Anal. Chem. 2006, 78, 556-560
Intein-Mediated Reporter Gene Assay for Detecting Protein-Protein Interactions in Living Mammalian Cells Akira Kanno,† Takeaki Ozawa,†,‡ and Yoshio Umezawa*,†
Department of Chemistry, School of Science, The University of Tokyo, Hongo Bunkyo-ku, Tokyo 113-0033, Japan, and Japan Science and Technology Corporation, Tokyo, Japan, Department of Molecular Structure, Institute for Molecular Science, Myodaiji, Okazaki, Aichi 444-8585, Japan
For nondestructive analysis of chemical processes in living mammalian cells, here we developed a new reporter gene assay for detecting cytosolic protein-protein interactions based on protein splicing of transcription factors with DnaE inteins. The protein splicing induces connection of a DNA-binding protein (modified LexA; mLexA) with a transcription activation domain of a herpes simplex virus protein (VP16AD). We thereby circumvented the limitation of earlier methods for monitoring proteinprotein interactions, including the two-hybrid systems, protein complementation systems (PCS), and protein reconstitution systems, and rather combined their advantages. To test the applicability of this method, we monitored epidermal growth factor (EGF)-induced interactions on cell membranes of a known partner, an oncogenic product Ras and its target Raf-1. Ras was connected with N-terminal DnaE and mLexA, while Raf-1 was connected with C-terminal DnaE and VP16AD. Upon stimulation with EGF, the interaction between Ras and Raf-1 triggered folding of the DnaEs, thereby inducing protein splicing to form mLexA-VP16AD fusion protein, and transcription of a reporter gene, firefly luciferase. The extent of RasRaf-1 interaction was quantified by measuring the luciferase activity. The interaction was not able to be monitored by two-hybrid systems nor by PCS of split firefly luciferases; however, by using the protein splicing elements and the reporter gene, we obtained the bioluminescence signals sufficient for evaluation of the interactions close to cell membranes. Protein-protein interactions play pivotal roles in many chemical processes in living cells, yet they have been among the most difficult aspects of molecular and cellular biology to be studied. Monitoring protein-protein interactions in living cells is important for screening and assaying chemicals that would increase or inhibit cellular signaling processes. To promote a greater understanding about the chemical processes, several methods have been developed for detecting protein-protein interactions. Available information about protein-protein interactions was yielded mostly * To whom correspondence should be addressed: (phone) +81-3-5841-4351; (fax) +81-3-5841-8349; (e-mail)
[email protected]. † The University of Tokyo. ‡ Institute for Molecular Science.
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by biochemical methods, but these methods were of destructive analysis, which did not provide us with live-cell dynamics. The yeast two-hybrid system1,2 and the mammalian two-hybrid system3,4 use a “bait” protein fused to a DNA binding domain with a nuclear localizing sequence (NLS) in order to find its “prey” protein connected to a transcription activation domain with a NLS. The interaction between the bait and the prey accumulates the transcription activation domain on a specific sequence of a DNA lying upstream of a reporter gene; thus, the reporter gene expression is transactivated. Although significant signals for detection are obtained with the two-hybrid systems, their limitations are that detectable protein-protein interactions occur only in the nucleus to transactivate the reporter gene.5-7 The splitubiquitin system8-10 for detecting an interaction between a membrane protein and a cytosolic protein also has a limitation that interactions between cytoplasm proteins or nuclear localizing proteins cannot be detected. To overcome these limitations, several methods have been reported, including the protein complementation system (PCS) such as dihydrofolate reductase,11,12 β-galactosidase,13,14 β-lacta(1) Fields, S.; Song, O. K. Nature 1989, 340, 245-246. (2) Chien, C. T.; Bartel, P. L.; Sternglanz, R.; Fields, S. