection, the cells (total 5 × 107 cells in 5 dishes) were washed with DMEM containing 2% FBS and infected either with Adv-β gal, as a control, or with Adv-5′flag-pdef at a multiplicity of infection (MOI) of 15 pfu/cell. The medium was replaced with DMEM containing 10% FBS 1 h postinfection. At 48 h postinfection, cells were washed twice with PBS and collected in lysis buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 and 1:30 diluted protease inhibitor cocktail [Roche Applied Science, Mammheim, Germany]). Cells were incubated for 20 min on ice and insoluble cell debris was removed by centrifugation at 14 000g for 20 min at 4 °C. The supernatant proteins (about 58 mg) were transferred to new tubes and combined with 20 µL of anti-FLAG-conjugated agarose beads (M2-agarose, Sigma, St. Louis, MO). Anti-FLAG antibody beads have high efficiency for capturing FLAG-tagged fusion proteins in the IP procedure. Following an overnight incubation, the agarose beads were washed five times with wash buffer 1328

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Cho et al. containing 25 mM Tris-HCl [pH 6.8], 2.7 mM KCl, and 137 mM NaCl. Immunoprecipitated proteins were dissociated from the agarose beads by incubating in the presence of 3× FLAG peptide (150 ng/µL final concentration; Sigma) or by addition of nonreducing SDS sample buffer (63 mM Tris-HCl [pH 6.8], 2% SDS, 10% glycerol and 0.005% bromophenol blue). The samples were boiled for 3 min in SDS sample buffer and separated by 15% SDS-PAGE. 2.4. Coomassie Blue Staining and In-Gel Digestion. Gels were washed three times with ddH2O for 5 min each and stained with Bio-Safe Coomassie Stain solution (Coomassie G250 Stain; Bio-Rad, Hercules, CA) for 1 h with gentle shaking at room temperature. The gels were then washed with ddH2O for 30 min, replacing ddH2O every 10 min. The gels were cut into 25 pieces per lane. The excised protein bands were destained with 75 mM ammonium bicarbonate/ 40% ethanol (1:1, v/v). After destaining, DTT solution (5 mM dithiothreitol/25 mM ammonium bicarbonate) was added to the tube to cover the gel pieces for reduction of proteins, and the tube was incubated at 60 °C for 30 min. The liquid was discarded, and the gels pieces cooled to room temperature. For alkylation, IAA solution (55 mM iodoacetamide/25 mM ammonium bicarbonate) was added to the tube, and the tube was incubated at room temperature for 30 min in the dark. The gel pieces were dehydrated in 100% acetonitrile (ACN), and then dried in a vacuum centrifuge. The gel pieces were rehydrated in 10 µL of 25 mM ammonium bicarbonate buffer containing 20 µg/mL modified sequencing grade trypsin (Roche Applied Science, Mammheim, Germany) and incubated for 45 min on ice. Trypsin digestion was continued overnight at 37 °C, and the supernatants were transferred to new tubes. 2.5. Nano-LC-ESI-MS/MS Analysis. For proteomic analysis, we used an LC-MS/MS system with reverse phase (RP)-LC comprised of a Surveyor MS pump (Thermo Electron, San Jose, CA) for LC, a Spark auto sampler (EMMEN, The Netherlands), and a Finnigan LTQ linear ion trap mass spectrometer (MS) (Thermo Electron, San Jose, CA) equipped with nanospray ion (NSI) sources. The trypsin digested peptides (12 µL) were injected directly into a peptide CapTrap cartridge for concentration and desalting and applied to the RP column, which was packed in house with 5 µm, 300 Å pore size C18, and separated. The mobile phase solutions A (ddH2O) and B (ACN) both contained 0.1% (v/v) formic acid. The flow rate was maintained at 200 nL/min. The gradient was started at 2% B and a linear gradient to 60% B was achieved in 53 min, and then ramped to 80% B in 7 min and to 100% A over the next 10 min. The mass spectrometer was operated in a data-dependent mode (in a range of m/z 400-1600) in which each full MS scan was followed by five MS/MS scans where the five most abundant peptide molecular ions were dynamically selected from the prior scan for collision-induced dissociation (CID) using a normalized collision energy of 35%. The temperature of the heated capillary and electrospray voltage were 200 °C and 2.1 kV, respectively. 2.6. Data Analysis and DTA Select. All MS/MS data were searched against the IPI human protein database (IPI v3.09, 8/03/2005) using the SEQUEST algorithm (Thermo Electron, San Jose, CA) incorporated into the BioWorks software (version 3.2). The database searches allowed for variable modifications on the cysteine residue (carboxyamidomethylation, 57 Da) and on the methionine residues (oxidation, 16 Da), using a peptide mass tolerance of 2 Da, and a fragment mass tolerance of 1

