PIG8, Using

An Analysis of an Interactome for Apoptosis Factor, Ei24/PIG8, Using the Inducible Expression System and Shotgun Proteomics Young Yil Bahk,*,† Jaehoon Lee,‡ Ick-Hyun Cho,§ and Han-Woong Lee*,| Department of Integrated OMICS for Biomedical Sciences, Graduate School, Yonsei University, Seoul 120-749, Korea, Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 446-746, Korea, Department of Dermatology, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul 135-710, Korea, and Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul 120-749, Korea Received June 3, 2010

ei24 (etoposide-induced 2.4 kb transcript, also designated PIG8 (p53-induced gene 8)), is a DNA damage response gene involved in growth suppression and apoptosis. ei24 gene expression is specifically induced by wild type p53, and its overexpression suppresses cell growth by inducing apoptotic cell death. Generally, the protein-protein interaction is known to regulate their targets, as well as to modify cell fates. In this study, using the established NIH/3T3, oncogenic H-Ras/G12V transformed NIH/3T3, and B16F10 cells, which are expressing mouse Ei24 proteins under the tight control of expression by doxycycline, a proteomic screening was conducted to identify the binding partners for Ei24. Immunoprecipitation of mEi24 and associated proteins was performed using the mEi24 expressing cell lysates. Isolated immuno-complexes were resolved by SDS-PAGE and analyzed by liquid chromatography tandem mass spectrometry. There were 61 novel potential mEi24 interacting proteins identified, among which are NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16F10/mEi24 cells; however, some mEi24 interacting proteins were specific to two NIH/3T3 related cells and to B16F10/mEi24 cells. This approach led to the identification of many interacting partners, and the discovery of these associated proteins will lead to a better understanding of the mechanisms underlying the physiological and cell biological roles of Ei24. Keywords: Ei24/PIG8 • apoptosis factor • inducible cell lines • interactome

Introduction

sion by monitoring DNA damage and executing pathways that negatively control cell growth.

p53 is a transcription activator that induces expression of numerous downstream target genes in response to intra- and extracellular stress signals, including DNA damage, oxidative stress, and activated oncogenes. The functions of p53 cover diverse aspects of cell biology, including cell cycle control, apoptosis, metabolism, fertility, differentiation, and cellular reprogramming. The role of p53 as the “gatekeeper” in the cellular defense against genotoxic damage and its function as a critical tumor suppressor, protecting the organism from potentially harmful cells which incurred DNA damage, are well documented.1,2 Clinical studies and mouse models have demonstrated that inactivation of the tumor suppressor p53 occurs in more than 50% of human cancers and is functionally inactivated in many more.3-5 p53 functions in tumor suppres-

Ei24 (Etoposide-induced 2.4 kb transcript, also designated PIG8 (p53-Induced Gene 8)) is a protein produced by ei24, a DNA damage response gene involved in growth suppression and apoptosis. ei24 gene expression is specifically induced by wild type p53, and overexpression suppresses cell growth by inducing apoptotic cell death. ei24 was first isolated by differential display in NIH/3T3 mouse embryonic fibroblasts as an etoposide-induced gene associated with apoptosis by etoposide.6 Etoposide, a cancer drug, inhibits the enzyme topoisomerase II, which unwinds DNA, and by doing so causes DNA strands to break. Cancer cells are less able to repair this damage than healthy cells. It is used as a form of chemotherapy for cancers such as Ewing’s sarcoma, lung cancer, testicular cancer, lymphoma, nonlymphocytic leukemia, and glioblastoma multiforme.7 ei24, which is located on human chromosome 11q24, a region frequently altered in cancers, and the proximal region of mouse chromosome 9,8 is highly conserved between mouse and human (98% homologous) suggesting an important biological role, and its expression in NIH/3T3 cells is induced following etoposide treatment. The ei24 gene is widely expressed in many human tissues including heart, liver, placenta, skeletal muscle, and pancreas.9 This gene has higher expression

* Corresponding authors. Young Yil Bahk, PhD. Phone: 82-2-2123-3866. Fax: 82-2-2123-8682. E-mail: [email protected]. Han Woong Lee, PhD. Phone: 82-2-2123-5698. Fax: 82-2-2123-8107. E-mail: [email protected]. † Graduate School, Yonsei University. ‡ Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine. § Department of Dermatology, Sungkyunkwan University School of Medicine. | College of Life Science and Biotechnology, Yonsei University.

5270 Journal of Proteome Research 2010, 9, 5270–5283 Published on Web 08/23/2010

10.1021/pr100552y

 2010 American Chemical Society

Interactomes of Ei24/PIG8 in wild type p53-expressing cells and is an immediate-early induction target gene of p53-mediated apoptosis.8,10 The protein encoded by this gene contains six putative transmembrane domains and may suppress cell growth by inducing apoptotic cell death through the caspase 9 and mitochondrial pathways, which is triggered at the endoplasmic reticulum when levels of Ei24 increase after induction by p53.11 Although Ei24 is indispensable for establishing the p53-triggered signaling events and is reported to be lost in aggressive breast cancer and to play a pivotal role in prevention of tumor spread in invasive breast tumors,12 very little biological progress and research attention on Ei24 have been made on its characterization, activity, and function, and the protein network in normal and malignant cells largely remains elusive. We have established NIH/3T3 mouse embryonic fibroblast, oncogenic H-Ras/G12V transformed NIH/3T3, and B16F10 mouse melanoma carrying an inducible form of mouse Ei24 (mEi24) and analyzed interacting proteins with Ei24. Although some proteins act primarily as single monomeric units, a significant portion of all proteins function in association with partner molecules or as components of large molecular assemblies. Thus, the protein complexes are critical in fulfilling the various cellular functions, and the protein complexes are more than the sum of the components and represent a fundamental level of integration of the information encoded by individual genes.13 Principally, detailed investigation of protein-protein interactions and their regulation is a prerequisite for gaining detailed insight into signaling pathways. Although it has become clear that no single technique would be sufficient to meet all the requirements of proteomics-based investigations, to date proteomics techniques are applied for the investigation of protein expression profiles, protein-protein interactions, and also protein quantification and have become more and more accepted as promising techniques in various research. Some of the proteomics results can be used to confirm interactions, which have already been assumed before, but others have delivered a huge number of novel and interesting correlations that might help in the development of novel strategies for the respective protein networks. Moreover, it is naturally agreed that systematic deciphering of proteinprotein interactions has potential to generate comprehensive and instructive signaling networks and to fuel new discovering strategies. Therefore, we used a proteomics approach that combined coimmunoprecipitation experiments, one-dimensional electrophoresis, and identification of interacting partner proteins by mass spectrometry using the established cell lines, which are expressing mouse Ei24 proteins under the tight control of expression by an antibiotic, doxycycline, to identify the novel interactors for Ei24. In this study, we arrived at some new insights regarding biological clues for unraveling the protein network directly and/or indirectly caused by p53 and Ei24.

Experimental Procedures Cell Culture and Establishment of Inducible Oncogenic Ei24-Expressing Cell Lines. NIH/3T3 mouse embryonic fibroblasts were bought from American Type Culture Collection (ATCC, CRL-1658, Bethesda, MD, USA), maintained in DMEM (Invitrogen, Carlsbad, CA, USA) containing 10% heat-inactivated bovine calf serum (Hyclone, Logan, UT, USA), and supplemented with antibiotics in a humidified incubator at 37 °C with 5% CO2. B16F10 mouse melanoma cells were kindly donated by Dr. Sung Hee Baek from the Department of