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9578-9582. (3) Dang, C. V.; Barrett, J.; Villagarcia, M.; Resar, L. M. S.; Kato, G. J.; Fearon, E. R. Mol. Cell. Biol. 1991, 11, 954-962. (4) Fearon, E. R.; Finkel, T.; Gillison, M. L.; Kennedy, S. P.; Casella, J. F.; Tomaselli, G. F.; Morrow, J. S.; Dang, C. V. Proc. Natl. Acad. Sci. U.S.A. 1992, 89, 7958-7962. (5) Flores, A.; Briand, J. F.; Gadal, O.; Andrau, J. C.; Rubbi, L.; Van Mullem, V.; Boschiero, C.; Goussot, M.; Marck, C.; Carles, C.; Thuriaux, P.; Sentenac, A.; Werner, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 7815-7820. (6) Ito, T.; Tashiro, K.; Muta, S.; Ozawa, R.; Chiba, T.; Nishizawa, M.; Yamamoto, K.; Kuhara, S.; Sakaki, Y. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 11431147. (7) Walhout, A. J. M.; Sordella, R.; Lu, X. W.; Hartley, J. L.; Temple, G. F.; Brasch, M. A.; Thierry-Mieg, N.; Vidal, M. Science 2000, 287, 116-122. (8) Johnsson, N.; Varshavsky, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1034010344. (9) Stagljar, I.; Korostensky, C.; Johnsson, N.; te Heesen, S. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5187-5192. (10) Dunnwald, M.; Varshavsky, A.; Johnsson, N. Mol. Biol. Cell 1999, 10, 329344. (11) Remy, I.; Michnick, S. W. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 53945399. (12) Pelletier, J. N.; Arndt, K. M.; Pluckthun, A.; Michnick, S. W. Nat. Biotechnol. 1999, 17, 683-690. (13) Rossi, F.; Charlton, C. A.; Blau, H. M. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 8405-8410. (14) Wehrman, T.; Kleaveland, B.; Her, J. H.; Balint, R. F.; Blau, H. M. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 3469-3474. 10.1021/ac051451a CCC: $33.50
© 2006 American Chemical Society Published on Web 12/07/2005
mase,15 green fluorescent protein (GFP)16 and its variants,17 firefly luciferase,18 and Renilla luciferase.19,20 We have previously proposed a novel concept, a protein reconstitution system (PRS) based on protein splicing, for detecting protein-protein interactions21-23 and protein localization in organelles.24,25 Although PCS and PRS allow us to monitor interactions between cytoplasm and membrane-proximal proteins, these systems suffer from low end point signals for the interactions, due to much lower activity of complemented or of reconstituted split reporters in comparison to intact reporter proteins. Here we describe a protein splicing-based reporter gene assay to monitor protein-protein interactions in mammalian cells. Protein splicing is a posttranslational autocatalytic processing in which an intein is excised out with the concomitant ligation of the flanking exteins.26-29 An important property of the protein splicing is that the substitution of exteins for different peptides does not interfere with the splicing process.30,31 We chose N- and C-terminal halves of an Ssp. DnaE intein as the protein splicing elements, and modified LexA (mLexA) and a transcription activation domain of a herpes simplex virus protein (VP16AD) were used as the couple of transcription factors. The present reporter gene assay allows us to detect epidermal growth factor (EGF)induced membrane-proximal Ras-Raf-1 interactions that could not be detected with the previous reporter gene assay, the two-hybrids method. In addition, the present reporter gene assay enabled us to obtain sufficient signals for the interactions that were not identified with the firefly luciferase complementation system. EXPERIMENTAL SECTION Materials. All reagents used were of the highest available purity. DNA-modifying enzymes were from Takara Bio Inc. (Tokyo, Japan). pCMV-Ras encoding Ras, pCMV-RasV12 encoding RasV12, and pCMV-Raf-1 encoding Raf-1 were from Clontech (Palo Alto, CA). pRL-TK encoding Renilla luciferase, pG5luc encoding firefly luciferase, and pACT encoding VP16AD were obtained from (15) Galarneau, A.; Primeau, M.; Trudeau, L. E.; Michnick, S. W. Nat. Biotechnol. 2002, 20, 619-622. (16) Magliery, T. J.; Wilson, C. G. M.; Pan, W. L.; Mishler, D.; Ghosh, I.; Hamilton, A. D.; Regan, L. J. Am. Chem. Soc. 2005, 127, 146-157. (17) Nyfeler, B.; Michnick, S. W.; Hauri, H. P. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 6350-6355. (18) Luker, K. E.; Smith, M. C. P.; Luker, G. D.; Gammon, S. T.; Piwnica-Worms, H.; Piwnica-Worms, D. P. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 1228812293. (19) Kaihara, A.; Kawai, Y.; Sato, M.; Ozawa, T.; Umezawa, Y. Anal. Chem. 2003, 75, 4176-4181. (20) Paulmurugan, R.; Gambhir, S. S. Anal. Chem. 2003, 75, 1584-1589. (21) Ozawa, T.; Nogami, S.; Sato, M.; Ohya, Y.; Umezawa, Y. Anal. Chem. 2000, 72, 5151-5157. (22) Ozawa, T.; Kaihara, A.; Sato, M.; Tachihara, K.; Umezawa, Y. Anal. Chem. 2001, 73, 2516-2521. (23) Ozawa, T.; Takeuchi, M.; Kaihara, A.; Sato, M.; Umezawa, Y. Anal. Chem. 2001, 73, 5866-5874. (24) Ozawa, T.; Sako, Y.; Sato, M.; Kitamura, T.; Umezawa, Y. Nat. Biotechnol. 