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Proteomic Analysis of a PDEF Ets Transcription Factor Da. The database search was limited to only those peptides that would be generated by tryptic cleavage. The SEQUEST results were filtered by Xcorr versus charge state. Xcorr was used for a match with g1.9 for singly charged ions, g 2.2 for doubly charged ions, and g3.75 for triply charged ions. We set Delta Cn g 0.1, Rsp e 4, and the probability score g 1.0 × 10-3. Protein identifications were made based on the corresponding peptide identification and the identity was validated by DTAselect v1.9. For more strict protein identification, proteins with two or more peptide identifications and Xcorr scores over 40 were considered positive identifications. 2.7. Development of a Statistic for PDEF Interactors. We used the three measures, the minimum shortest path, lmin, the degree, k, and the clustering coefficient, C, for each protein to select statistically significant potential PDEF interactors. The definitions of the minimum shortest path, the neighbor interaction degree coefficient, and clustering coefficient have been introduced previously.15,16 The minimum shortest path between protein i (i ) 1, 2,..., 230) and the remaining selected PDEF interactors can be expressed as follows: lmin,i )

min {lij}

j)1,2,...,230 j*i

where lij is the shortest path between proteins i and j. Note that a true interactor is likely to have a small minimum shortest path, a large neighbor interaction degree coefficient and a large clustering coefficient. To make the direction of all the measures the same for true interactors, we modified the minimum shortest path as follows: ˆl min,i ) R - lmin,i, where R ) max {lmin,i} i)1,2,...230

As a result, a high value of the individual measures indicates a high probability of being an interactor of PDEF. To combine these three measures, we then used the statistic, di, the Euclidean distance from the origin to protein i in the 3-D space spanned by the three measures, 2 di ) √ˆl min,i + ki2 + Ci2, where i ) 1, 2, ...,230

Before di was computed, each measure was scaled to values between 0 and 1 using the minimum and maximum value of the measure in both the identified protein set and a randomly sampled set, as described below, in order to prevent di from being biased toward measures with large values. The 3-D scatter plot of the three measures is shown in Supporting Information Figure S1. 2.8. Computation of p-Value. Let d and d0 be the sets of statistics computed from the identified 230 proteins and randomly sampled 230 proteins (100 times), respectively. The probability density function (PDF) of d0 representing the joint PDF of the three measures for random samples was considered as an empirical null hypothesis (H0). A p-value for protein i was computed by: pi )

number of d0 larger than di total number of d0

)

#{d0 g di} #{d0}

As shown in Supporting Information Figures S2 and S3, since more than 95% of d0 have values less than 1, proteins with di larger than 1 were selected as statistically significant interactors of PDEF at the p ) 0.05 level of significance. 2.9. Western Blot Analysis. After SDS-PAGE, gels were transferred to nitrocellulose membranes (Whatman, Germany), Western blot analyses were conducted using standard protocols