research articles Biological Sciences, Creative Research Initiative Center for Chromatin Dynamics, Seoul National University, Seoul, Korea. Cells were subcultured after they became 85% confluent. Tetracycline-inducible cell lines were established as previously described.14,15 The established mEi24 inducible NIH/3T3, oncogenic H-Ras/G12V transformed NIH/3T3 (H-Ras/G12V/NIH/ 3T3), and B16F10 cell lines were designated as NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16F10/mEi24, respectively. The mEi24 was tagged with Flag sequence in the C-terminal region of the cDNA. The sequence fidelities were verified with an ABI Prism dye terminator cycle sequencing kit (Applied Biosystems, Foster City, CA, USA). Each pTRE-Ei24/Flag-IRES enhanced green fluorescence protein (EGFP) construct was cotransfected with pEF-Puro-Tet-On into each NIH/3T3, H-Ras/ G12V/NIH/3T3, or malignant B16F10 cells using FuGENE 6 transfection reagent (Roche, Mannheim, Germany) according to the previously described procedures.14,15 After puromycin selection (10 µg/mL) and microscopic screening assays based on the simultaneous expression of EGFP, individual puromycinresistant clones were selected, amplified, and screened by Western blotting for inducible expression (with or without doxycycline; Clontech Laboratories, Palo Alto, CA, USA) of mEi24 with Flag tagging antibody (M2 monoclonal antibody from Sigma, St. Louis, MO, USA). Individual clones are referred to collectively as NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/ mEi24, and B16F10/mEi24. At least three independent sets of cultures of each mEi24-expressing cell were individually assessed for density of culture and Ei24 expression based on the microscopic assay for green fluorescence protein expression and the Western blot analyses with Flag M2 antibody. Immunoblot Analysis and Immunoprecipitation Assays. Proteomic screenings were performed using immunocomplexes immunoprecipitated from total cell lysates of NIH/3T3, H-Ras/ G12V/NIH/3T3, and B16F10 cells inducibly expressing mEi24/ Flag for 72 h, respectively. Cells were lysed using a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 containing protease inhibitor cocktail (Roche)). Protein concentrations were normalized using the Bradford colorimetric method or BCA reagent against known concentrations of BSA. For immunoblot analysis, equal amounts of protein (30 µg) were resolved on 10% SDS-PAGE gels and separated by the standard SDS-PAGE method. Proteins were electrophoretically transferred to the NC membrane (PROTEAN, Whatman GmbH, Dassel, Germany) using Semi-Dry Transfer Blotter (Amersham Biosciences, Buckinghamshire, UK) and blocked with 5% nonfat dry milk in TBS. To detect mEi24 expression, the blots were probed with the primary antibody directed against Flag epitope followed by HRP conjugated secondary antibodies (BioRad Laboratories, Hercules, CA, USA). The detection of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) with a rabbit polyclonal antibody as the primary antibody (AbFrontier, Seoul, Korea) was used for an internal control. The immunoreactive proteins on the membrane were detected by chemiluminescence using the West-Save substrate (AbFrontier) onto X-ray film (Agfa-Gevaert N.V., Mortsel, Belgium). For reprobing the blots, the blots were washed in 1X TBS three times to remove chemiluminescent substrate and incubated with BlotFresh Western Blot Stripping Reagent (SignaGen Laboratories, Gaithersburg, MD, USA) for 15 min at room temperature with vigorous agitation according to the manufacturer’s instruction. For immunoprecipitation assay and protein identification with mass spectrometry for interacting proteins, lysates in the lysis buffer containing 10 mg of total protein from the mEi24 Journal of Proteome Research • Vol. 9, No. 10, 2010 5271

research articles induced cells were precleared using the appropriate isotype IgG antibody followed by mixing 20 µg of anti-Flag M2 antibody conjugated into protein A agarose (Sigma, A-2220) and then incubating with gentle shaking for 5 h at 4 °C. For coimmunoprecipitation for the interacting proteins with mEi24, 1 mg of cell culture lysates and 20 µL of anti-Flag M2 antibody conjugated into protein A agarose (50% bead slurry) were used. Antibodies used for coimmunoprecipitation were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA): karyopherin β1 (sc-11367), Bcl-2 (sc-7382), ATP 5B (sc-166443), calnexin (sc-11397), prohibitin (sc-56346), proteasome (prosome, macropain), 26S subunit non-ATPase 2 (PSMD2) (sc-68352), and caveolin-1 (sc-894), as well as anti-Flag antibody (M2) for a positive control. For immunoprecipitation of karyopherin β1, mitochondrial ATP synthase β subunit (ATP 5B), and prohibitin, the precleared cell lysates (500 µL in volume) in the lysis buffer containing protease inhibitor cocktail were mixed with appropriate primary antibodies directed against individual proteins followed by incubating with gentle rocking for 5 h at 4 °C. Then, 25 µL of Protein A agarose beads (50% bead slurry, Peptron Inc., Daejeon, Korea) was added into the above immunocomplexes and incubated with gentle rocking overnight at 4 °C. The immunocomplexes were washed with the cold lysis buffer three times, and the antibody-selected proteins were eluted from the antibody protein A beads by boiling in 2X SDS-loading buffer (100 mM Tris-HCl, 10% glycerol, 2% SDS, and 0.05% bromophenol blue) for 5 min. The sample was resolved on a 10% SDS-PAGE and transferred to NC membranes as described above. Similar conditions with the same antibodies without induced expression of mEi24 were used for the negative control lane. For mass spectrometry analysis, after staining with colloidal Commassie brilliant blue, lanes were excised and cut into 10 equivalent slices, destained, digested, and extracted using the previously described procedure.15 For immunoblot analysis for the immunocomplex, the membrane was then used with the same procedure as that mentioned above. Indirect Immunofluorescence Assay. Cells cultured on coverslips were treated with 2 µg/mL of doxycycline for 36 h and then fixed in 10% formalin and permeabilized with 0.5% Triton X-100. After blocking for 1 h with 5% BSA in PBS at room temperature, the cells were incubated with primary antibodies in 2.5% BSA at 4 °C overnight. Primary antibodies used were as follows: rabbit anti-Bcl-2 (Santa Cruz Biotech), 1:100; rabbit anticalnexin (Santa Cruz Biotech), 1:100; rabbit antikaryopherin β1 (Santa Cruz Biotech), 1:100; and mouse anti-M2 Flag (Sigma), 1:2000. Alexa Fluor 488- or 568-conjugated secondary antibodies (Invitrogen) were added for 1 h at RT or room temperature followed by nuclear staining with 4′-6-diamidino2-phenylindole (DAPI, Sigma). The images were obtained by a confocal fluorescence microscope with a 100× immersion lens (Zeiss, Exton, PA). In-Gel Trypsin Digestion and Mass Spectrometry Analysis. In an adapted in-gel digestion procedure,16 the evenly excised Coomassie Blue stained protein lanes were destained with 50% acetonitrile in 25 mM NH4HCO3 and dried in a vacuum centrifuge. The dried gel pieces were then rehydrated at 4 °C for 45 min with 20 ng/µL of trypsin (modified, sequencing grade, Roche) in 25 mM NH4HCO3. After replacement of the supernatant with 20 µL of 25 mM NH4HCO3, the samples were incubated overnight at 37 °C. The tryptic digested samples were analyzed by LC-ESI-MS/MS as described previously. A Dionex LC Packings nano HPLC system (LC-Packings, Amsterdam, the 5272

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Bahk et al. Netherlands), comprising a FAMOS autosampler, a Switchos 10-port valve unit, and an UltiMatePLUS nanoflow pumping unit, online coupled to a QSTAR Pulsar ESI-hybrid Q-TOF tandem mass spectrometer (Applied Biosystems, Foster City, CA) was used. The samples were desalted on a precolumn (2 cm × 200 µm i.d.; Zorbax 300SB-C18, 5 µm, Agilent technologies, Santa Clara, CA, USA) in a flow of 0.1% formic acid for 10 min at 4 µL/min and separated on an analytical column (15 cm × 75 µm i.d.; Zorbax 300SB C18, 5 µm, Agilent technologies, Santa Clara, CA, USA) by application of a gradient from 0 to 32% acetonitrile in 0.1% formic acid over 110 min at 200 nL/ min. The column outlet was coupled directly to the highvoltage ESI source (typically 2.3 kV), and peptides eluting from the column were sprayed directly into the orifice of the mass spectrometer. Information-Dependent Acquisition (IDA) mode was performed to acquire MS/MS spectra based on an inclusion mass list and dynamic assessment of relative ion intensity. MS spectra acquisition time was set at 3 s over the mass range of 400-1500 amu. For CID, nitrogen gas was used at a setting of 4, and the collision energy was set to automatic, allowing increased energy with increasing ion mass. A dynamic exclusion window was applied to prevent the selected peptide from further selection over 2 min. Database Searching. Peak lists of MS/MS spectra were processed using Analyst QS (v1.1, Applied Biosystems, Foster City, CA) software and searched against the International Protein Index (IPI) protein database version 3.20 (European Bioinformatics Institute, Hinxton, UK), the NCBI-nonredundant (nr) (version 14_08_2006, 486696 mammalian entries), and EST and other databases using Mascot17 operating on a local server (version 2.1, Matrix Science). Initial search parameters were the following: allowance of tryptic missed cleavages, 2; fixed modification, carbamidomethylation of cysteines; variable modification parameters, oxidation Met; peptide tolerance, 1.0 Da; MS/MS tolerance, 0.8 Da; default charge state, +2 and +3; and asparagine/glutamine (formally “deamidation”, but due to deisotoping artifact during data extraction). The candidate proteins with at least two peptides, whose probability MOWSE score is statistically meaningful (p < 0.05), were only selected using MSQuant, version 1.4.16a (www.cebi.sud.dk). Protein identifications with a single significant peptide were manually verified by the inspection of the MS/MS fragment ion spectrum. Different isoforms of the protein reported were verified by identification of at least one unique peptide. Only the highest scoring identification was selected where multiple protein’s IDs were listed.