2003, 21, 287-293. (25) Kim, S. B.; Ozawa, T.; Watanabe, S.; Umezawa, Y. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 11542-11547. (26) Hirata, R.; Ohsumi, Y.; Anraku, Y. FEBS Lett. 1989, 244, 397-401. (27) Kane, P. M.; Yamashiro, C. T.; Wolczyk, D. F.; Neff, N.; Goebl, M.; Stevens, T. H. Science 1990, 250, 651-657. (28) Noren, C. J.; Wang, J. M.; Perler, F. B. Angew. Chem., Int. Ed. 2000, 39, 451-466. (29) Paulus, H. Annu. Rev. Biochem. 2000, 69, 447-496. (30) Cooper, A. A.; Chen, Y. J.; Lindorfer, M. A.; Stevens, T. H. Embo J. 1993, 12, 2575-2583. (31) Chong, S. R.; Xu, M. Q. J. Biol. Chem. 1997, 272, 15587-15590.
Promega Co. (Madison, WI). pEG202 encoding LexA and pSH1834 encoding LexA operator were purchased from OriGene Technologies, Inc. (Rockville, MD). Mammalian expression vectors, pcDNA3.1(+) and pcDNA3.1(+)/myc-His (B), were obtained from Invitrogen (Groningen, Netherlands). Ham’s F-12 medium, fetal bovine serum (FBS), and a LipofectAMINE 2000 reagent were obtained from Gibco BRL (Rockville, MD). An alkaline phosphatase-labeled anti-mouse IgG antibody was purchased from Jackson ImmunoResearch Laboratories, Inc. A chemiluminescent substrate, CDP-STAR, was obtained from New England Biolabs, Inc. (Beverly, MA). Construction of Plasmids for Mammalian Cell Expression. The Escherichia coli strain DH5R was used as the bacterial host for all plasmid construction. For protein expressions in mammalian cells, we used pcDNA3.1(+), which has a human cytomegalovirus immediate-early (CMV) promoter. DNA sequences of constructs are shown in Figure 1b. According to the previous report,32 we made two amino acid substitutions, R157G and K159E, to delete the nuclear localization signal. The detailed schemes are available on request. The sequences of all plasmids were verified by sequencing with a genetic analyzer ABI prism 310 (PE Biosystems, Tokyo, Japan). Cell Culture and Transfection. CHO-EGFR cells were cultured in Ham’s F-12 medium supplemented with 10% heatinactivated FBS, 100 units/mL penicillin, and 100 µg/mL streptomycin at 37 °C in 5% CO2. The CHO-EGFR cells were seeded in 12-well culture plates with penicillin- and streptomycin-free Dulbecco’s modified Eagle’s medium (Sigma) and grown to 8090% confluence before transient transfection using LipofectAMINE 2000 reagent. All cells to be assessed by dual-luciferase reporter assay33 were transfected with 0.5 µg of the reporter plasmid pX8luc and 5 pg of pRL-TK vector. Eight hours after the transfection, the medium was replaced with Ham’s F-12 medium supplemented with 10% FBS, penicillin, and streptomycin, and then the cells were incubated at 37 °C for 24 h. Expression of fusion proteins was confirmed with western blotting. Evaluation of Firefly Luciferase Activities. Cells are transfected under the same experimental conditions at different wells on culture plates; however, variation such as the number of cells and transfection efficiency is not negligible in each replicate. To eliminate these errors, dual-luciferase reporter assay was performed,33 in which firefly (Photinus pyralis) and Renilla (Renilla reniformis, also known as sea pansy) luciferases were measured sequentially from a single sample. Firefly luciferase activity was monitored after adding the substrate molecule for firefly luciferase, beetle luciferin. After quantifying the firefly luminescence, this reaction was quenched, and Renilla luciferase activity was measured with the substrate molecule for Renilla luciferase, coelenterazine. The luminescence from firefly luciferase (LF) was measured for the first 10 s, and the luminescence coming from Renilla luciferase (LR) was measured for another 10 s. The firefly luminescence normalized against the Renilla luminescence was termed as relative light unit (RLU; RLU ) LF/ LR). All measurements of the luminescence were performed with a Minilumat (32) Rhee, Y.; Gurel, F.; Gafni, Y.; Dingwall, C.; Citovsky, V. Nat. Biotechnol. 2000, 18, 433-437. (33) Lorenz, W. W.; McCann, R. O.; Longiaru, M.; Cormier, M. J. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 4438-4442.