with appropriate primary and horseradish peroxidase (HRP)conjugated secondary antibodies or a HRP-conjugated antiFlag monoclonal antibody (1:1000 dilution, Sigma, St. Louis, MO). Dilutions of 1:1000 were used for the primary antibodies of monoclonal mouse anti-p62 (BD Transduction Laboratories, Bedford, MA) and p120 catenin (catenin δ-1; Cell Signaling, Danvers, MA). HRP-conjugated anti-mouse IgG and HRPconjugated anti-rabbit IgG (Cell Signaling, Danvers, MA) were each used at a dilution of 1:10 000. ECL (Amersham Biosciences, U.K.) was used for signal detection. 2.10. Immunofluorescent Staining. MDA-MB-231 cells (ATCC, Manassas, VA) were transiently infected with recombinant adenovirus Adv-5′flag-pdef in a chamber-slide (Nalge Nunc International, Rochester, NY). Two days after viral infection, the cells on the slide were washed twice with Tris Buffered Saline (TBS). The cells were then fixed with freshly prepared methanol/acetone (1:1, v/v) for 1 min at room temperature and washed with TBS four times. The cells were incubated with FITC-conjugated anti-FLAG M2 at 10 µg/mL (Sigma, St. Louis, MO) in TBS at room temperature for 1 h. After washing cells twice with TBS, the slide was coverslipped with Vectashield Mounting Medium containing 4′-6-Diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA) to counterstain nuclei. Fluorescence images were captured using a fluorescence microscope (Zeiss, Exton, PA).

3. Results 3.1. Isolation of a PDEF Interacting Protein Complex. To identify proteins interacting with PDEF, FLAG-tagged PDEF was overexpressed in the MDA-MB-231 human breast cancer cell line by adenovirus-mediated gene delivery. MDA-MB-231 cells do not express endogenous PDEF,17 and generally exhibit a low transfection efficiency. Therefore, we used adenovirus-mediated Pdef gene delivery to express PDEF. After Adv-5′flag-pdef infection of cells, PDEF-associated proteins were isolated by coimmunoprecipitation using anti-FLAG conjugated to agarose beads (Figure 1). To monitor the overexpression levels of Pdef in Adv-5′Flagpdef infected cells, the expression levels of PDEF after the infection of Adv-5′Flag-pdef were evaluated by comparing them with MDA-MB-453 cells which are known to express Pdef at high levels. RT-PCR analysis showed that the Pdef expressions after the adenoviral infection were very similar to or a little bit higher to the MDA-MB-453 cells (Figure 2). After infection with Adv-5′flag-pdef or Adv-β-gal, MDA-MB-231 cells were lysed and PDEF expression was confirmed by Western blot analysis of total cell lysates (TCL) and anti-FLAG immunoprecipitates. As shown in Figure 3A, by Western blot analysis using Anti-FLAG antibodies, PDEF protein was detected in the TCL and 3× Flag peptide-eluted samples of the immunoprecipitates from cells infected with Adv-5′flag-pdef, but not Adv-β-gal (control). PDEF was detected as two main bands at approximately 35 and 45 kDa in MDA-MB-231 cells (Figure 3A), although PDEF is detected as one band (approximately 45 kDa) in MCF-7 breast cancer cells (data not shown). These data suggest that alternative splicing variants or different post-translational modifications may occur in the MDA-MB-231 cells. PDEF-interacting proteins, which coprecipitated with PDEF, were separated on SDS-PAGE and visualized by Coomassie blue staining (Figure 3B). Numerous protein bands were observed in samples immunoprecipitated from Adv-5′flag-pdef- infected cells, but not Adv-β-gal-infected cells. Two independent experiments of Journal of Proteome Research • Vol. 8, No. 3, 2009 1329

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Figure 1. Schematic diagram of the experimental procedure. PDEF was overexpressed using an Adv-5′flag-pdef vector and PDEF bound protein complexes were isolated using anti-Flag M2Agarose beads. The proteins were then separated by SDS-PAGE, and peptides generated by in-gel digestion were analyzed by nanoLC-MS/MS.