Results and Discussion Establishment of NIH/3T3, Oncogenic H-Ras/G12V Transformed NIH/3T3, and B16F10 Cell Lines for an Apoptosis Factor, Mouse Ei24. After transfection of mEi24 cloned into the pTRE-IRES-EGFP vector, several candidates were primarily chosen based on their antibiotics selection (10 µg/mL of puromycin) and on their microscopic appearance for EGFP and were finally selected using Western blot analysis with anti-Flag M2 antibody in all of NIH/3T3 (Figure 1A), oncogenic H-Ras/ G12V transformed NIH/3T3 fibroblasts (Figure 1B), and malignant B16F10 melanomas (Figure 1C). This system appears to be close to ideal in some respects for this experiment. First, it is relatively easy to create. Second, in this biological context, recruitment of mEi24 expression is only responding to the cellular and physiological functions of this mEi24 protein level in these cells. Its expression of the reverse tetracycline repressor

Interactomes of Ei24/PIG8

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Figure 1. Establishment of three mEi24 expressing inducible NIH/3T3, H-Ras/G12V/NIH/3T3, and B16F10 cells. The expression levels of mEi24 inducible cells (NIH/3T3/mEi24 (A), H-Ras/G12V/NIH/3T3/mEi24 (B), and B16F10/mEi24 (C)) treated with or without 2 µg/mL of doxycycline for the indicated periods of time were monitored by immunoblot analyses with the specific antibody raised against the Flag sequence. For reverted cells, the cells were treated with the inducer for the indicated periods and, after replacement with fresh medium, allowed to grow for the indicated periods of time without the inducer. Background mEi24 expression for the uninduced cells was undetectable even after long exposure. The quantity of the applied protein was normalized by Western blot analysis with anti-GAPDH polyclonal antibody. (D) Morphology of parental NIH/3T3 and oncogenic H-Ras expressing transformed NIH/3T3 cells. (E) Microscopic assays for the EGFP expression in mEi24 inducible cells. Journal of Proteome Research • Vol. 9, No. 10, 2010 5273

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Bahk et al. 18,19

is driven by the EF-1a promoter. mEi24 Western blots gave no band on NC paper in parental cells (NIH/3T3 and B16F10) and uninduced cells in spite of very long exposure onto X-ray film, and an increase in mEi24 protein expression was only shown in response to doxycycline depending on the induction time with doxycycline. In the process of induction time and mEi24 protein expression, the appearance of cells with smaller and apoptotic morphology was also observed as previously described by Zhao et al.,11 and these cell physiologies might have been due to mEi24 protein expression. However, after treatment of doxycycline for the indicated periods of time, when the cells were detached and replated on plastic culture dishes with fresh medium without inducer, the mEi24 expression and the smaller and apoptotic morphology of the cells reverted to the original form (data not shown). Therefore, we used three different kinds of cell systems to purify mEi24 in complex with interacting proteinssNIH/3T3 mouse embryonic fibroblasts, oncogenic H-Ras/G12V transformed NIH/3T3 fibroblasts, and malignant B16F10 mouse melanoma cellssthat express mEi24 under the tight control of an antibiotic, doxycycline, as an inducer. Each cell inducibly expressing mEi24 (designated as NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16F10/mEi24) expresses mEi24 at high level with the inducer monitored by Western blot with the Flag M2 antibody from Sigma Co. (Figure 1) and by microscopic assays for EGFP expression (Figure 1E) and is a suitable, unique, and useful system for identifying mEi24 interactome. Purification of mEi24 Immuno-Complexes and Identification of Copurifying Proteins in the Interactome of mEi24. The identification of protein-protein interactions is key to probing the specificity and fidelity of many cellular physiological processes. Recently, proteomic technology combining affinity purification of native protein complexes, separation on onedimensional SDS-PAGE, and mass spectrometry is powerful for revealing interacting partners which are directly or indirectly associated with a protein of interest. In the present approach, a large-scale immunoprecipitation allowed us to identify, by mass spectrometry, with high confidence, many interacting partners for mEi24, most of which are novel interactors. In this study, we used three different systems to purify mEi24 in complex with interacting proteins, normal NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and malignant B16F10/mEi24. The usefulness of the inducible protein expressing cell systems to validate the proteome profile changes and to elucidate the signaling network that can be detected- by one and/or twodimensional polyacrylamide gels and MS analysis has been demonstrated previously,14,15,20-22 and our coimmunoprecipitation approach has advantages of identifying direct or indirect interactions with mEi24 that are relevant in these specific biological contexts. Cell extracts from all three cell clones, which induced the mEi24 expression for 72 h with doxycycline, were collected. Solubilization of the protein of interest without disrupting interactions with potential partners is a first step in characterizing an interactome. The ionic strength and nature of the detergent can have profoundly different effects on the solubilization of different proteins. Our use of a low-ionic strength lysis buffer for these proteomic studies allowed us to efficiently solubilize mEi24 with only Triton X-100, a relatively mild detergent, hopefully with minimal disruption of the mEi24 interactome. The Flag tagged mEi24 proteins, as well as any associated protein components, were recovered by immunoprecipitation. Basically, among the cell clones, the mEi24 complex remained relatively invariant (Figure 2B and C). mEi24 5274


was immunoprecipitated from total cell lysates of three expressing systems with anti-Flag M2 antibody conjugated with Protein A agarose, and immuno-complexes immobilized in Protein A beads were removed using 2X Laemmli buffer solution, resolved on SDS-PAGE, and subjected to tryptic digestion followed by LC-MS/MS analysis. In parallel, as a negative control, the same procedure was undertaken but using the uninduced cell lysates (Figure 2B and 2C). As shown in Figure 2, lots of proteins specifically coimmunoprecipitated with Flag-tagged mEi24 from the total cell lysates of three cell systems. The lanes, which are evenly cut as 10 equivalent slices, were excised from the gel in three of the immunoprecipitation lanes as well as in the parallel negative controls (uninduced NIH/3T3/mEi24 and B16F10/mEi24 cells) for the mass spectrometry by destaining and in-gel digestion followed by peptide extraction. The tryptic peptides obtained from each gel slice were analyzed by LC-MS/MS running on the Q-STAR Pulsar ESI-hybrid Q-TOF instrument. Proteins identified in the mEi24 immunoprecipitation lane with two or more peptides and a confidence level more than 95% (more than 40 Probability based Mowse Score), but absent in the negative control lanes of uninduced cell lysates, can be considered as a specific interactome for mEi24, as listed in Tables 1, 2, and 3. In the duplicate of this experiment, the isolated immuno-complexes were not eluted from Protein-A agarose. Analysis of the SDSPAGE lanes obtained from the immunoprecipitation resulted in a total of 162 proteins. These proteins, along with NCBI gene identification numbers, molecular weight, the number of identified peptides, the probability, and only those proteins found in duplicate experiments were included in the final summary. Basically, two cell systems, NIH/3T3/mEi24 and H-Ras/G12V/NIH/3T3/mEi24 cells, showed the totally same protein identities from the mass spectrometry analysis. There were 61 mEi24 interacting proteins identified in each of NIH/ 3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16F10/mEi24 cells; however, some mEi24 interacting proteins were specific to two NIH/3T3-based cells (43 proteins) and to B16F10/mEi24 cells (58 proteins) (Figure 3A). The commonly identified proteins from three cell lines were categorized based on the Panther Database (http://www.pantherdb.org), for which the main functional categories were ribosome assembly (ribosomal proteins), ATP synthase subunits, nuclear transport, and heat shock proteins (Figure 3B). On the other hand, main functional categories for NIH/3T3-based cells were cell architecture and structure, heat shock proteins, chromatin packaging and remodeling, and protein modification process, and those for B16F10/mEi24 cells were proteasome-mediated proteolysis process, transport (vesicle mediated), heat shock proteins, GTPase-mediated signaling process, and kinases and kinase modulators (Figures 3C and 3D). Thus, from these results, we have demonstrated that mEi24 forms different complexes in different cell types or cell status. The identification of these cell-specific interacting partners may imply a unique role for mEi24 in those cell types. For example, a relationship between ribosomal proteins and ubiquitination has been reported. It has been reported by a large-scale proteomics study of ubiquitinated proteins that ribosomal proteins are ubiquitinated.23 Moreover, some ribosomal subunits were suggested to contribute to regulation of the translation machinery and cell cycle through the ubiquitination procedure.24 However, despite these intriguing and promising findings, little progress has been made on the characterization of EI24/PIG8, and its activity and function in normal and cancer cells remain largely to be elusive.