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Table 1. Activities of Firefly Luciferase in the Presence or Absence of Transcription Factorsa RLU mLexA-VP16AD CHO-EGFR reporter mLexA-DnaEn DnaEc-VP16AD
189 ( 16 0.112 ( 0.015 0.203 ( 0.021 0.334 ( 0.030 0.216 ( 0.030
a CHO-EGFR cells were cultured in 12-well plates and separately transfected with 0.5 µg of plasmid, pmLV (mLexA-VP16AD), pmLDn (mLexA-DnaEn), or pDcV (DnaEc-VP16AD). All cells to be assayed were transfected with 5 pg of the control vector pRL-TK and 0.5 µg of the reporter gene pX8luc. The cells were incubated for 48 h, and the luminescence intensities were measured. As their control, intact CHOEGFR cells (CHO-EGFR) and CHO-EGFR cells transfected only with 0.5 µg of pX8luc (reporter) were incubated under otherwise identical conditions.
Figure 1. (a) Principle for the intein-mediated reporter gene assay. DnaEn (amino acids 1-123) and DnaEc (amino acids 1-36) are connected with mLexA (amino acids 1-229) and VP16AD (amino acids 411-456), respectively. Interested protein X and Y are linked to the ends of DnaEs. Interaction between X and Y accelerates the folding of DnaEn and DnaEc, and protein splicing results. mLexA and VP16AD are linked together by a peptide bond to obtain a transcriptional activity. (b) The schematic structures of the constructs. c-Myc is myc epitope tag, EQKLISEEDL. The sequence of the GS linker is (GGGGS)6. MCSs are multiple cloning sites. Ras conveys an extracellular signal to its target of effector proteins, Raf-1. cDNAs encoding each fusion protein are inserted into pcDNA3.1(+). For the construction of pX8luc, five GAL4 binding sites in a pG5luc vector are replaced with eight-repeated LexA operators. “Promoter” indicates a major late promoter of adenovirus.