Figure 2. RT-PCR analysis of PDEF mRNA expression. MDA-MB231 cells infected with Adv-β-Gal or Adv-5′flag-pdef vector and MDA-MB-453 parent cells were lysed 48 h after infection. Total RNAs isolated were treated with DNase I and cDNAs were synthesized for the RT-PCR analysis as shown in Materials and Methods. The PDEF mRNA expression levels from the MDA-MB231 cells infected with Adv-5′Flag-pdef were very similar to or a little bit higher to the MDA-MB-453 cells known to express PDEF at high levels.

infection and LC-MS/MS were performed and representative data are presented. 3.2. Identification of PDEF Binding Proteins. To identify all possible proteins interacting with PDEF, we used highthroughput LC-MS/MS proteomics technology. Coimmunoprecipitates from both β-gal and PDEF cell lysates were separated by SDS-PAGE and the gel lanes were excised and divided into 25 protein bands. Each band was in-gel trypsin digested and further separated by reverse-phase liquid chromatography using a column directly coupled with an electrospray ionization (ESI) tandem mass spectrometer. Precursor mass and CID fragmentation mass data for the tryptic peptides were searched against a human IPI proteome database (IPI v3.09, 8/03/2005). After filtering the data, as described in Materials and Methods, we first identified 299 proteins in the 1330

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Figure 3. Isolation of a PDEF-interacting protein complex. MDAMB-231 cells infected with Adv-5′flag-pdef vector were lysed 48 h after infection. The whole-cell lysates were immunoprecipitated using anti-FLAG M2 agarose bead. (A) The lysates were immunoblotted with an anti-FLAG M2-peroxidase conjugated antibody. (B) SDS-PAGE gels of the cell lysates after infection were stained with Coomassie G250. TCL, total cell lysates; F/Th, flow through.

Proteomic Analysis of a PDEF Ets Transcription Factor

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PDEF sample and 10 proteins in the β-gal sample. Some of the proteins in the β-gal control were keratins, which are likely to have been introduced during sample handling. The 4 major keratins detected were keratin type I cytoskeletal 10, keratin 9, keratin type II cytoskeletal 1, and keratin type II cytoskeletal 2 epidermal. Some proteins appeared in both control and PDEF lanes. These proteins were considered to be proteins nonspecifically trapped in the M2 anti-FLAG antibody-conjugated agarose beads. Thus, these proteins were not analyzed further. We searched DTAselect-passed proteins with low scores in the control lane and identified 3 more proteins that were present in both the control and PDEF samples. After subtracting these 3 proteins, a total of 286 unique proteins were present in the PDEF sample (Table 1). Additional information for the 286 proteins is available in Supporting Information Table 1. Although there were 7 unique proteins in the β-gal control sample, their spectral counts were very low (2 or 3). Without capture of a FLAG-tagged bait protein (in our case, FLAGPDEF), these 7 proteins may have had access to the M2 antiFLAG antibody-conjugated agarose beads and remained bound after washing. 3.3. Selection of Potential PDEF Interactors. To remove nonspecific binding false positives from the 286 candidate proteins identified as potential PDEF interactors, we used the previously published protein-protein interaction (PPIs) database from the NCBI (ftp://ftp.ncbi.nlm.nih.gov). True interactors are more likely than noninteractors to exhibit the following characteristics: (1) interact densely with each other (high neighbor interaction degree coefficient15,16), (2) have small distances between them (minimum shortest path), and (3) form highly connected local networks (high clustering coefficient15,16) in the global PPI network reconstructed using all known PPIs. On the basis of these characteristics, we developed a statistical approach to estimate the probability for each identified potential PDEF interactor of belonging to a set of the same size of randomly selected interactors by chance as follows. (1) A total of 230 proteins having interactions in the known PPI network were selected. (2) Three measures,15,16 the minimum shortest path between the protein and remaining selected PDEF interactors, the neighbor interaction degree coefficient and the cluster coefficient, were computed. (3) A set of the same number of proteins were randomly selected 100 times from the known PPI network, and the three measures were computed for all proteins in each random sampling. (4) A joint probability density function was estimated using 100 sets of the three measures computed from step 3. (5) For each of the observed PDEF interactors, a p-value determining whether the protein belongs to a random set of interactors was computed using the joint distribution from step 4. (6) Finally, 121 proteins with p-values less than 0.05 were selected as potential true PDEF interactors (Table 2). Note that 56 proteins (Supporting Information Table 3) for which no associated interactions are available in the known PPI network were not evaluated by the above analysis, and thus, no p-values were computed for these proteins. See Supporting Information for further details of the computational procedure. 3.4. Reconstruction of a PDEF Network. We reconstructed a hypothetical protein network to predict the potential roles of PDEF by investigating in which cellular events the selected PDEF interactors are involved. After reconstructing an initial network with the first neighbors of the 121 selected interactors, we removed the proteasome and nuclear transport network modules. These modules were considered less meaningful