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Figure 2. Affinity-purification of mEi24-interacting proteins. (A) Schematic diagram of the strategy used to elucidate mEi24 interacting proteins. Cell lysates from NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16F10/mEi24 of induced or uninduced were incubated with Flag M2 Agarose beads. The affinity-purified proteins were resolved onto SDS-PAGE. The lanes, which are evenly cut as 10 equivalent slices, were excised from the gel, subjected to trypsin digestion, and analyzed by LC-MS/MS running on the Q-STAR Pulsar ESI-hybrid Q-TOF instrument. (B) Proteins purified from total lysates of NIH/3T3/mEi24 and H-Ras/G12V/NIH/3T3/mEi24 cells induced with or without doxycycline were resolved by SDS-PAGE gel. The proteins were visualized by colloidal Coomassiae solution and subjected to further investigation. (C) Proteins from B16F10/mEi24 cells induced with or without inducer were subjected to the same experimental procedures.

While these studies were not quantitative, the spectral counts identified in proteomics screens are related to the abundance of the protein in the sample. Therefore, in an initial effort to rank protein hits, we analyzed each set of proteomics data based on the enrichment of spectral counts in NIH/3T3-based cell lines and B16F10 cells carrying an inducible expression system for mEi24 (Figure 4). Figure 4A represents some proteins that are reduced in the number of spectra in immunocomplex from the B16F10/mEi24 cells, and Figure 4B shows those increased in complex from the B16F10/mEi24 cells compared to the NIH/3T3-based cells. Cellular functions and signaling networks are the result of the coordinated action of several proteins in macromolecular assemblies. Protein complex composition varies with time to adapt to changing cellular requirements. Therefore, the analysis of protein complex composition is an important step in elucidating the cellular physiology and signaling network of a cell. Our proteomics tools have proven to be successful in the identification of multicomponent complexes formed under native conditions. Although it will be important to further investigate the biochemical basis for assembly of these complexes and their biological roles, it is shown that, from our results, protein composition of mEi24 immuno-complexes reflects the specific cellular physiology for normal NIH/3T3 and malignant B16F10 cells. Validation of Some Novel mEi24 Interacting Proteins by Immunoprecipitation Followed by Western Blot Analysis. Some proteins have been reported to bind Ei24 through direct interaction with Ei24.11 To further validate the proteins identified by MS/MS peptide matching to be components of the Ei24 interactome, we performed Western blot analysis on both the

total cell lysate and the Ei24 immunoprecipitate of the three cell lines, NIH/3T3/mEi24, H-Ras/G12V/NIH/3T3/mEi24, and B16/F10/mEi24. Of the many mEi24 interacting proteins identified, we first selected Bcl-2, which has been previously reported to directly interact with Ei2411 and is a death regulator known to reside in mitochondria, ER, and nuclear envelope, and 7 proteins that represent novel mEi24-interacting proteins, karyopherin β1, mitochondrial ATP synthase β subunit (ATP 5B), calnexin, prohibitin, calveolin-1, and proteasome (prosome, macropain) 26S subunit non-ATPase 2 (PSMD2). Five proteins chosen for immunoprecipitation except caveolin-1 and PSMD2 were identified as the interacting proteins in three kinds of cell lines. As shown in Figure 5, mEi24 protein expression was observed in both the total cell lysate and mEi24 immunoprecipitate but not in the parental cell lysates and the negative control lysates from the uninduced cells. For a further validation of any interaction and owing to the efficiency of immunoprecipitation, we performed the reciprocal immunoprecipitation with mEi24 in B16F10/mEi24 cell lysates, and the associated complexes were analyzed for the presence of expressed mEi24, ATP 5B, prohibitin, and karyopherin β1 (Figure 6A-6C). From these experiments, we demonstrate that six proteins were immmunoprecipitated with the inducibly expressed mEi24 in three kinds of cells. In addition, of the many novel mEi24 interacting proteins identified, we chose to validate caveolin-1 as an interacting partner in two NIH/3T3-based cell systems carrying an inducible form of mEi24 and PSMD2 in B16F10/mEi24 melanoma cells. To confirm that caveolin-1 is associated with mEi24 in NIH/3T3-based cells and that PSMD2 is in B16F10 cells, we performed immunoprecipitation experiJournal of Proteome Research • Vol. 9, No. 10, 2010 5275

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Table 1. Identification of Proteins Commonly Recovered from the Flag-mEi24 Tandem Affinity Purification in NIH/3T3-Based Cells and B16F10/mEi24 Cells Carrying an Inducible Form of mEi24a accession number

protein ID

peptide protein no. mass (Da) scoreb

gi:6680748

NP_0311531

15

59716

985

gi:23272966 gi:16307541 gi:20454828 gi:7949005

AAH37127 AAH10319 Q9CQQ7 NP_058035

14 4 2 2

56632 108338 28930 12489

911 218 78 122

gi:11602916 NP_065640

2

32831

127

gi:12847456 gi:263673 gi:12057236 gi:30851368 gi:10048438 gi:11342591 gi:15186758 gi:21595190 gi:45477093 gi:15186756 gi:14573323 gi:903309

BAB27577 AAB24947 AAG45965 AAH52438 NP_065252 CAC17143 AAK91128 AAH31900 Q7TMY7 AAK91127 AAK68050 BAA04548

2 2 8 15 3 7 13 2 2 2 7 2

17619 8230 123694 97122 129882 119509 115948 113258 116975 115963 136883 73416

147 105 426 893 67 548 748 118 56 114 332 152

gi:695625

CAA85521

2

59518

141

gi:1526541 gi:683793 gi:12963511 gi:6755354 gi:6755350 gi:3097244 gi:56270278 gi:13592069 gi:198578 gi:227256 gi:57102362

BAA13422 AAA62450 NP_075622 NP_035420 NP_035417 CAA69615 AAH86897 NP_112371 AAA16796 1617167A XP_534008

2 2 5 4 3 2 2 3 3 3 3

28181 64971 16078 32591 24901 16291 17576 18904 17730 269665 26657

80 117 237 175 84 97 95 202 176 174 172

gi:6806903

NP_033852

9

109682

510

gi:62234487 gi:47847430 gi:53754 gi:53454 gi:28916685 gi:6681289 gi:56971731

NP_080758 BAD21387 CAA46522 CAA30538 NP_803129 NP_031941 AAH87944

4 3 4 6 3 6 2

134662 155428 70598 76677 26048 40838 52735

215 190 200 284 150 361 147

gi:18605662 gi:809561 gi:41322904 gi:11528490 gi:33990013 gi:6678722

AAH23120 CAA31455 NP_035247 NP_071292 AAH56365 NP_032541

2 8 2 4 5 2

88604 40992 519466 144712 135580 71259

143 445 45 251 351 122

gi:17391324 AAH18556 gi:22256949 O54734

3 5

52078 48983

160 237

gi:16359229 AAH16080 gi:11692645 AAG39913 gi:31980806 NP_058063

4 2 3

68486 77272 21681

169 129 173

gi:54915 CAA40624 gi:33438248 BAC65475 gi:55153885 AAH85315

3 2 6

85677 192355 35751

162 118 256

gi:54887356 AAH37009

7

82591

379

gi:53551 gi:13937353 gi:6755302 gi:54777 gi:60360164 gi:7657011 gi:12846758 gi:55291

2 2 2 3 4 6 13 7

89505 29802 37432 57108 135175 126772 49608 53597

112 104 102 148 200 378 873 483

CAA44079 NP_114039 NP_036122 CAA29759 BAD90301 NP_056550 BAB27292 CAA35803

biological functionsc

description +

ATP synthase, H transporting, mitochondrial F1 complex, R subunit, isoform 1 ATP 5B protein ATP 1a1 protein ATP synthase B chain, mitochondrial precursor ATP synthase, H+ transporting, Mitochondrial F0 complex subunit F ATP synthase, H+ transporting, mitochondrial F1 complex, γ subunit ATP synthase δ chain, mitochondrial precursor F1F0-ATPase subunit ε Ran binding protein 5 (Ran BP5)/Importin 5 Karyopherin (Importin) β1 Exportin 4 Ran binding protein 7 (Ran BP 7)/Importin 7 Importin 9 isoform 2 Importin 11 protein Importin 8 (Ran binding protein 8, Ran BP 8) Importin 9 isoform 1 Ran binding protein 21 (Ran BP21) stress-70 protein (PBP74/CSA) CCTθ, θ subunit of the chaperoin containing TCP-1 (CCT) 14-3-3 η Calnexin 40S ribosomal protein S19 ribosomal protein L6 ribosomal protein L10A ribosomal protein S14 ribosomal protein L29 40S ribosomal protein S10 ribosomal protein Talin PREDICTED: Similar to 40S ribosomal protein S3 sarcoplasmic/endoplasmic reticulum calcium ATPase 2 isoform b plasma membrane calcium ATPase 1 mFLJ00137 protein, WD40 Poly(A) binding protein Nucleolin Apoptosis factor Bcl-2 Etoposide induced 2.4 (Ei24) 5031425D22Rik protein, Serum amyloid A-like 1 component of oligomeric golgi complex 4 γ-actin Plectin 1 isoform 1 protein flightless I homologue cullin-associated and neddylation-dissociated leucine-rich repeat flightless interacting protein 1 isoform 2 Ppm1b protein, PP2Cc Dolichyl-diphosphooligosacchar ide-protein glycosyltransferase 48 kDa subunit precursor Ribophorin 1 aspartyl-β-hydroxylase progesterone receptor membrane component transferrin receptor mKIAA0034 protein, Clathrin similar to glyceraldehydes-3-phosphate dehydrogenase hydroxyacyl-CoA dehydrogenase/ 3-ketoacyl-CoA thiolase/ enoyl-CoA hydratase P1.m protein Prohibitin Reticulocalbin thyroid hormone binding protein mKIAA4153 protein, Reticulon damage specific DNA binding protein unnamed protein product unnamed protein product