LB9506 luminometer (Berthold GmbH & Co. KG, Wildbad, Germany) and were made in triplicate with different wells of culture plates. RLU values in Table 1 and in Figures 2-4 were averages with standard deviations. Immunoblot Analysis. Lysate of cells transfected with given plasmids was separated by SDS-PAGE and electrophoretically transferred onto a nitrocellulose membrane. The membrane was probed with monoclonal anti-LexA antibody (Santa Cruz, CA) or monoclonal anti-c-Myc antibody (Santa Cruz, CA) and then with alkaline phosphatase-labeled anti-mouse IgG antibody. The secondary antibody was visualized with a chemiluminescence system of CDP-STAR by using a LAS-100 plus image analyzer (Fuji Film Co., Tokyo, Japan). RESULTS AND DISCUSSION Design of Fusion Proteins for Monitoring Protein-Protein Interactions. The strategy for the present method is based on 558 Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
our previous reports, the split GFPs system21,23 and the split firefly luciferases system,22 in which a protein-protein interaction triggers a protein splicing reaction, and results in reconstitution of split GFPs and split firefly luciferases, respectively. Here we used mLexA and VP16AD as the two functional proteins to perform the reporter gene assay based on protein splicing, which can evaluate juxtamembrane protein-protein interactions. LexA, an E. coli protein, has a DNA binding domain that recognizes the specific DNA sequence called as LexA operator (LexA op). The LexA protein is known to have an intrinsic NLS that leads the protein into the nucleus. To delete this NLS without the loss of the DNA binding ability, we modified the LexA protein as described in the previous report.32 mLexA thereby allows us to prevent a LexA-fused protein from localizing into the nucleus. VP16AD, a transcription-activating protein descended from a herpes simplex virus, activates transcription of a reporter gene when VP16AD and mLexA are spliced together. As the reporter gene, we designed a plasmid pX8luc, which was constructed by substitution of five GAL4 binding sites in pG5luc with LexA binding sites. pX8luc consists of a major late promoter of adenovirus, eight-repeated LexA ops, and the firefly luciferase gene downstream of the repeats (Figure 1b). In this study, we used DnaEs as the protein splicing element. We constructed a cDNA encoding a fusion protein consisting of mLexA, the N-terminus of DnaE (DnaEn), and a protein of interest (protein “X”) (Figure 1a). Its target protein (protein “Y”) and the C-terminus of DnaE (DnaEc) were fused to VP16AD. The flexible linkers (GGGGS)6 were inserted between the interacting proteins and the inteins in each DnaE to not disturb the interactions between the target proteins. To yield efficient splicing reaction, two particular amino acid sequences (KFAEY fused to DnaEn and CFNLSH fused to DnaEc) were inserted at the splicing junctions of each cDNA. These two cDNAs were separately inserted into multiple cloning sites of pcDNA3.1(+) expression vectors, and we named these plasmids as pmLDn (encoding a mLexA-DnaEn fusion protein) and pDcV (encoding a VP16AD-DnaEc fusion protein) (Figure 1b). The principle of the present method is shown in Figure 1a. When X interacts with Y, the DnaEs are brought in proximity and undergo correct folding, which induces protein splicing. Consequently, mLexA and VP16AD directly link to each other by a peptide bond. Until mLexA is ligated with VP16AD, the firefly
luciferase reporter gene is not transcribed into mRNA. Because the molecular weight of mLexA-VP16AD is ∼ 30 kDa, the ligated mLexA-VP16AD can be subjected to passive diffusion toward the nucleus. As a result, the extent of the protein-protein interaction is evaluated by measuring the magnitude of the observed luminescence intensity originating from firefly luciferase translated from the mRNA. Transcriptional Activity of Spliced mLexA-VP16AD. To confirm whether the reporter gene works only when mLexA and VP16AD are spliced, pX8luc-transfected CHO-EGFR cells were separately transfected with pmLDn or pDcV, which does not possess any cDNAs of interacting proteins. The result is shown in Table 1. These cells displayed low luminescence intensities. Meanwhile, cells transfected with pX8luc and a pcDNA3.1(+) vector carrying the cDNA of mLexA-VP16AD fusion protein (pmLV) showed significantly high luminescence intensities, indicating that the reporter gene was not transcribed until mLexA and VP16AD were spliced. Transcription Factors as a Probe for Protein-Protein Interaction. For the proof of principle, we examined three pairs of proteins: (i) Ras and Raf-1, (ii) RasV12 and Raf-1, and (iii) Ras and delRaf-1. Ras interacts with Raf-1 upon EGF stimulation, while RasV12, a constitutively active mutant of Ras, binds to Raf-1 without EGF stimulation. delRaf-1 lacks its Ras-interacting domain