Array MAPPIT: High-Throughput Interactome Analysis in Mammalian

Recent large-scale interaction mapping efforts are beginning to provide insight in the extent of the interactome network and it seems that most protei...
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Array MAPPIT: High-Throughput Interactome Analysis in Mammalian Cells Sam Lievens,†,‡ Nele Vanderroost,†,‡ Jose´ Van der Heyden,†,‡ Viola Gesellchen,†,‡ Marc Vidal,§,| and Jan Tavernier*,†,‡ Department of Medical Protein Research, VIB, A. Baertsoenkaai 3, 9000 Ghent, Belgium, Department of Biochemistry, Ghent University, A. Baertsoenkaai 3, 9000 Ghent, Belgium, Center for Cancer Systems Biology (CCSB) and Department of Cancer Biology, Dana-Farber Cancer Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115 Received July 9, 2008

Physical interactions between proteins play a key role in probably every cellular process. Efforts to chart the protein interaction networks are ongoing in a number of model organisms using a diversity of approaches. The resulting genome-wide interaction maps will provide a scaffold for further detailed functional analysis. We developed MAPPIT, a mammalian two-hybrid approach that allows identification and analysis of mammalian protein-protein interactions in their native environment. Here, we introduce an efficient MAPPIT assay that permits high-throughput screening of arrayed collections of proteins and complements a previously published cDNA library screening approach. We validated both methods in screens for interaction partners of the Cullin-based E3 ubiquitin ligase subunits SKP1 and Elongin C. In addition to a number of known interactors, novel SKP1 and Elongin C binding proteins were identified. The array assay is an important addition to the MAPPIT suite of technologies that is expected to significantly increase its utility as a toolbox to screen for novel interactors of proteins or small molecules. Keywords: interactome • protein-protein interaction • two-hybrid • MAPPIT • screening

Introduction Protein-protein interactions are at the heart of cellular function. Virtually every cellular process relies on these physical associations, from the brief encounters of proteins involved in signal transduction cascades to the assembly of large and more stable protein complexes that make up molecular machines such as ribosomes, proteasomes or nuclear pore complexes. Recent large-scale interaction mapping efforts are beginning to provide insight in the extent of the interactome network and it seems that most proteins are, at some time point in a cell’s lifespan, involved in complex formation with one or more interaction partners.1-3 A wide variety of strategies is available to map these networks and to analyze in detail the edges between individual proteins.4-6 These can roughly be divided in biochemical methods, generally involving purification of protein complexes from cell lysates, and genetic approaches, based on complementation of genetically encoded hybrid bait and prey proteins which leads to functional restoration of a reporter system in living cells. Many of these technologies offer unique opportunities. For example, yeast two-hybrid and tandem affinity purification approaches have shown to be well-suited for large-scale screens * To whom correspondence should be addressed. Jan Tavernier, A. Baertsoenkaai 3, B-9000 Ghent, Belgium. Tel, +32-9-2649302; fax, +32-92649492; e-mail, [email protected]. † Department of Medical Protein Research, VIB. ‡ Ghent University. § Dana-Farber Cancer Institute. | Harvard Medical School. 10.1021/pr8005167 CCC: $40.75

 2009 American Chemical Society

and have been applied to draw the first pathway-specific and genome-wide interaction maps in different model organisms,7-9 and both fluorescent protein complementation systems and FRET-type assays allow real-time measurement of designated protein-protein interactions.10,11 We developed MAPPIT, for MAmmalian Protein-Protein Interaction Trap (www.mappit.be), a cytokine receptor-based two-hybrid system that operates in mammalian cells12 (Figure 1a). Through its ease of use, this method has proven to be valuabletodetectandstudydesignatedmammalianprotein-protein interactions in their native environment (e.g., see refs 13-15) and to validate data from high-throughput yeast two-hybrid mapping efforts.16-18 MAPPIT constitutes a suite of interaction analysis tools, also including ReverseMAPPIT, where a positive read-out is generated upon disruption of the association between two proteins, allowing screening for compounds (or proteins) that interfere with protein-protein interactions.19 In addition, MASPIT (MAmmalian Small molecule Protein Interaction Trap) is a three-hybrid variant that enables analyzing and screening for interactions between organic molecules and proteins.20 The main assets of the technology stem from the mammalian background it operates in, which ensures proper post-translational modification of the examined proteins and enables evaluation of the effect on protein interactions of alterations of the cellular physiology induced by exogenous stimuli (e.g., activation of a signaling cascade, addition of a (therapeutic) compound or induction of a stress response). Journal of Proteome Research 2009, 8, 877–886 877 Published on Web 01/21/2009