ATP synthase subunit ATP ATP ATP ATP

synthase synthase synthase synthase

subunit subunit subunit subunit

ATP synthase subunit ATP synthase subunit ATP synthase subunit nuclear transport nuclear transport nuclear transport nuclear transport nuclear transport nuclear transport nuclear transport nuclear transport nuclear transport HSP70 family chaperone (stress response) chaperoin chaperone, signal transduction Chaperone (Calcium binding protein) ribosomal protein ribosomal protein ribosomal protein ribosomal protein ribosomal protein ribosomal protein ribosomal protein ribosomal protein ribosomal protein ion channel (calcium homeosis) ion channel (calcium homeosis) mrna splicing RNA binding protein RNA binding protein (RNA metabolism) apoptosis apoptosis transporter transporter actin and actin related protein actin binding cytoskeletal protein nonmotor actin binding protein transcription factor transcription factor (RNA binding protein) protein phosphatase protein glycosylation protein glycosylation protein modification receptor receptor receptor mediated endocytosis metabolism carbohydrate metabolism DNA replication cell cycle control, DNA replication calcium binding protein membrane traffic protein DNA repair

a Bolded proteins in the table represent those validated using coimmunoprecipitation with mEi24. b Ions score is -10 log(P), where P is the probability that the observed match is a random event. Individual ion scores >42 indicate identity or extensive homology (p < 0.05). Protein scores are derived from ion scores as a nonprobabilistic basis for ranking protein hits. c Based on the Panther Database (http://www.pantherdb.org).

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Table 2. Identification of Proteins Commonly Recovered from the Flag-mEi24 Tandem Affinity Purification in NIH/3T3-Based Cells Carrying an Inducible Form of mEi24a accession number

protein ID

peptide no.

mass (Da)

protein score

gi:13242237 gi:40556608 gi:31981722

NP_077327 NP_032328 NP_071705

14 11 9

70827 83229 72378

843 580 438

gi:37360114 gi:6679567

BAC98035 NP_033012

15 3

146622 43927

796 159

gi:26986198

CAD59182

5

134190

261

gi:29789347

NP_598547

2

146803

149

gi:13879386

AAH06673

3

127377

116

gi:22165349

NP_036169

2

88006

90

centromere/kinetochore zw10

gi:975689

CAA62503

2

31421

88

gi:22324938

NP_082720

2

27837

76

erythrocyte band 7 integral membrane protein, protein 7.2B. Stomatin protein kinase δ binding protein

gi:452528

BAA04234

4

42953

167

gi:21410529 gi:986918 gi:6705981

AAH31297 AAB67986 BAA89463

4 2 3

72696 51416 7411

201 124 125

gi:20137006

NP_071855

2

226217

131

gi:62526118 gi:2078001

NP_78060 CAA69019

10 3

63654 51533

535 186

myosin, heavy polypeptide 9, nonmuscle isoform 1 cytoskeleton-associated protein 4 Vimentin

gi:56207519 gi:55153941 gi:1374782

CAI24271 AAH85278 BAA12803

4 2 2

4276 32568 109899

212 138 137

Reticulon 4 Nucleophosmin 1 possible ubiquitin protein ligase, Nedd4

gi:19882207 gi:6754222

NP_608219 NP_034578

2 2

40556 30812

113 93

gi:2645205 gi:33243895 gi:51452 gi:38174226 gi:20071177 gi:51653 gi:28972407 gi:72535978 gi:1870428

AAC39954 AAQ01516 CAA37653 AAH60711 AAH26845 CAA24153 BAC85657 AAZ73088 AAB48774

2 2 2 2 2 3 2 2 2

151860 31713 58833 209793 128606 27863 158705 75361 11011

70 75 150 150 124 163 140 109 127

gi:26344105 gi:76326597 gi:12848873 gi:21311891 gi:26344105 gi:12834876 gi:54927 gi:27369888 gi:26328849 gi:1177528

BAC35709 BAC27042 BAB28118 NP_083042 BAC35709 BAB23076 CAA34316 NP_766204 BAC28163 CAA58028

2 3 2 2 2 2 2 2 2 2

69891 71395 33465 44755 69891 30144 11714 104659 78679 324231

115 123 122 119 115 88 84 71 71 58

gi:26350301

BAC38790

4

51470

223

farnesyl diphosphate synthetase heterogeneous nuclear ribonucleoprotein A/B p160 myb-binding protein voltage-dependent anion channel 2 unnamed protein product Nucleophorin 205 protein Nucleophorin 133 protein unnamed protein product mKIAA0804 protein NDC1 anti-DNA immunoglobulin light chain IgM unnamed protein product unnamed protein product unnamed protein product transmembrane protein 43 unnamed protein product unnamed protein product unnamed protein product hypothetical protein LOC224171 unnamed protein product antigen identified by monoclonal antibody Ki-67 unnamed protein product

a

description heat shock cognate 71 kDa protein heat shock protein 90 β heat shock 70 kDa protein 5 (glucose-regulated protein) mKIAA0829 protein polymerase I and transcript release factor SMC2 protein SMC4 structural maintenance of chromosomes 4-like 1 2810406C15Rik protein, Ncapd2 protein

magnesium-dependent protein phosphatase b-2, PP2C Jak1 protein A10 protein Caveolin-1

biological functions heat shock protein heat shock protein heat shock protein transcription factor transcription factor chromatin packaging and remodeling chromatin/chromatin binding protein chromatin packaging and remodeling microtubule family cytoskeletal protein cytoskeletal protein signal transduction, protein phosphorylation protein phosphatase nonreceptor tyrosine kinase amino acid biosynthesis G-protein modulator, Structural protein cell structure cell structure intermediate filament structural protein membrane traffic protein chaperone, rRNA metabolism ubiquitin-protein ligase, ubiquitin proteasome system cholesterol metabolism ribonucleoprotein, mRNA splicing DNA-directed DNA polymerase ion channel

Bolded proteins in the table represent those validated using coimmunoprecipitation with mEi24.

ments with the inducibly expressed mEi24, caveolin-1, and PSMD2 proteins. The associated complexes were analyzed by immunoblot for the presence of flag tagged mEi24, caveolin1, and PSMD2 (Figure 5H and 5I). Our proteomic analysis of Ei24 interacting proteins also identified a number of proteins that have not been previously demonstrated. These may represent novel proteins that interact with Ei24 and may be proteins that are either substrates or regulators of its function. Indeed, it is demonstrated that the tested proteins are present in the Ei24 immuno-complexes. Taken together, these findings demonstrate the robustness of our approach and the useful implication of the resulting data. Immunofluorescence Colocalization of the Identified Novel mEi24 Interacting Proteins with mEi24. The distribution and colocalization relationships of some mEi24 interacting

proteins, Bcl-2, calnexin and karyopherin β1, and mEi24, were examined by double immunofluorescence labeling of these proteins in cultures of NIH/3T3/mEi24 and B16F10/mEi24 cells. As an initial step, whether novel interacting proteins and mEi24 were at least partially colocalized within the cells was determined. To accomplish this goal, two kinds of cells were seeded on coverslips and induced the flag tagged mEi24 with 2 µg/ mL of doxycycline. A time period of 36 h after induction, cells were fixed and subsequently stained with primary antibodies followed by Alexa Fluor 488 or 568-conjugated secondary antibodies. Nuclei were stained with DAPI and were then analyzed under a confocal microscope. Dual staining showed that Bcl-2, calnexin, and karyopherin β1 (red color) colocalized (cf. yellow color) with flag tagged mEi24 (green color) in NIH/3T3/mEi24 (Figure 7A) and in Journal of Proteome Research • Vol. 9, No. 10, 2010 5277