and is expected to display no interaction with Ras. CHO-EGFR cells were transfected with pmLDn and pDcV carrying (i) Ras and Raf-1, (ii) RasV12 and Raf-1, or (iii) Ras and delRaf-1. The cells were stimulated with 100 nM EGF for 24 h. We measured signals arising from these harvested cells. Although the difference in the amount of spliced mLexA-VP16AD between the cells treated with EGF and the nontreated cells was not detected on the western blotting pattern (data not shown), the significant difference in RLU was detected with the present method. The result concludes that the present method is superior to the western blot as to the sensitivity of the method. Regardless of whether the cells were stimulated with EGF, the cells harboring a pair of RasV12 and Raf-1 showed 10-15-fold higher RLU than the cells containing Ras and delRaf-1. After the cells containing the pair of Ras and Raf-1 were stimulated with EGF, these cells showed 5-fold higher RLU than the cells containing the same pair of proteins in the absence of EGF (Figure 2). From these results, we concluded that the maximum RLU due to protein-protein interactions in CHO-EGFR cells is ∼40, and the background RLU without a particular protein-protein interaction is less than 10. The cells carrying the noninteracting pair showed a subtle RLU. The signal may be due to that the endogenous weak interaction between DnaEn and DnaEc brought about autosplicing reaction,23 and therefore, mLexA-VP16AD was produced. In addition, basal kinase reactions may, regardless of EGF starvation, induce RasRaf-1 interactions, which trigger protein splicing. As a result, the RLU ratio of RasV12-Raf-1 to Ras-delRaf-1 was slightly higher than that of the EGF-treated cells to the nontreated cells. Firefly Luciferase Translated in an EGF ConcentrationDependent Manner. To demonstrate the quantitative detection of EGF-induced protein-protein interactions in CHO-EGFR cells, RLU was assessed as to EGF concentration dependence. After CHO-EGFR cells were transfected with pmLDn•Ras and pDcV•Raf-1, the cells were stimulated with differing concentra-
Figure 2. Firefly luciferase activities upon protein-protein interactions. CHO-EGFR cells were cultured in 12-well plates and transfected with 0.5 µg of pmLDn•Ras and pDcV•delRaf-1 (open bars), pmLDn•RasV12 and pDcV•Raf-1 (gray bars), or pmLDn•Ras and pDcV•Raf-1 (solid bars). All cells to be assayed were transfected with 0.5 µg of pX8luc. To normalize the observed luminescence from firefly luciferase to that from Renilla luciferase, CHO-EGFR cells in each well were cotransfected with 5 pg of the pRL-TK. The cells were subject to stimulation with 100 nM EGF for 24 h, and the luminescence was measured.
Figure 3. Concentration dependence of EGF on RLU. CHO-EGFR cells were cultured in 12-well plates and transfected with 0.5 µg of the pmLDn•Ras and pDcV•Raf-1 (solid squares). All cells to be assayed were transfected with 0.5 µg of pX8luc. To normalize the observed luminescence from firefly luciferase to that from Renilla luciferase, CHO-EGFR cells in each well were cotransfected with 5 pg of the pRL-TK. The cells were subject to stimulation with EGF for 24 h, and the luminescence was measured. The concentrations of EGF ranged from 1.0 × 10-13 to 2.0 × 10-7 M.
tions of EGF for 24 h and then the cells were lysated to measure luminescence intensities. Figure 3 shows that RLU increased with increasing EGF concentrations from 1.0 × 10-9 to 1.0 × 10-7 M. No change in the RLU was observed at EGF concentrations lower than 1.0 × 10-10 M. The result in Figure 3 indicates that this assay system can be used for the quantitative analysis of the extent of Ras-Raf-1 interactions in proximity to plasma membranes. Comparison of Intein-Mediated Reporter Gene Assay with Previous Methods in Detection of Ras-Raf-1 Interactions on Cell Membranes. To compare the sensitivity or the detection limit of the present method with that of the two-hybrid system in monitoring EGF-induced Ras-Raf-1 interactions, we constructed cDNAs encoding the mLexA•Ras fusion protein and the VP16AD•Raf-1 fusion protein; they do not have any inteins. The two cDNAs were separately inserted into pcDNA3.1(+), and the resultant two vectors were named pmL•Ras and pV•Raf-1 (the Analytical Chemistry, Vol. 78, No. 2, January 15, 2006
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Raf-1 (Figure 4). This result indicates that the present method enabled us to detect EGF-induced Ras-Raf-1 interactions, which were not detected by the firefly luciferase complementation method. This observed difference in the sensitivity between these two methods may be attributed to the fact that the present reporter gene assay has two amplification steps: (i) accumulation of mLexA-VP16AD fusion proteins by protein splicing and (ii) transcription of firefly luciferase by the mLexA-VP16AD fusion proteins.