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Figure 1. MAPPIT concept and validation of the screening tools. (a) In MAPPIT, a bait protein is C-terminally fused to a chimeric receptor consisting of the extracellular part of a type I cytokine receptor (CR) such as EpoR or LR and the transmembrane and intracellular domains of the murine leptin receptor (LR) that is made deficient in STAT3 (Signal Transducer and Activator of Transcription 3) recruitment through replacement of receptor tail tyrosines Y985, Y1077 and Y1138 by phenylalanine (F) residues (LR-F3). When coexpressed with a prey that is fused to a receptor fragment containing functional STAT3 recruitment sites (here a fragment of the gp130 receptor chain), the receptor complex is functionally complemented, and upon cytokine ligand stimulation (L), signaling is restored. STAT3 molecules are activated, migrate to the nucleus and induce transcription of a STAT3-reponsive reporter gene. More detailed information can be found on www.mappit.be. (b) FACS analysis of bait expression. TRex44 cells (filled histogram) and TRex44 cell pools stably expressing the bait chimera (clear histogram) for SKP1 (TRex44 pCLL-SKP1; left panel) and Elongin C (TRex44 pCELElonginC; right panel) were stained with an antibody recognizing the extracellular domain of the chimera, anti-LR (left panel) and anti-EpoR (right panel), and analyzed by FACS. (c) MAPPIT assay to evaluate the functionality of the stably expressed SKP1 and Elongin C baits. TRex44 pCLL-SKP1 or TRex44 pCEL-ElonginC cell pools stably expressing the SKP1 or Elongin C bait chimera, respectively, were transiently transfected with the rPAP1-luciferase reporter plasmid and either the SVT prey encoding a subdomain of the SV40 large T protein as a negative control, or a positive control prey, FBXW11 for SKP1 bait and SOCS2 for Elongin C bait. Transfected cells were left untreated or stimulated with the appropriate ligand for 24 h before measuring luciferase activity. Values shown are the average of triplicate samples, error bars correspond to the standard deviation.

For identifying (novel) protein binding partners, we previously developed an open-ended FACS-based screening procedure that allows probing complex cDNA libraries in MAPPIT or MASPIT assays.20,21 Here, we describe a versatile MAPPIT assay format that enables rapid screening of an arrayed prey collection. We evaluate both methods using SKP1 (S-phasekinase-associated protein-1) and Elongin C as a bait, proteins which participate in SCF-(SKP-Cullin-F-box) and ECS-(ElonginC-Cullin-SOCS-box) type E3 ubiquitin ligase complexes, respectively.22

Materials and Methods Plasmid Constructs. The bait vector pCLL-SKP1 containing full-length human SKP1 cloned 3′ to the full-length mutant (Y985F, Y1077F, Y1138F) LR was cloned in the SacI-NotI sites of the pCLL vector as previously described.23 The bait vector pCEL-ElonginC containing full-length mouse Elongin C has been described earlier.24 Preys for transient MAPPIT assays were cloned in the EcoRI-NotI sites of pMG1 or pMG2 plasmids.14 For retroviral transfer, the preys were cloned in the EcoRI-NotI sites of pLN-CD90-CMV-gp130-ccdB.20 For reverse 878

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transfection in the array assay format, prey constructs containing full-length human ORFs were obtained from the hORFeome v3.1 collection.25 Inserts were transferred from Gateway entry vectors (Invitrogen) to the Gateway compatible version of the MAPPIT prey vector pMG1 as described.25 The Gateway compatible pMG1 was derived from pMG1 by cloning Gateway Reading Frame Cassette B from the Gateway Vector Conversion System (Invitrogen) in the EcoRI-NotI sites of pMG1. In addition, a stop codon was inserted immediately after the attR2 site by site-directed mutagenesis using the QuickChange SiteDirected Mutagenesis Kit (Stratagene) with primers 5′-CTTGTACAAAGTGGTTTGATGGCCGCACTAGAGAAAAAACCTCCC3′ and 5′-GGGAGGTTTTTTCTCTAGTGCGGCCATCAAACCACTTTGTACAAG-3′. The E-tagged SKP1 expression construct used in the coimmunoprecipitation assays was obtained by subcloning full-length human SKP1 from pCLL-SKP1 into a pMET7-E-tag vector backbone derived from pMET7-E-CIS which was described earlier.26 pCLL-SKP1 was cut with SalI, blunted by Pfu DNA Polymerase (Stratagene) and cut further with XhoI. pMET7-E-CIS was linearized using NotI, blunted with Pfu DNA Polymerase and cut with XhoI to allow ligation