because they are related to general processes that a transcription factor undergoes. For instance, the interactors which are subunits in the proteasome (PSMA7, PSMC1/2/3/5, and PSMD2) may be captured while PDEF is being degraded and those comprising nuclear transport complexes (importins and RAN) may be captured while the PDEF is being transported into the nucleus. Thus, after applying this biological filter, we focused on the remaining network modules shown in Figure 4: EGFR signaling, mRNA processing, cell cycle, DNA repair, tRNA biosynthesis, and cytoskeleton organization. The heat shock proteins were included because they interact with some of the network modules (e.g., EGFR signaling). Note that each network module is associated with a biological process (e.g, RNA processing), as determined by examining the GO Biological Processes (GOBPs) database. 3.5. Characteristics of PDEF-Interacting Proteins. To further characterize the 121 proteins identified, we classified them by biological processes. Biological process ontologies for the 121 proteins were found in the Web site http://genomewww5.stanford.edu/cgi-bin/source/sourceResult (Figure 5). Of the total 121 proteins, 5% take part in protein catabolism, 7.4% are involved in transport and 4.1% in tRNA aminoacylation. Some of the proteins have roles in protein import into the nucleus and protein folding. Higher fractions of the proteins were allotted to cytoskeleton organization (17.3%), cell cycle and DNA metabolism (13.2%), and mRNA processing (9.9%). Some of transcription factors interact not only with DNA, but also with single-stranded RNAs and associated-proteins.18 Thus, it is not surprising that some mRNA-associated proteins such as poly(A)-binding protein 1 interact with PDEF. Cellular localization of the proteins was also analyzed. The network analysis indicated that the 121 candidate proteins were primarily cytoplasmic or nuclear (Supporting Information Table 2). Since the Ets transcription factor protein is synthesized in the cytoplasm, it may interact with signaling molecules and other proteins and, once activated, may be translocated into the nucleus where it binds to its target DNA. However, whether PDEF exists in the cytoplasm and is activated by signaling pathways and then translocates into nucleus is not known. Thus, we overexpressed FLAG-tagged PDEF using the adenoviral vector in MDA-MB-231 cells and immunocytochemical staining of the cells was performed using anti-FLAG to localize PDEF. PDEF was more predominant in the nucleus than in the cytoplasm of the cells (Figure 6). However, there was moderate staining in the cytoplasm suggesting that some PDEF is also present in the cytoplasm. 3.6. Confirmation of the PDEF Interaction with Catenin δ-1AB and p60. Immunoprecipitation and Western blot (IPWB) analysis were performed to confirm the association of some components with PDEF (Figure 7), in particular, a 1AB isoform of catenin δ-1, which is an efficient tyrosine kinase substrate in ligand-induced receptor signaling through the EGF, ERBB2 receptors (Figure 7A) and p60 (Sequestosome 1, p62) which is a serine/threonine kinase (Figure 7B). Although p60 was not in the 121 selected proteins after network analysis, our IP-WB experiment revealed a direct or indirect interaction between p60 and PDEF.