5278


AAH27378 Q8BTM8 NP_084514 NP_082509 AAH82548 NP_064393 NP_038609 AAH03936 NP_059506 NP_203534 AAH65173

AAB06943 CAA65761 AAH04017 NP_705771 Q11011 NP_666329 NP_742012 NP_758512 AAH06857

AAH42443 NP_766277 AAH18353 NP_031592 I84737 BAC39609 NP_0311557 NP_076014

BAB27120 BAC98244 BAB24848 BAB23538 AAH57688 AAH25526 AAA51041 AAM22159 BAB24696 NP_033438 BAC40527 AAH94679 CAA05361 BAC41426 BAC41401 BAC65757 NP_938034

gi:20072723 gi:38257560 gi:13384738 gi:21389320 gi:52139023 gi:9910228 gi:15011849 gi:13278190 gi:8567340 gi:15426055 gi:40787837

gi:1515359 gi:1405933 gi:13278412 gi:23956316 gi:249903 gi:34610207 gi:29789383 gi:31559887 gi:13905134

gi:27502768 gi:45598377 gi:17390825 gi:10048468 gi:2119280 gi:26351945 gi:6671622 gi:51093867

gi:12846314 gi:37360532 gi:12840425 gi:12836175 gi:34785917 gi:19343754 gi:559059 gi:20453849 gi:2978383 gi:6678407 gi:26353794 gi:66267550 gi:2598562 gi:28394195 gi:2598562 gi:28394195 gi:26006117

4 3 5 4 3 2 2 2 2 5 5 4 9 10 13 6 7

2 3 2 2 4 2 5 4

3 2 4 6 8 5 2 4 2

2 11 12 6 2 2 2 2 2 2 3

6 4 2 3 2 2 2

peptide no. 8 2 2 2 4 2

22222 125733 32849 117901 50556 58078 60475 273639 35049 20047 56590 166417 72433 224058 190970 184585 125147

103251 105811 87863 528089 100601 51811 33253 243084

106102 57878 38276 101500 103286 106841 144165 105648 19036

22205 281018 531690 147331 143544 288552 108920 32762 97617 106998 58300

60903 83180 92418 59586 157184 188638 23692

mass (Da) 129206 22248 290360 47252 203573 66780

183 116 294 174 157 130 107 81 74 274 273 264 535 531 729 432 325

124 183 98 99 243 185 206 196

70 104 247 290 310 212 70 181 87

150 563 518 240 107 71 67 93 89 71 152

341 191 101 114 65 83 120

protein score 403 122 85 125 160 96

description 1810009A 16Rik protein, Ubr4 protein proteasome (prosome, macropain) 26S subunit ATPase 2 ubiquitin specific protease 9, X chromosome proteasome 26S ATPase subunit 4 proteasome-associated protein ECM29 homologue proteasome (prosome, macropain) 26S subunit, non-ATPase 2 (PSMD2) heat shock protein 65 heat shock protein hsp84 tumor rejection antigen gp96 (endoplasmin) CCT (chaperoin containing TCP-1) ε subunit triple functional domain (PTPRF interacting) Trio protein IQ motif containing GTPase activating protein 1 PREDICTED: similar to Rab5B, member Ras oncogene family isoform 1 Rab5c protein filamin A (a-filamin, filamin 1), endothelial actin-binding protein dynein, cytoplasmic, heavy chain 1 leucine-rich PPR motif-containing protein Gcn1/1 protein FK506 binding protein 12, Rapamysin associated protein 1 component of oligomeric golgi complex 1 Yipf4 protein coatomer protein complex, subunit γ2 coatomer protein complex, subunit β1 solute carrier family 3 (activator of bibasic and neutral amino acid transport) lysosomal R-glucosidase M2-type pyruvate kinase enolase 1 protein (EG433182 protein) aldehyde dehydrogenase 1 family, member L2 puromycin-sensitive aminopeptidase (PSA) alanyl-tRNA synthetase isoleucine-tRNA synthetase methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1 like endoplasmic reticulum protein ERp19 (thioredoxin domain containing 12 (endoplasmic reticulum) SCY1-like 2 (S. cerevisiae) DEAD(Asp-Gln-Ala-Asp)/H box polypeptide RIG-1 heterogeneous nuclear ribonucleoprotein U baculoviral IAP repeat-containing 6 kinesin heavy chain- mouse (fragment) unnamed protein product B-cell receptor-associated protein 37 carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, dihydrorotase unnamed protein product mKIAA1738 protein unnamed protein product unnamed protein product lgh-4 protein Nucleoporin 188 putative protein IGF2R unnamed protein product tumor protein D52 isoform 5 unnamed protein product EPRS protein BiP mKIAA0462 protein mKIAA0219 protein mKIAA1244 protein Testis expressed gene 2

Bolded proteins in the table represent those validated using coimmunoprecipitation with mEi24.

CAA38762 AAA37866 NP_035761 CAA83430 AAH60724 NP_057930 XP_531627

gi:51455 gi:194027 gi:6755863 gi:468550 gi:38511847 gi:7710042 gi:73968313

a

protein ID AAH40468 AAB51069 NP_033507 NP_036004 NP_759013 AAH23049

accession number gi:26251739 gi:1914825 gi:6678523 gi:7110701 gi:37718970 gi:27692965

protein kinase like nucleic acid metabolism, apoptosis ribonucleoprotein protease inhibitor, inhibition of apoptosis

glycogen metabolism glycolysis glycolysis carbohydrate metabolism metalloprotease amino acid synthetase amino acid synthetase amino acid biosynthesis sulfur redox metabolism

small GTPase, signal transduction nonmotor actin binding protein microtubule binding motor protein serine/threonine protein kinase receptor kinase modulator nonreceptor serine/threonine protein kinase transporter general vesicle transporter vesicle coat protein (transport) vesicle coat protein (transport) amino acid transport

heat shock protein heat shock protein heat shock protein chaperoin G-protein mediated signaling G-protein modulator, signal transduction small GTPase, signal transduction

biological functions ubiquitin-protein ligase proteolysis proteolysis proteolysis proteolysis proteolysis

Table 3. Identification of Proteins Commonly Recovered from the Flag-mEi24 Tandem Affinity Purification in B16F10/mEi24 Cells Carrying an Inducible Form of mEi24a

research articles Bahk et al.

Figure 3. Protein numbers identified in NIH/3T3-based mEi24 expressing cell lines and B16F10/mEi24 cells. (A) The total number of identified proteins with LC-MS/MS was 162. All identified proteins are described in Tables 1-3. Fourty-three only identified proteins (NIH/3T3-based mEi24 expressing cells) and 58 only proteins from B16F10/mEi24 cells were identified, and 61 proteins were overlapped in NIH/3T3-based mEi24 expressing cells and B16F10/mEi24 melanomas. (B) Functional categorization of the overlapped identified proteins in NIH/ 3T3-based mEi24 expressing cell lines and B16F10/mEi24 cells. (C) Functional categorization of the identified proteins only from NIH/3T3-based mEi24 expressing cells. (D) Functional categorization of the identified proteins only from B16F10/mEi24 cells.


research articles

Journal of Proteome Research • Vol. 9, No. 10, 2010 5279

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Figure 4. Each set of proteomics data based on the enrichment of spectral counts in NIH/3T3-based cell lines and B16F10 cells carrying an inducible expression system for mEi24. We have chosen 13 proteins found in the immunocomplex from both of NIH/3T3-based cells and B16F10/mEi24 cells. The spectral numbers observed from the selected proteins that are reduced (A) and increased (B) in malignant B16F10/mEi24 cells. 9, The numbers of spectral counts for the identified protein from NIH/3T3-based cells. 0, Those from malignant B16F10/mEi24 cells.