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Array MAPPIT Interactome Analysis of the SKP1 containing fragment. The mock prey construct expressing only the gp130 receptor fragment was obtained by cutting out the prey insert of a prey-containing pMG1 vector using EcoRI and XhoI, blunting the vector backbone through Pfu DNA Polymerase and self-ligation. Cell Lines, Transfection, Transduction, FACS, and Reporter Assays. Culture conditions, transfection procedures and luciferase reporter assays were as previously described.27 In a typical transient MAPPIT experiment, cells were transfected 24 h after seeding in 6-well plates. After overnight transfection, cells were transferred to 96-well plates and either left unstimulated or treated with the appropriate ligand (100 ng/mL leptin for pCLL-type bait vectors or 10 ng/mL Epo for pCEL-type bait vectors) for 24 h before measuring luciferase activity. Cell pools stably expressing the SKP1 or Elongin C baits were generated by recombining the pCLL-SKP1 or pCEL-ElonginC vectors into the FRT site of the TRex44 screening cell line, respectively,21 according to the protocol of the manufacturer (Invitrogen). Retroviral plasmids were packaged into viral particles using the Plat-E packaging cell line as described.28 Transduction of the bait-expressing cell pool was carried out overnight in the presence of 2.5 µg/mL Polybrene. Bait expression analysis was done by staining cells with rat anti-mouse LR or goat antihuman EpoR antibodies and an appropriate Alexa488-conjugated secondary antibody.21 hIL5RR∆cyt expression was analyzed by staining with the R16-4 mouse monoclonal antibody21 and a secondary PE-conjugated goat anti-mouse IgG1 antibody (Invitrogen). Stained cells were analyzed on a FACSCalibur flow cytometer (BD Biosciences). Coimmunoprecipitation and Western Blot Analysis. HEK293T cells were transfected with Flag-tagged (and E-tagged) expression constructs and lysed in modified RIPA buffer (200 mM NaCl, 50 mM Tris-HCl, pH 8.0, 0.05% SDS, 2 mM EDTA, 1% Nonidet P-40, 0.5% deoxycholic acid and Complete protease inhibitor cocktail (Roche Applied Science)). Lysates were centrifuged and supernatants were precleared using protein G-sepharose beads (GE Healthcare). Prey protein complexes were precipitated by incubation with 4 µg/mL anti-Flag mouse monoclonal antibody (Sigma) and protein G-sepharose. Precipitated proteins were separated by SDS-PAGE and interactions were detected by immunoblotting using anti-Flag M2 (Sigma), anti-E (GE Healthcare) or anti-ElonginC (Santa Cruz Biotechnology) antibodies. cDNA Library Screening. The cDNA library screening procedure used here was adapted from previously described protocols.20,21 TRex44 pCLL-SKP1 cells (250 × 106) were infected with a HEK293T-derived prey cDNA library20 in medium containing 2.5 µg/mL Polybrene. Medium was replaced with fresh medium after 16 h. One day later, cells were ligand stimulated (100 ng/mL mouse leptin; R&D systems). Infection efficiency was determined by staining for expression of CD90 using a PE-conjugated anti-CD90 antibody (2 µg/mL; BD Pharmingen). At 48 h after stimulation, cells were stained for IL5RR expression using the R16-4 monoclonal antibody21 at a final concentration of 1 µg/mL. Cells were stained with a secondary PE-conjugated goat anti-mouse IgG1 antibody (1.5 µg/mL; Invitrogen) and enriched after labeling with anti-PE magnetobeads (StemCell Technologies) using LS columns (Miltenyi). Column eluate was expanded and restimulated for 24 h with ligand. Cells were stained again with R16-4 and PEconjugated goat anti-mouse IgG1 and positive cells were sorted in bulk. After expanding the sorted pool again, positive cells were stained and FACS sorted individually into the wells of

microtiter plates. Sorting steps were done on a MoFlo cell sorter (Beckman Coulter). Cell clones were analyzed for prey and ligand-dependent hIL5RR expression using dot blot with anti-FLAG (Sigma) and R16-4 antibodies, respectively. The prey-cDNA of validated clones was amplified by RT-PCR and sequenced. RNA was isolated with an RNeasy kit (Qiagen), cDNA was prepared with Superscript II RT (Invitrogen) and PCR was performed using primers flanking the cDNA insert (5′-GGCATGGAGGCTGCGACTG-3′ forward and 5′-GTTACTTAAGCTAGCTTGC-3′ reverse). Array Screening. Reverse transfection mixes were prepared using a slightly modified version of the previously described ‘lipid method’ using the Effectene transfection kit (Qiagen).29 Prey plasmid DNA (1 µg) and pXP2d2-rPAP1-luciferase reporter plasmid (0.2 µg)12 were resuspended in EC DNA condensation buffer supplemented with 0.4 M sucrose to a final volume of 15 µL. This mixture was incubated with 1.5 µL of enhancer solution, and next, 5 µL of Effectene transfection reagent was added. Finally, 1 vol (21.5 µL) of 0.1% gelatin was added and the lipid-DNA mixture was diluted with water up to a final volume of 200 µL. Five or four microliters of these mixtures was spotted into black 96-well (Nunc no. 137101) or white 384well (BD Falcon no. 353276) tissue-culture treated microtiter plates, respectively, and dried overnight at room temperature. The whole procedure was automated on a Tecan Freedom Evo robotic platform. To screen the plates, pCLL-SKP1 bait or pCEL-ElonginC bait expressing cells were added at 20 000 cells/well (96-well format) or 5000 cells/well (384-well format); 24 h later, cells were stimulated with ligand (100 ng/mL leptin for pCLL-SKP1 bait expressing cells or 10 ng/mL Epo for pCEL-ElonginC bait expressing cells) or left unstimulated, and after another 24 h, luciferase activity in cell lysates was measured using a TopCount (96-well format) or EnVision (384-well format) reader. For both 96- and 384-well format screens, all preys were assayed in duplicate in both unstimulated and ligand stimulated conditions (4 data points per prey for each screening experiment). In 384-well format, duplicates of unstimulated and stimulated wells were on the same plate, and the average of unstimulated and stimulated luciferase values was normalized for the plate median unstimulated or stimulated value, respectively. For the 96-well format, duplicates and unstimulated and stimulated wells were on separate plates processed in parallel, and values obtained for luciferase activity were normalized for each plate median value first and then averaged between the normalized value of the duplicate plates. A threshold was set to filter out unreliable data: when one of the luciferase data points for a given prey was more than 10× lower than the median value of that plate, or when the CV (coefficient of variation) of either the unstimulated or the stimulated values was more than 1, the data for that prey was removed from the analysis. In addition, preys binding to the MAPPIT receptor system itself, as indicated by their recurrence in different screening experiments using different and unrelated baits, were left out of the analysis.