4. Discussion We used proteomic and bioinformatic approaches to analyze a PDEF interacting protein complex. We originally identified 299 potential interacting proteins and then narrowed down the potential candidates to 121 proteins as PDEF binding partners Journal of Proteome Research • Vol. 8, No. 3, 2009 1331

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Table 1. Total 286 Proteins Originally Identified by LC-MS/MS Analysis

in MDA-MB-231 cells. To identify proteins nonspecifically binding to anti-FLAG-agarsose beads, we infected cells with Adv-β-gal and examined lysates from these cells by mass 1332

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spectrometry analysis and database searching. Only 13 proteins, primarily keratins, were identified in this control. Keratins are common contaminants in mass spectrometry-based proteom-

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Proteomic Analysis of a PDEF Ets Transcription Factor a

Table 2. Selected 121 Proteins by the Analysis of Published Protein-Protein Interactions

a

The categories are regrouped by GO biological processes.

ics studies. These proteins were removed from the list of potential PDEF interactors, leaving 286 candidate PDEFinteractor proteins. These proteins include cytoplasmic and nuclear proteins involved in EGFR signaling, mRNA processing, cell cycle, DNA repair, tRNA biosynthesis, and cytoskeleton organization. Integration of our data with the protein-protein interaction data from the Human Proteome Reference Database allowed us to build a comprehensive interactome map for PDEF interactions. This interactome map consists of several modules, suggesting that PDEF may have versatile functions in cells. The ErbB family of receptor tyrosine kinases consists of four cell surface receptors: EGFR/ErbB1/HER1, ErbB2/HER2, ErbB3/ HER3, and ErbB4/HER4. These ErbB receptors are well-known mediators of cell proliferation and differentiation. EGFR and

ErbB2 are often overexpressed or amplified in cancers.19 In particular, HER2/neu is a tyrosine kinase belonging to the EGFR/ErbB family and is overexpressed in approximately 15-35% of breast cancers.20-23 The overexpression of HER2 is associated with an increased tendency to metastasize and respond poorly to hormonal and chemotherapeutic agents and a poor prognosis for the patients. Our candidate PDEFinteracting proteins include EGFR and possibly its associating signaling proteins such as erbin and catenin-δ1. Interestingly, ErbB receptors have been detected in the nucleus and may also have transcriptional functions within the nucleus of the cell.24 PDEF may receive Her2 signals through Stat1 and cateninδ1. In cDNA microarray and Real-Time PCR assays, two genes, catenin-δ1 (delta-Catenin; CTNND1) and prostate-specific Journal of Proteome Research • Vol. 8, No. 3, 2009 1333

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Figure 4. A hypothetical protein interaction network for PDEF. Shaded circles indicate the proteins belonging to the 121 selected PDEF interactors and hollow circles are the proteins added to the network as first neighbors during network reconstruction (see Materials and Method for further details on network reconstruction). The undirected edges and the arrows denote known PPIs and signal transduction interactions between proteins in the network, respectively. The potential roles of PDEF may be predicted by the interaction of PDEF with the shaded proteins in the network modules (see Results).

Figure 5. Physicochemical characteristics and GO biological process distribution of the selected 121 proteins. Percent distribution of GO biological process for the selected PDEF interactome is presented. Detailed information is available in Supporting Information Table 1.

membrane antigen (PSMA), were significantly overexpressed in prostate cancer compared to benign prostate hyperplasia.25 1334

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Additionally, in a Her2 signal transduction pathway analysis by MS-based quantitative proteomics, many phosphoproteins were identified, including many known Her2 signaling molecules as well as known EGFR signaling proteins. Stat1, Dok1, and catenin-δ1 were also included in these EGFR signaling proteins which had not been previously linked to Her2.26 Catenin-δ1 may be overexpressed in cancers, and in breast cancer may be activated by Her2 signaling. Our data also revealed interactions between Stat1, ErbB2 interacting protein, and catenin-δ1 and PDEF (Figure 4). These signaling pathways are distinct from the classical Her2 signaling Erk/MAPK pathway and are involved in cooperation with PDEF to promote breast cancer cell motility and invasion.11 Thus, these observations strongly suggest that PDEF is affected by Her2 signal transduction in certain breast cancer cells and are consistent with PDEF expression profiles in breast cancer cells.12,27,28 SkBr-3 breast cancer cells, which show high levels of Her2 expression and no metastatic property in xenografts, highly express PDEF mRNA; MCF-7 cells, which show intermediate levels of Her2 expression and no metastasis, have intermediate levels of PDEF expression, and MDA-MB-231 cells, which do

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Figure 6. PDEF is expressed predominantly in the nucleus. Immunofluorescent staining was conducted to localize PDEF within the cell. In MDA-MB-231 cells virally infected with Adv-5′Flag-PDEF, PDEF was detected using an anti-FLAG M2 FITC conjugate after 48 h. The micrographs show that PDEF is more predominantly localized in the nucleus rather than in the cytoplasm. However, there was moderate staining in the cytoplasm, suggesting that some PDEF is also present in the cytoplasm. (A) DAPI staining of the nucleus, (B) anti-Flag of PDEF, (C) merge of DAPI and anti-Flag staining.