Figure 4. Comparison of the present method with the firefly luciferase complementation and the two-hybrid methods as to their sensitivities. CHO-EGFR cells were cultured in 12-well plates and transfected with 0.5 µg of the pmLDn•Ras and pDcV•Raf-1 (solid squares), pNluc•Raf-1 and pCluc•Ras (solid triangles), or pmL•Ras and pV•Raf-1 (open circles). In the two-hybrid assay, all cells to be assayed were transfected with 0.5 µg of pX8luc. To normalize the observed luminescence from firefly luciferase to that from Renilla luciferase, CHO-EGFR cells in each well were cotransfected with 5 pg of the pRL-TK. In the two-hybrid assay or the firefly complementation assay, the cells were subject to stimulation with EGF for 24 h or 5 min, respectively. The concentrations of EGF ranged from 1.0 × 10-13 to 2.0 × 10-7 M.
vector constructs are shown in Figure 1b). After CHO-EGFR cells were transfected with pmL•Ras and pV•Raf-1, the cells were stimulated with various EGF concentrations for 24 h. Figure 4 and Table 1 indicate that the RLU from the cells was as low as that of CHO-EGFR cells transfected with pX8luc alone. In this case, although mLexA-Ras interacted with VP16AD-Raf-1 on cell membranes,34 increase in the RLU was not observed. The results in Figure 4 demonstrate that DnaEn and DnaEc are necessary for splicing mLexA and VP16AD; this splicing triggers the transcription of firefly luciferase gene. Ras-Raf-1 interaction induced in proximity to membranes was not detected by the conventional two-hybrid method, while the interaction was monitored by the present method. Next, to compare the sensitivity of the present method with that of the previous firefly luciferase complementation method,18 we constructed and inserted cDNAs encoding the Raf-1-Fluc(2-416) protein and the Fluc(398-550)-Ras protein into pcDNA3.1(+) vectors (named pNluc-Raf-1 and pCluc-Ras, respectively). Fluc(2-416) and Fluc(398-550) represent the N- and the C-half terminus of full-length firefly luciferase, respectively. After transient transfection with pNluc-Raf-1 and pCluc-Ras, the CHOEGFR cells were stimulated with differing concentrations of EGF for 5 min and harvested. The RLU from the cells was extremely lower than that of the cells carrying pmLDn-Ras and pDcV(34) Inouye, K.; Mizutani, S.; Koide, H.; Kaziro, Y. J. Biol. Chem. 2000, 275, 3737-3740.
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CONCLUSION A novel protein splicing-based reporter gene assay was developed as a method for detecting protein-protein interactions in mammalian cells. This method is based on the protein splicing of naturally split Ssp DnaE inteins, which produces a peptide bond linking between mLexA and VP16AD. The use of the inteins together with the transcription factors has overcome the limitation of the two-hybrid systems,1-4 which detect interactions only in proximity to the nucleus: we quantitatively monitored EGFinduced juxtamembrane Ras-Raf-1 interactions. The present method has two signal amplification steps, (i) accumulation of mLexA-VP16AD and (ii) transcription of the reporter gene. This dual-amplification allowed us to observe the Ras-Raf-1 interactions, which were not identified with the conventional firefly luciferase complementation method. Although this study aims at demonstrating the detection of the Ras-Raf-1 interaction in the EGF signaling pathway, other protein-protein interactions can be detected with the present method such as cytosolic protein-protein interactions. Because of the feasibility of the present method for detecting other protein-protein interactions, there are many possible applications of this method. If the firefly luciferase reporter gene is replaced with the cDNA of GFP, we can perform high-throughput screening for identifying interactions of membrane-proximal or cytosolic proteins with a fluorescence-activated cell sorter. Moreover, quantitative evaluation of interactions between cell membrane proteins, screening of agonist-like chemicals for the EGF signaling pathway, and identification of candidates interacting with membraneanchored proteins are some of potential applications. ACKNOWLEDGMENT This work was supported by grants from the Japan Science and Technology Agency (JST), and Japan Society for the Promotion of Science (JSPS). The Grant-in-aid for the 21st Century COE Program for Frontiers in Fundamental Chemistry from the Ministry of Education, Culture, Sports, Science and Technology is also acknowledged. Received for review August 11, 2005. Accepted November 7, 2005. AC051451A