Results Construction of the Screening Cell Lines. MAPPIT is a two-hybrid technology based on functional complementation of a cytokine receptor signaling pathway (Figure 1a). MAPPIT bait receptors are designed as chimeras consisting of the extracellular part of the erythropoietin receptor (EpoR) or the leptin Journal of Proteome Research • Vol. 8, No. 2, 2009 879

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Figure 2. MAPPIT cDNA library screening. (a) Layout of the cDNA library screening protocol. A cell pool stably expressing a MAPPIT bait chimera is infected with a retrovirally packaged prey cDNA library which enters the cell through a murine ecotropic virus receptor (mEcoR). The retroviral vector expresses both the prey chimera and the CD90 membrane tag for evaluation of infection efficiency. Upon addition of the appropriate cytokine ligand, cells that harbor an interacting bait-prey pair express the STAT3-dependent rPAP1 driven reporter-gene leading to expression of the hIL5RR-∆cyt membrane tag. Using a high affinity antibody against the tag, these cells are coupled with magnetobeads and isolated using MACS. After expanding the recovered cell fraction, cells are restimulated with the cytokine ligand and positive cells are isolated using FACS. The latter cycle may be repeated if necessary, and a depletion step where false positive cells that express the membrane tag without prior cytokine stimulation are sorted out can be incorporated in the protocol. (b) FACS evaluation of the rPAP1-hIL5RR-∆cyt reporter read-out. SKP1 bait expressing TRex44 pCLL-SKP1 cells and a derived cell pool infected with retrovirally packaged FBXW11 prey that had been either ligand stimulated for 24 h or left unstimulated were stained with an anti-hIL5RR antibody and analyzed by FACS. The percentage of cells that exhibit IL5RR-∆cyt expression above background levels is indicated.

receptor (LR), the transmembrane and intracellular portion of a STAT3 recruitment-deficient mutant LR with a C-terminally attached bait, whereas the prey protein is tethered to a gp130 receptor fragment containing functional STAT3 recruitment sites. Bait-prey interaction leads to restoration of the signaling pathway, whereby phosphorylation of the prey chimeras enables recruitment and activation of STAT3 molecules, which stimulate STAT3-dependent reporter activity.12 The Elongin C bait receptor used in this study contains the EpoR extracellular domain and has previously been shown to 880

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allow interaction mapping in MAPPIT.24 The SKP1 bait chimera was coupled with the LR extracellular domain, and was functionally validated in transient MAPPIT assays using a prey corresponding to the F-box protein FBXW11 as a positive control (data not shown). Cell pools stably expressing the SKP1 or Elongin C bait chimeras were generated through Flpmediated recombination in the TRex44 MAPPIT screening cell line. This HEK293-derived cell line expresses a murine ecotropic virus receptor to allow retroviral delivery of a prey cDNA library and carries the STAT3-responsive rPAP1-hIL5RR∆cyt reporter

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Array MAPPIT Interactome Analysis Table 1. F-Box (-Like) Proteins Identified in the SKP1 cDNA Library Screena symbol

description

number of clones (fusions)

FBXL8 FBXL15 FBXW5 FBX044 FBXO2 CDCA3 FBXL6 FBXW9

F-box and leucine-rich repeat protein 8 F-box and leucine-rich repeat protein 15 F-box and WD domain protein 5 F-box protein 44 F-box protein 2 cell division cycle associated 3 F-box and leucine-rich repeat protein 6 F-box and WD-40 domain protein 9

9(5) 1(1) 12(5) 9(7) 1(1) 1(1) 3(1) 5(3)

a For each protein identified, the number of distinct in-frame preycDNA fusions is indicated in parentheses.