Figure 7. PDEF interacts with Catenin δ-1 and p60. MDA-MB-231 cells were infected with Adv-5′flag-pdef and subjected to IP using anti-FLAG M2 agarose beads. Immunoprecipitated complexes were analyzed by Western blotting using anti-catenin δ-1 (A) and anti-p60 (B). These data confirm the interaction of PDEF with catenin δ-1, and p60 directly or indirectly, as identified by IPLC-MS/MS analysis.

not express Her2 and have metastatic property in xenografts, have little or no expression of PDEF mRNA. Our PDEF interactome list showed the interaction of cell cycle related molecules such as Proliferating cell nuclear antigen (PCNA). There have been reports that PDEF promoted anchorage-independent growth of ErbB2-expressing cells. With the use of laser capture microdissection, Gunawardane et al. found that PDEF mRNA is overexpressed in breast tumor epithelia throughout tumor progression. These findings suggest that the transcription factor PDEF may play positive roles in breast tumorigenesis.11 In the immunohistologic examination, Ets-1 was expressed in the proximal tubules and coexpressed with PCNA. Furthermore, overexpression of Ets-1 promoted the cell cycle and increased the promoter activity and protein expression of cyclin D1 in LLC-PK1 cells.29 Thus, our data also implies that the expression of PDEF in breast cancer cells might promote the growth of cancer cells by interacting with PCNA. However, it has also been reported that the expression of PDEF in breast cancer cells leads to inhibition of invasion, migration, and growth and the growth-suppressive effects of PDEF expression are mediated in part by a G(0)-G(1) cell cycle

arrest associated with elevated p21 levels.12 Gu et al. also reported that reduced expression of PDEF leads to a morphologic change, increased migration and invasiveness in prostate cancer cells.17 Our list showed the interaction of cell cycle related molecule CDC2, which is pivotal in regulating the G2/M cell cycle transition checkpoint. It has been demonstrated that one of Ets factors, Ets-2, plays a direct role in the regulation of cdc2 expression and may participate in the coordinated regulation of cdc2-cyclin A expression which is essential for the modulation of cdc2-regulated processes.30 It has also been found that Cdk10, a CDC2-related kinase, binds to Ets2 in vitro and in vivo and inhibits Ets2 transactivation in mammalian cells.31 However, recent report showed that binding of murine cdk10 to Ets2 transcription factors in vitro did not show a direct involvement in the G2/M transition and, therefore, did not affect the proliferation rate of the cell lines analyzed.32 According to the breast cancer cell line properties, high expression of HER2 results in high PDEF proteins and low metastatic properties. Also, our PDEF-CDC2 interaction data and reports seem to support that PDEF might have inhibitory effect on the tumorigenesis. However, since it is still not clear that PDEF binding to CDC2 will have stimulating or inhibiting effect on the tumorigenesis of breast cancer cells, further studies are necessary to clarify it. Thus, our current results imply both aspects of the roles of PDEF in the invasion, migration and growth of breast cancer cells. Therefore, it needs further experiments to clarify the roles of PDEF in the tumorigenesis. PDEF-interacting proteins are localized not only in the nucleus, but also in the cytoplasm, as well as in other subcellular compartments. Similar phenomena have been observed in other protein interaction studies based on global proteomics analysis. For example, the transmembrane receptor beta-arrestin interacts with nuclear proteins as well as proteins from other subcellular compartments.33 Other cytosolic proteins found to be associated with PDEF, such as tRNA synthases, cytoskeletal motors, HSPs, ubiquitin-protein ligase E3C, and proteasome subunits, may bind to PDEF in the process of PDEF protein synthesis, movement, modification, degradation, and/or activation or deactivation by signaling cascades (Table 1). Although it is still unknown whether PDEF can be ubiquitinated and degraded in the proteasome, our data strongly suggest that PDEF may be processed by the ubiquitinationproteasome pathway. Although numerous transcription factors shuttle between the cytoplasm and nucleus, whether PDEF may Journal of Proteome Research • Vol. 8, No. 3, 2009 1335