B16F10/mEi24 (Figure 7B) cells. The endogenous Bcl-2, calnexin, and karyopherin β1 proteins were detected by confocal immunofluorescence in NIH/3T3/mEi24 and B16F10/mEi24 cell lines. These three proteins, detected with the rabbit polyclonal antibodies directed against each of the three proteins, were localized in the cytosol and nucleus as the punctuate signals (the third columns in Figure 7A and B). The induced mEi24, detected with the mouse monoclonal antibody against the Flag tag, was mostly cytosolic with some minor nuclear aggregation (the second columns in Figure 7A and B). The tested proteins partially presented some overlap of their signals mainly responsible for the endogenous novel protein expression patterns (the fourth and merged columns in Figure 7A and B). The partially punctuated distribution and extensive colocalization of Bcl-2, calnexin, and karyopherin β1 with the induced mEi24 indicates their association. ATP Synthase Complex As a Coimmunocomplex Member for mEi24 Protein. Some approaches were used to independently confirm the interactions between mEi24 and ATP synthase. Indeed, several subunits of ATP synthase were coimmunoprecipitated with mEi24 from total cell lysate in lysis buffer, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 5280


and 1% Triton X-100 containing protease inhibitor cocktail. This coimmunoprecipitation of ATP synthase with mEi24 from total cell lysate suggested the physical association of mEi24 with the assembled ATP synthase. Additional ATP synthase subunits that were detected by MS analysis for the coimmunocomplex giving only one matched peptide with high confidence are listed in Table 4. In spite of only one peptide sequence for these identified subunits, based on the protein score values for their identification with MS analysis and the coappearance of other ATP synthase subunits in the immuno-complex which had more than two peptides and more than 40 probability-based Mowse score values (Table 1 and 4), as well as the validation of ATP 5B in immuno-complex with mEi24, we conclude that mEi24 physically associates with the assembled ATP synthase. Reversely, the interaction of mEi24 with the ATP synthase was analyzed by coimmunoprecipitation immunoblot utilizing an antibody raised against a subunit of the ATP synthase (ATP 5B). To detect the newly synthesized flag tagged mEi24, a flag epitope monoclonal antibody was utilized, which as expected recognized tagged mEi24 either in the starting material or after the ATP synthase was coimmunoprecipitated (Figure 5A). However, mEi24 and ATP synthase subunits were not detected

research articles


Figure 5. Coimmunoprecipitation using anti-Flag tag antibody with total lysates of NIH/3T3-based cells and B16F10/mEi24 cells. Total lysates from the appropriate cells were incubated with anti-Flag-M2 antibody conjugated into Protein A agarose, washed with the lysis buffer, and separated onto 10% SDSPAGE. The gels were then immunoblotted for each protein with the specific antibody: A, Flag; B, Bcl-2; C, karyopherin β1; D, ATP 5B; E, calnexin; F, prohibitin; G, caveolin-1; and H, PSMD2. Controls are 30 µg of total lysate of NIH/3T3 cells. (IP: immunoprecipitation.)

Figure 6. Coimmunoprecipitation using specific antibodies directed against ATP 5B, prohibitin, and karyopherin β1. Total lysates from the parental B16F10 and uninduced and induced B16F10/mEi24 cells were mixed with the appropriate primary antibody raised against individual proteins followed by incubating with gentle rocking. Then, 50% bead slurry of protein A agarose was added into the immunoprecipitates, incubated with gentle rocking, washed, and separated onto 10% SDS-PAGE. The gels were then immunoblotted with anti-Flag antibody. For reprobing the blots after detection of Flag-mEi24, the blots were incubated with BlotFresh Western Blot Stripping Reagent and reprobed with the specific antibodies directed against ATP 5B (A), prohibitin (B), and karyopherin β1 (C). Controls are 30 µg of total lysate of B16F10 cells. (IP: immunoprecipitation.)

in coimmunoprecipitated samples derived from extracts of parental B16F10 melanomas and uninduced B16F10/mEi24 cells, demonstrating the specificity of this approach. These interactions demonstrate that mEi24 may be involved in functions and/or compartmentalization in the cell other than localization in the endoplasmic reticulum, and the ATP synthase complex as the interacting partners found in the present study is a potential implication for suggesting the behavior of mEi24 in energy metabolism. The F1F0 ATP synthase is a multisubunit enzyme comprised of a soluble F1 portion which catalyzes the synthesis of ATP from ADP and inorganic phosphate through a rotary motion driven by coupling of F1 to the central stalk of the F0 portion.25 Practically, the F0 is a proton channel which generates power through the translocation of protons moving down their gradient. In 1994, the ATP synthase has been found to be located in the unlikely and extracellular site of the plasma membrane on human tumor cells26 and discovered to reside on the cell surface of many additional cell types, with implications in angiogenesis, cellular immunity, cholesterol uptake, cellular pH regulation, and additional fields.27 Nucleocytoplasmic Transport Factors as Ei24 Interacting Proteins. Various kinds of nucleocytoplasmic transport receptors including exportin 4 and karyopherin β-family members, which are involved in the active nuclear transport, were detected in mEi24 immuno-complexes isolated from all kinds of tested cells (Table 1). They include karyopherin β1 and importin 1, 7, 8, 9 (isoforms 1 and 2), and 21. We confirmed the presence of karyopherin β1 in immuno-complexes isolated only from the mEi24 induced NIH/3T3-based cells and B16F10/ mEi24 cells, whereas it was not detected in the two parental cells and the uninduced cells (Figure 5C). In addition, this interaction of karyopherin β1 with mEi24 was further confirmed by reciprocal coimmunoprecipitation using the mEi24 induced B16F10/mEi24 cells (Figure 6C) and a confocal immunofluorescence detection for some novel proteins (Bcl-2, calnexin, and karyopherin β1) with the induced mEi24. In eukaryotic cells, events taking part in DNA and RNA, such as replication and transcription, are spatially separated from protein synthesis by the nuclear envelope. Thousands of macromolecules are transferred between the nuclear and cytoplasmic compartments at interphase.28,29 Nucleocytoplasmic transport occurs through the nuclear pore complex (NPC), a large assembly that spans the nuclear envelope. The NPCs are impermeable to most macromolecules, with the notable exception of nucleocytoplasmic transport receptors. The majority of nucleocytoplasmic transport receptors belongs to the family of karyopherin β proteins, also known as importin β-like proteins. Importin-β-related and exportin nuclear transport receptors, which shuttle between nucleus and cytoplasm and interact with NPCs and recognize and bind cargo molecules, mediate many of the nucleocytoplasmic transport events. The directionality of transport appears to be determined by the RasGTP gradient.30,31 The karyopherin protein family shares weak sequence homology overall except the N-terminal half, where they share the ability to interact with the regulator RanGTP.29 Thus, we suggest here that Ei24 interacts with various importin family proteins and exportin 4 and might be involved in the nucleocytoplasmic transport event.

Concluding Remarks In summary, we used an in vivo, coimmunoprecipitation approach, coupled to shotgun proteomics using tandem mass Journal of Proteome Research • Vol. 9, No. 10, 2010 5281

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Figure 7. Double immunofluorescence labeling of the novel mEi24 interacting proteins with mEi24 in NIH/3T3/mEi24 (A) and B16F10/ mEi24 cells (B). Confocal immunofluorescence microscopy of endogenous mEi24 interacting proteins (the third column of the figure, red color), the induced mEi24 (the second column, green color), and DAPI (the first column, blue color) was monitored in NIH/3T3/ mEi24 and B16F10/mEi24 cells. Yellow color indicates colocalization of both antibodies. Shown in the fourth column are the merged images of the red (the novel proteins, Alexa Fluor 568) and of the green (the induced mEi24, Alexa Fluor 488) antibody stains. Table 4. ATP Synthase Subunits That Were Identified in the Copurifying Proteins in the Interactome of mEi24, But Giving Only One Matched Peptide with High Confidence of More than 40 Probability-Based Mowse Score Values accession number

protein id

peptide no.

mass (Da)

protein score

gi:2623222 gi:20070412 gi:3913897 gi:16741459 gi:13385484 gi:32892270 gi:59808056

AAB86421 NP_613063 O35143 AAH16547 NP_080259 AAP89027 AAH89550

1 1 1 1 1 1 1

56344 23349 12137 18752 5834 7761 8231

89 52 50 45 41 40 70

spectrometry, to identify putative, novel Ei24 interacting proteins. Importantly, this work demonstrates a powerful system that can be applied to novel protein targets to rapidly and selectively identify novel interactomes. We focused on mEi24 inducing cell systems in NIH/3T3, H-Ras/G12V/NIH/ 3T3, and B16F10 cells to identify protein-protein interactions 5282


description

ATP synthase β-subunit ATP synthase, H+ transporting, mitochondrial ATPase inhibitor, mitochondrial precursor ATP synthase, H+ transporting, mitochondrial ATP synthase, H+ transporting, mitochondrial ATP synthase F0 subunit 8 ATP synthase, H+ transporting, mitochondrial subunit e (Atp5k protein)

F1 complex O subunit F0 complex subunit d F1 complex, ε subunit F1F0 complex,

that are contextually important. We have described here the use of proteomics technology and some immunochemical techniques to evaluate the in vivo response of and interactomes of some cell lines to mEi24 expression and improve our understanding of the molecular signaling processes responsible for Ei24-mediated apoptotic cell death. However, it is true that,

research articles

Interactomes of Ei24/PIG8 under the physiological states, Ei24 expression is induced as a DNA damage response gene and an immediate-early induction target gene of p53-mediated apoptosis involved in growth suppression and apoptosis. Although these proteomic and some immunochemical approaches for Ei24’s interactome using the inducible overexpression system might not represent the accurate physiological relevance in vivo, the present findings provide the possibility for new avenues not only to explore the protein networks evoked by Ei24 but also to elucidate the molecular mechanism for the cell death by etoposide treatment. The discovery of these novel interacting proteins will provide to a better understanding of cellular physiology of Ei24 and may lead us to the identification of new targets. Abbreviation: mEi24, mouse etoposide-induced 2.4 kb transcript; EGFP, enhanced green fluorescence protein; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; ATP 5B, mitochondrial ATP synthase β subunit; PSMD2, proteasome (prosome, macropain) 26S subunit non-ATPase 2; NPC, nuclear pore complex; NIH/3T3/mEi24, mEi24 inducible NIH/3T3; H-Ras/G12V/NIH/3T3/mEi24, mEi24 inducible, oncogenic H-Ras/G12V transformed NIH/3T3; B16F10/mEi24, mEi24 inducible B16F10; DAPI, 4′-6-diamidino-2-phenylindole.