which, upon activation, leads to expression of a selectable membrane tag used in the cDNA library screening protocol (Figure 2a).21 Bait expression was confirmed by FACS using an antibody against the extracellular part of the bait receptor chimera (Figure 1b). Functionality of the stably expressed SKP1 and Elongin C baits was verified by transient transfection of FBXW11 or SOCS2 positive control preys respectively, showing strong stimulation-dependent luciferase signals for both interactions (Figure 1c). MAPPIT cDNA Library Screening for Interactors of SKP1. Having confirmed the ability to obtain robust MAPPIT signals using the luciferase reporter, we next checked the functionality of the hIL5RR∆cyt reporter system that is used in the cDNA library screening approach. MAPPIT analysis using the rPAP1-hIL5RR∆cyt reporter read-out was evaluated by infecting the SKP1 bait expressing TRex44 cell pool with a retrovirally packaged FBXW11 prey construct (Figure 2b). Approximately 13% of the infected cell pool displayed liganddependent hIL5RR∆cyt reporter expression, well in accordance with the infection rate that was determined to be around 14% (data not shown). No cells of the uninfected parental TRex44 cell line expressing the SKP1 bait stained positive. As a first approach to identify protein binding partners of SKP1, we applied a prey cDNA library screening protocol (Figure 2a).20,21 The SKP1 bait-expressing cell pool was infected with a retrovirally packaged HEK293T derived prey cDNA library. Infection efficiency was around 16% as determined by staining for the CD90 membrane tag, which was constitutively expressed by the prey-encoding retrovirally transferred sequences (data not shown). On the basis of their liganddependent expression of the hIL5RR∆cyt membrane tag, cells harboring a prey that interacts with SKP1 were first enriched through a magnetobead assisted cell sorting (MACS) step and two consecutive rounds of FACS sorting (data not shown). Starting from 250 × 106 library infected cells, 3 × 106 were recovered in the MACS eluate. After bulk sorting of the expanded and restimulated MACS eluate as a second enrichment step, a population corresponding to cells expressing high to intermediate levels of hIL5RR∆cyt after ligand stimulation as compared to an unstimulated control sample was isolated in the final single cell FACS sort. Individually sorted cells were expanded and ligand-dependent hIL5RR∆cyt reporter expression was confirmed in dot blot experiments (data not shown). Prey identity of the clones showing ligand-inducible hIL5RR∆cyt staining was revealed by sequencing prey-specific RT-PCR products (Table 1). From the 113 clones that gained an RT-PCR amplification product, 93 gave a readable sequence corresponding to a MAPPIT prey as

determined by the presence of gp130-specific sequences. For 33 clones, the sequence of which corresponded to 6 distinct proteins, the prey was a known interactor of SKP1. These include CDCA3, an F-box like protein that participates in E3 ligase complexes involved in cell cycle regulation, and a number of F-box proteins (FBXL8, FBXL15, FBXW5, FBXO44 and FBXO2), all of which are SCF E3 ligase subunits implicated in recognition of substrates targeted for ubiquitination. In addition, 8 clones contained preys corresponding to 2 other F-box proteins not previously reported to be SKP1 interactors (FBXL6 and FBXW9). It is of note that for many of the preys, multiple cell clones were identified containing distinct cDNA fragments and thus expressing different in-frame prey fusion proteins (Table 1). Array MAPPIT Screening for interactors of SKP1 and Elongin C. MAPPIT cDNA library screening assures a wide coverage of the proteome, but due to the large complexity of the libraries involved, the procedure to isolate the small fraction of positive cells harboring an interacting bait and prey is lengthy and prey identification is laborious. We sought a way to develop a more efficient MAPPIT screening approach taking advantage of the availability of a large collection of full-length human ORFs which can easily be transferred to a MAPPIT prey vector by recombinatorial cloning.25 We developed a method based on reverse transfection,29 where mixtures of the prey plasmids and the luciferase reporter are complexed with a lipofection agent and spotted in microtiter plates. By simply culturing bait-expressing cells in these plates, they are (reverse) transfected resulting in a cell array where each well contains cells expressing a different prey (Figure 3). We generated a microtiter plate array containing a subset of 1879 full-length human ORF preys which were selected from the available human ORF collection based on their association with the Gene Ontology annotation30 ‘signal transduction’ (Supporting Information Table 1). The arrayed prey collection was screened for interactors of SKP1 by covering the plates with the SKP1 bait-expressing cell pool previously used in the cDNA screening experiment, stimulating the plates with the appropriate ligand and measuring luciferase activity. To evaluate reproducibility of the assay, two independent screening experiments were performed with the SKP1 bait. The combined results of both screenings are presented in Figure 4a as a dot plot of normalized luciferase values of ligand-stimulated versus unstimulated wells. When a threshold of 10-fold ligand-induced reporter gene induction was used, the same set of 5 preys was positive in both experiments. All of these preys correspond to F-box proteins. Three (BTRC, FBXL8 and FBXW11) are known and 2 (FBXW9 and FBXO46) are novel SKP1 binding partners. When lowering the threshold, none of the preys scored positive in both screens. Also, none of these corresponded with a protein that, based on the well-characterized organization and function of SCF-type E3 ubiquitin ligases, would be a valid candidate direct interaction partner of SKP1, such as, for example, an F-box protein. Two of the preys scoring positive in the array were also identified in the cDNA library screen (FBXL8 and FBXW9). To validate the MAPPIT screening data, the specific interaction with SKP1 of FBXW9 and FBX046 was confirmed in coimmunoprecipitation experiments (Figure 5). The same arrayed collection of preys was also screened for proteins binding to Elongin C, by overlaying the set of plates covering the collection with the Elongin C bait expressing cell pool (Figure 4b). Five preys scored positive when the threshold was set at 10-fold induction. Four of these (SOCS2, RAB40B, Journal of Proteome Research • Vol. 8, No. 2, 2009 881

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Figure 3. Array MAPPIT screening concept. Schematic overview of the array MAPPIT procedure. A collection of prey plasmids together with a reporter plasmid is mixed with a transfection reagent and spotted in microtiter plates to generate prey arrays. Sets of these are reverse transfected by overlaying them with a bait-expressing cell line and treated with ligand or left unstimulated before read-out of the luciferase activity. Parallel sets covering the complete prey collection could be differentially stimulated by exogenous treatments to evaluate their effect on the interaction pattern.