research articles exist in the cytoplasm, interact with other signaling molecules, and then become activated and translocated into the nucleus is currently unclear. Our immunocytochemical staining results show that PDEF is located primarily in the nucleus, but some PDEF proteins are present in the cytoplasm (Figure 6). This result suggests that PDEF may interact with other partner proteins in the cytoplasm and can be activated by, or cotranslocated into nucleus with, these proteins. Interestingly, our data showed the differences of the molecular identity of PDEF proteins after infection with Adv-FLAGPDEF into two different breast cancer cell lines. Both MDAMB-231 cells and MCF-7 cells are breast cancer-origin ones. But, it is known that MCF-7 cells are ER positive and have no metastasis in animal model, but MDA-MB-231 cells are ER negative and have metastasis. Currently, it is not clear whether these characteristics of cell context have some relationships with different molecular appearances of PDEF proteins in these cells. Further studies are required to reveal how these differences are produced. Although not included in our final PDEF-interacting network analysis, p60 (sequestosome 1 or p62) was originally found in the PDEF complex and the interaction was confirmed by IPWB analysis. p60 is a multifunctional cytoplasmic protein able to noncovalently bind ubiquitin and several signaling proteins. A previous report has indicated that p60 is overexpressed in breast cancer and that PDEF stimulates p60 promoter activity.34 Thus, it is quite interesting that PDEF also binds directly or indirectly to p60. Since p60 can noncovalently bind free ubiquitin, serving as an ubiquitin sink,35 in breast cancer cells, elevated p60 may be involved in ubiquitin-proteasome mediated-degradation of certain proteins, including PDEF. Our PDEF interactome data showed some nuclear proteins such as cell cycle and DNA repair proteins, but not many transcriptional factors are found (Table 1). This might be due to the fact that we used total cell lysates, instead of nuclear enriched proteins, to see the PDEF interactomes in whole cell context. We expect that, in the future studies, nuclear enrichment and liquid separation of proteins, instead of in-gel method, will help to identify more transcription factors interacting with PDEF. Although our PDEF interactome data for breast cancer cells did not indicate an interaction with Nkx3.1 or the androgen receptor, as previously shown in prostate cells,8,36 our data revealed the possible involvement of Her2 signaling in PDEF activation through ERBB2IP, catenin-δ1, and Stat1. Many other interacting proteins, such as MYB binding protein (p160), CDC2, PCNA, thymopoietin, and nucleophosmin, may also play a role in PDEF activation in breast cancer cells, and the roles of these proteins need to be further analyzed in the future.

Acknowledgment. This work was supported by a Korea Science and Engineering Foundation (KOSEF) grant (M10649000184-08N4900-18410), a grant from the 21C Frontier Functional Proteomics Project (FPR08A2-120), and, partially, by a KOSEF Grant (M1AN 29-2007-03990) from the Korean government (MEST). Supporting Information Available: 3-D scatter plots of measures before and after the modification of the minimum shortest path and scaling of the three measures, distribution of d0, and that of the observed d, and the histogram of p-values of 230 identified proteins. Supplementary Table 1, descriptive names, IPI numbers, coverage %, peptide numbers (#) and SEQUEST scores of the original 286 PDEF-associated proteins; 1336

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Cho et al. Supplementary Table 2, detailed information of the selected and categorized 121 PDEF interactor proteins; Supplementary Table 3, the presence of absence of the interactions in the known PPI network and their p-values of the original 286 PDEFassociated proteins. This material is available free of charge via the Internet at http://pubs.acs.org.

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