Acknowledgment. This research was supported by grants from Korean Research WCU grant (R31-2008-00010086-0) (Y.Y.B.). The work was also supported by the National Research Foundation of Korea (NRF) grants funded by the Korea government (MEST) (2009-0083365, 2009-0081177, 2008-2005805, 2009K001284) and National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs (MoHWFA), Republic of Korea (0520220) (H.W.L.).

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(12) (13) (14) (15)

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References (1) Prives, C.; Hall, P. A. The p53 pathway. J. Pathol. 1999, 186, 112– 26. (2) Rich, T.; Wyllie, R. I.; Wyllie, A. H. Defying death after DNA damage. Nature 2000, 407, 777–783. (3) Marine, J. C.; Francoz, S.; Maetens, M.; Wahl, G.; Toledo, F.; Lozano, G. Keeping p53 in check: essential and synergistic functions of Mdm2 and Mdm4. Cell Death Differ. 2006, 13, 927– 934. (4) Malkin, D. The role of p53 in human cancer. J. Neurooncol. 2001, 51, 231–243. (5) Hollstein, M.; Sidransky, D.; Vogelstein, B.; Harris, C. C. p53 mutations in human cancers. Science 1991, 253 (5015), 49–53. (6) Lehar, S. M.; Nacht, M.; Jacks, T.; Vater, C. A.; Chittenden, T.; Guild, B. C. Identification and cloning of Ei24, a gene induced by p53 in etoposide-treated cells. Oncogene 1996, 12, 1181–1187. (7) Sparano, J. A.; Lee, S.; Chen, M. G.; Nazeer, T.; Einzig, A.; Ambinder, R. F.; Henry, D. H.; Manalo, J.; Li, T.; Von Roenn, J. H. Phase II trial of infusional cyclophosphamide, doxorubicin, and etoposide in patients with HIV-associated non-Hodgkin’s lymphoma: an Eastern Cooperative Oncology Group Trial (E1494). J. Clin. Oncol 2004, 22 (8), 1491–1500. (8) Gu, Z.; Flemington, C.; Chittenden, T.; Zambetti, G. P. ei24, a p53 response gene involved in growth suppression and apoptosis. Mol. Cell. Biol. 2000, 20 (1), 233–241. (9) Su, A. I.; Wiltshire, T.; Batalov, S.; Lapp, H.; Ching, K. A.; Block, D.; Zhang, J.; Soden, R.; Hayakawa, M.; Kreiman, G.; Cooke, M. P.; Walker, J. R.; Hogenesch, J. B. A gene atlas of the mouse and human protein-encoding transcriptomes. Proc. Natl. Acad. Sci. U.S.A. 2004, 101 (16), 6062–6067. (10) Gentile, M.; Ahnstro¨m, M.; Scho¨n, F.; Wingren, S. Candidate tumour suppressor genes at 11q23-q24 in breast cancer: evidence

(23) (24) (25)

(26) (27)

(28) (29) (30) (31)

of alterations in PIG8, a gene involved in p53-induced apoptosis. Oncogene 2001, 20 (53), 7753–7760. Zhao, X.; Ayer, R. E.; Davis, S. L.; Ames, S. J.; Florence, B.; Torchinsky, C.; Liou, J. S.; Shen, L.; Spanjaard, R. A. Apoptosis factor EI24/PIG8 is a novel endoplasmic reticulum-localized Bcl2-binding protein which is associated with suppression of breast cancer invasiveness. Cancer Res. 2005, 65 (6), 2125–2129. Mork, C. N.; Faller, D. V.; Spanjaard, R. A. Loss of putative tumor suppressor EI24/PIG8 confers resistance to etoposide. FEBS Lett. 2007, 581, 5440–5444. Alberts, B. The cell as a collection of protein machines: preparing the next generation of molecular biologists. Cell 1998, 92, 291– 294. Kim, S.-y.; Kim, Y. S.; Bahk, Y. Y. Proteome changes induced by expression of tumor suppressor PTEN. Mol. Cells 2003, 15 (3), 396– 405. Kim, S.; Lee, Y. Z.; Kim, Y. S.; Bahk, Y. Y. A proteomic approach for protein-profiling the oncogenic ras induced transformation (H-, K-, and N-Ras) in NIH/3T3 mouse embryonic fibroblasts. Proteomics 2008, 8, 3082–3093. Matsumoto, R.; Nam, H. W.; Agrawal, G. K.; Kim, Y. S.; Iwahashi, H.; Rakwal, R. Exploring novel function of yeast Ssa1/2p by quantitative profiling proteomics using nanoESI-LC-MS/MS. J. Proteome Res. 2007, 6, 3465–3474. Perkins, D. N.; Pappin, D. J.; Creasy, D. M.; Cottrell, J. S. Probabilitybased protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 1999, 20, 3551–3567. Gossen, M.; Freundieb, S.; Bender, G.; Muller, G.; Hillen, W.; Bujard, H. Transcriptional activation by tetracyclines in mammalian cells. Science 1995, 268, 1766–1769. Zhu, H.; Iaria, J.; Sizeland, A. M. Smad7 differentially regulates transforming growth factor β-mediated signaling pathways. J. Biol. Chem. 1999, 274, 32258–32264. Park, J. W.; Kim, S.; Bahk, Y. Y. A Proteomic Approach for Dissecting H-Ras Signaling Networks in NIH/3T3Mouse Embryonic Fibroblast Cells. Proteomics 2006, 6, 2433–2443. Park, J. W.; Kim, S.; Lim, K. J.; Simpson, R. J.; Kim, Y. S.; Bahk, Y. Y. A proteomic approach for unraveling the oncogenic H-Ras protein networks in NIH/3T3 mouse embryonic fibroblast cells. Proteomics 2006, 6 (4), 1175–1186. Lim, Y.; Han, I.; Jeon, J.; Park, H.; Bahk, Y. Y.; Oh, E. S. Phosphorylation of focal adhesion kinase at tyrosine 861 is crucial for ras transformation of fibroblasts. J. Biol. Chem. 2004, 279 (28), 29060– 29065. Matsumoto, M.; Hatakeyama, S.; Oyamada, K.; Oda, Y.; Nishimura, T.; Nakayama, K. I. Large-scale analysis of the human ubiquitinrelated proteome. Proteomics 2005, 5, 4145–4151. Spence, J.; Gali, R. R.; Dittmar, G.; Sherman, F.; Karin, M.; Finley, D. Cell cycle-regulated modification of the ribosome by a variant multiubiquitin chain. Cell 2000, 102, 67–76. Das, B.; Mondragon, M. O.; Sadeghian, M.; Hatcher, V. B.; Norin, A. J. A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines. J. Exp. Med. 1994, 180, 273–281. Walker, J. E. The Mechanism of F1F0-ATPase, special issue. Biochim. Biophys. Acta 2000, 458, 2-3. Arakaki, N.; Nagao, T.; Niki, R.; Toyofuku, A.; Tanaka, H.; Kuramoto, Y.; Emoto., Y.; Shibata, H.; Magota, K.; Higuti, T. Possible role of cell surface H+ -ATP synthase in the extracellular ATP synthesis and proliferation of human umbilical vein endothelial cells. Mol. Cancer Res. 2003, 1, 931–939. Kuwesten, S.; Ohno, M.; Mattaj, I. W. Nucleocytoplasmic transport: Ran, beta and beyond. Trends Cell Biol. 2001, 11 (12), 497–503. Cook, A.; Bono, F.; Jinek, M.; Conti, E. Structural biology of nucleaocytoplasmic transport. Annu. Rev. Biochem. 2007, 76, 647– 671. Rexach, M.; Blobel, B. Protein importin into nucleic association and dissociation reactions involving transport substrate, transport factors and nucleoporins. Cell 1995, 83, 683–692. Lipowsky, G.; Bischoff, F. R.; Schwarzmaier, P.; Kraft, R.; Kostka, S.; Hartmann, E.; Kutay, U.; Go¨rlich, D. Exportin 4: a mediator of a novel nuclear export pathway in higher eukaryotes. EMBO J. 2000, 19 (16), 4362–4371.

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