ASB9 and ASB1) correspond to proteins known to bind to Elongin C. Lowering the cutoff enabled the detection of additional established Elongin C interaction partners but, at the same time, also increasing numbers of proteins that have not been correlated to Elongin C. When the threshold was set at 3-fold induction, 27 preys were identified in total, 10 of which correspond to known Elongin C binding proteins. All other preys were evaluated by transient MAPPIT retests (data not shown) and validation by coimmunoprecipitation, identifying two additional preys as true interactors of Elongin C (SPSB2 and SPSB4; Figure 5).

Discussion In this report, two conceptually different MAPPIT screening approaches were applied to identify protein interaction partners of SKP1 and Elongin C, key adaptor subunits of SCF and ECS complexes, respectively, two Cullin-based types of ubiquitin E3 ligases involved in marking proteins for degradation by the proteasome machinery.22 SCF (SKP-Cullin-F-box) ligases are multisubunit complexes held together by the Cullin subunit CUL1. At its carboxyl terminus, this molecular scaffold protein interacts with the RBX1 RING-finger protein which is involved in recruiting a specific E2 ubiquitin-conjugating enzyme that carries the ubiquitin unit which is to be transferred to the protein targeted for degradation. SKP1 bridges between the amino terminus of CUL1 and an F-box protein, the variable component of SCF complexes which is responsible for substrate recognition and thus determines target specificity.31 Elongin C performs a similar function in ECS (Elongin C-CullinSOCS-box) type E3 ubiquitin ligases, where it links a central CUL2 subunit to SOCS (Suppressor Of Cytokine Signaling)-box containing substrate receptor proteins.22 In a first approach, a cDNA library screening protocol was applied to identify proteins binding to the SKP1. Screening a HEK293T derived prey cDNA library yielded 6 known F-box or F-box-like interaction partners. In addition, 2 F-box proteins were identified that had not previously been reported to interact with SKP1 (FBXL6 and FBXW9). For some of these preys, multiple clones were selected and different clones harboring distinct gp130-prey fusions were found, indicative of the authenticity of the interaction. Both the total number of 882

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preys and the number of distinct fusions identified for a particular prey is likely to increase with increasing coverage of the complexity of the cDNA library screened, which was limited in the experiment reported here (around 10%). The cDNA library screening procedure had already been successfully used previously to screen for protein targets small molecules using the three-hybrid MASPIT setup. Using the ABL kinase inhibitor PD173955 as a bait, in addition to the known target ABL itself, a number of other kinases were identified, the interaction of which could be confirmed using independent methods.20 Thus, MAPPIT cDNA library screening is a valid approach for the discovery of protein interactors of both proteins and organic molecules. One of the main advantages of a cDNA library approach is the wide coverage of the proteome, as it includes multiple splice variants. In addition, cDNA libraries also encode protein fragments, which may reveal interactions that would not be detected with the corresponding full-length protein due to intramolecular interactions between protein subdomains. The downside, however, is that screening these highly complex libraries involves a multistage procedure and identification of the prey sequence requires a number of additional experimental steps. Array MAPPIT was developed to complement the openended cDNA screening protocol with an efficient procedure to screen a fixed collection of preys. The approach is very fast (3 days from seeding cells on premade arrays to signal readout) and easily automated, and prey identity is simply decoded by its position in the array. Screening an array of 1879 full-length human ORFs with the same SKP1 bait-expressing cell pool as applied in the cDNA library screening experiment, we obtained 5 positive hits corresponding to 3 known and 2 novel SKP1 interacting proteins. As for the known interactors, the novel interaction partners (FBXW9 and FBXO46) were members of the F-box protein family. In an array MAPPIT screen using an Elongin C bait, a total of 12 interaction partners were identified, 10 that were previously reported and 2 novel binding proteins, SPSB2 and SPSB4. The latter belong to the SPSB family of proteins that contain a SOCS box, the domain that is crucial for the interaction with Elongin C in ECS-type E3 ubiquitin ligase complexes.

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Figure 4. Array MAPPIT screening experiments. (a) A MAPPIT prey array containing 1879 preys was covered with a cell pool stably expressing the SKP1 chimeric bait protein (TRex44 pCLL-SKP1), stimulated with ligand or left unstimulated, and luciferase activity was measured. The dot plot shows normalized values of two independent screens performed in 96-well plate format (squares) and 384-well plate format (triangles). The dashed line indicates the threshold of 10-fold induction of ligand stimulated over unstimulated values. Known and novel interaction partners are shown in blue and green, respectively. (b) The same array collection as in (a) was screened by overlaying the plates with a Elongin C bait expressing cell pool (TRex44 pCEL-ElonginC). Normalized values of a 384-well format screening experiment are presented, with known and new proteins binding the Elongin C bait chimera shown in blue and green, respectively. Different induction factor cutoffs are marked by the dashed lines. Journal of Proteome Research • Vol. 8, No. 2, 2009 883

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Figure 5. Coimmunoprecipitation experiments confirming the novel interactions detected in the array MAPPIT screening experiments. HEK293-T cells were transfected with E-tagged SKP1 expression construct combined with either mock (empty prey vector only expressing the gp130 receptor fragment as negative control), FBXW11 (positive control), FBXW9 or FBXO46 Flag-tagged prey constructs (left panel) or with mock (negative control), SOCS2 (positive control), SPSB2 or SPSB4 Flag-tagged prey contructs alone (right panel). Preys were precipitated using an anti-Flag antibody. SKP1 was detected using an anti-E antibody (left panel) and endogenous Elongin C was revealed with an anti-Elongin C antibody. Upper panels show total lysates, lower panels correspond to immunoprecipitated protein fractions. Arrowheads mark the bands corresponding to the precipitated prey proteins.

Reproducibility of the array MAPPIT assay was evaluated by performing the SKP1 screening experiment using two independent sets of prey arrays. Applying the same stringency cutoff, both screenings identified the same set of F-box proteins, illustrating the robustness of the technology. The Elongin C screen illustrates the inverse correlation between screening stringency and false positive rate. Applying a high stringency threshold of 10-fold induction results in the identification of only true interactors. Lowering the cutoff gradually increases the number of false positives demonstrating that screening efficiency can be adjusted by choosing the appropriate threshold. Apart from false positives, every method also suffers from false negatives. In the SKP1 screen, no known interactions were missed according to the currently available interactome data (Ingenuity Knowledge Base (version 1602, Ingenuity Systems), BIND, BIOGRID, Cognia, DIP, INTACT, MINT and MIPS databases). In the case of the Elongin C screen, however, the arrayed collection contained a number of proteins that have previously been shown to directly interact with Elongin C and that did not score positive, even using the lower threshold (CIS, WSB2, SOCS3, SOCS4, SOCS6 and ASB3). As in any of this type of assays, there are different sources of false negatives in MAPPIT. A common source of failure to detect interactions in two-hybrid methods is steric hindrance of the interaction interface due to the fusion with components of the complementation system, the intracellular LR tail and the gp130 receptor fragment in the case of MAPPIT. In addition, since the bait receptor is anchored to the plasma membrane in MAPPIT, the assay depends on the prey being present in the submembrane cytoplasmic region. Incompatible prey localization can be overcome by removing the responsible localization signals (e.g., removal of the NLS for nuclear proteins) or by using protein subdomains (e.g., using only the intracellular domain of a membrane protein). In the current assay setup, we are bound to full-length proteins, but future arrays could be optimized by using protein subdomains. Some proteins can interfere with the assay itself, which is the case for SOCS3, a false negative in this screen. SOCS3 is a protein that associates with the LR 884

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and inhibits activation of JAK2, and was previously shown to be incompatible with MAPPIT for this reason.32 A recent study, aimed at putting together a toolkit for confidence scoring of novel interactions identified in largescale, high-throughput, yeast two-hybrid screening campaigns, compared a number of genetic interaction mapping technologies, including yeast two-hybrid and MAPPIT.33 These data show that the tested methods perform comparably well, with a sensitivity between 20 and 35% (see also ref 17). The overlap between the subsets of interactions detected by the different methods, however, is markedly limited, indicating that each method has specific limitations as to which type of protein interaction it is able to detect, and illustrating that these methods are highly complementary.

Conclusion Because of its simplicity, the array assay format greatly expands the utility of the MAPPIT toolbox as an in-cell trap to screen for novel protein binding partners either involved in protein-protein or in small molecule-protein interactions using MAPPIT or MASPIT, respectively. In the near future, we will expand the MAPPIT prey collection by transferring additional full-length ORFs from the CCSB-DFCI collection, which in version 5.1 contained 15 483 clones. In a later stage, also splice variants and subdomains can be incorporated. In addition, the assay can be downsized using high-density arrays on glass slides.29 As it operates in intact mammalian cells, MAPPIT enables protein interaction analysis under conditions induced by external stimuli that mimic a specific physiological state of the cell, for example, activation of signaling pathways by addition of a ligand or a compound or by applying (a)biotic stress factors, in specific cases after engineering the cells in order to make them responsive to the applied stimulus. The array assay format opens up the opportunity of rapid parallel comparison of interaction networks under different physiological conditions or between different baits (e.g., different splice variants of a given gene) across the whole prey

Array MAPPIT Interactome Analysis collection (Figure 3). This approach could potentially complement the extensive but static networks yielded by ongoing large-scale interactome mapping projects with information on the functional dynamics of the protein interaction network.34

Acknowledgment. We thank N. Kley for a sample of the HEK293T-derived MAPPIT prey cDNA library. This research was supported by grants from The Fund of Scientific Research-Flanders (No. G.0031.06), from Ghent University (GOA No. 12051401) and from the IUAP-6 (No. P6:28). Supporting Information Available: The complete list of preys used in the array MAPPIT screening experiments can be found in Supplementary Table 1. This material is available free of charge via the Internet at http://pubs. acs.org. References (1) Albert, R. Scale-free networks in cell biology. J. Cell Sci. 2005, 118 (Pt 21), 4947–4957. (2) Vidal, M. A biological atlas of functional maps. Cell 2001, 104 (3), 333–339. (3) Gavin, A. C.; Superti-Furga, G. Protein complexes and proteome organization from yeast to man. Curr. Opin. Chem. Biol. 2003, 7 (1), 21–27. (4) Kocher, T.; Superti-Furga, G. Mass spectrometry-based functional proteomics: from molecular machines to protein networks. Nat. Methods 2007, 4 (10), 807–815. (5) Lievens, S.; Lemmens, I.; Montoye, T.; Eyckerman, S.; Tavernier, J. Two-hybrid and its recent adaptations. Drug Discovery Today: Technol. 2006, 3, 317–324. (6) Suter, B.; Kittanakom, S.; Stagljar, I. Interactive proteomics: what lies ahead. BioTechniques 2008, 44 (5), 681–691. (7) Bouwmeester, T.; Bauch, A.; Ruffner, H.; Angrand, P. O.; Bergamini, G.; Croughton, K.; Cruciat, C.; Eberhard, D.; Gagneur, J.; Ghidelli, S.; Hopf, C.; Huhse, B.; Mangano, R.; Michon, A. M.; Schirle, M.; Schlegl, J.; Schwab, M.; Stein, M. A.; Bauer, A.; Casari, G.; Drewes, G.; Gavin, A. C.; Jackson, D. B.; Joberty, G.; Neubauer, G.; Rick, J.; Kuster, B.; Superti-Furga, G. A physical and functional map of the human TNF-alpha/NF-kappa B signal transduction pathway. Nat. Cell Biol. 2004, 6 (2), 97–105. (8) Rual, J. F.; Venkatesan, K.; Hao, T.; Hirozane-Kishikawa, T.; Dricot, A.; Li, N.; Berriz, G. F.; Gibbons, F. D.; Dreze, M.; Ayivi-Guedehoussou, N.; Klitgord, N.; Simon, C.; Boxem, M.; Milstein, S.; Rosenberg, J.; Goldberg, D. S.; Zhang, L. V.; Wong, S. L.; Franklin, G.; Li, S.; Albala, J. S.; Lim, J.; Fraughton, C.; Llamosas, E.; Cevik, S.; Bex, C.; Lamesch, P.; Sikorski, R. S.; Vandenhaute, J.; Zoghbi, H. Y.; Smolyar, A.; Bosak, S.; Sequerra, R.; Doucette-Stamm, L.; Cusick, M. E.; Hill, D. E.; Roth, F. P.; Vidal, M. Towards a proteomescale map of the human protein-protein interaction network. Nature 2005, 437 (7062), 1173–1178. (9) Stelzl, U.; Worm, U.; Lalowski, M.; Haenig, C.; Brembeck, F. H.; Goehler, H.; Stroedicke, M.; Zenkner, M.; Schoenherr, A.; Koeppen, S.; Timm, J.; Mintzlaff, S.; Abraham, C.; Bock, N.; Kietzmann, S.; Goedde, A.; Toksoz, E.; Droege, A.; Krobitsch, S.; Korn, B.; Birchmeier, W.; Lehrach, H.; Wanker, E. E. A human proteinprotein interaction network: a resource for annotating the proteome. Cell 2005, 122 (6), 957–968. (10) Jares-Erijman, E. A.; Jovin, T. M. Imaging molecular interactions in living cells by FRET microscopy. Curr. Opin. Chem. Biol. 2006, 10 (5), 409–416. (11) Kerppola, T. K. Visualization of molecular interactions by fluorescence complementation. Nat. Rev. Mol. Cell Biol. 2006, 7 (6), 449– 456. (12) Eyckerman, S.; Verhee, A.; der Heyden, J. V.; Lemmens, I.; Ostade, X. V.; Vandekerckhove, J.; Tavernier, J. Design and application of a cytokine-receptor-based interaction trap. Nat. Cell Biol. 2001, 3 (12), 1114–1119. (13) Erkeland, S. J.; Aarts, L. H.; Irandoust, M.; Roovers, O.; Klomp, A.; Valkhof, M.; Gits, J.; Eyckerman, S.; Tavernier, J.; Touw, I. P. Novel role of WD40 and SOCS box protein-2 in steady-state distribution of granulocyte colony-stimulating factor receptor and G-CSFcontrolled proliferation and differentiation signaling. Oncogene 2007, 26 (14), 1985–1994.

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