Anal. Chem. 2001, 73, 2516-2521
Split Luciferase as an Optical Probe for Detecting Protein-Protein Interactions in Mammalian Cells Based on Protein Splicing Takeaki Ozawa,†,‡ Asami Kaihara,‡ Moritoshi Sato,†,‡ Kazunari Tachihara,‡ 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 (JST), Tokyo, Japan
We describe a new method for detecting protein-protein interactions in intact mammalian cells; the approach is based on protein splicing-induced complementation of rationally designed fragments of firefly luciferase. The protein splicing is a posttranslational protein modification through which inteins (internal proteins) are excised out from a precursor fusion protein, ligating the flanking exteins (external proteins) into a contiguous polypeptide. As the intein, naturally split DnaE from Synechocystis sp. PCC6803 was used: The N- and C-terminal DnaE, each fused respectively to N- and C-terminal fragments of split luciferase, were connected to proteins of interest. In this approach, protein-protein interactions trigger the folding of DnaE intein, wherein the protein splicing occurs and thereby the extein of ligated luciferase recovers its enzymatic activity. To test the applicability of this split luciferase complementation, we used insulin-induced interaction between known binding partners, phosphorylated insulin receptor substrate 1 (IRS-1) and its target N-terminal SH2 domain of PI 3-kinase. Enzymatic luciferase activity triggered by insulin served to monitor the interaction between IRS-1 and the SH2 domain in an insulin dose-dependent manner, of which amount was assessed by the luminescent intensity. This provides a convenient method to study phosphorylation of any protein or interactions of integral membrane proteins, a class of molecules that has been difficult to study by existing biochemical or genetic methods. High-throughput drug screening and quantitative analysis for a specific pathway in tyrosine phosphorylation of IRS-1 in insulin signaling are also made possible in this system. Specific interactions between proteins in mammalian cells play key roles in many essential biological processes. Typical examples are protein-protein interactions that are involved in the assembly of enzymes and other protein homodimers and heterodimers in the regulation of intracellular transport pathways, gene expression, receptor-ligand interactions, and the therapeutic or toxic effects of administered drugs. To increase our understanding of these * To whom correspondence should be addressed: (phone) +81-3-5841-4351; (fax) +81-3-5841-8349; (e-mail)
[email protected]. † Japan Science and Technology Corp. ‡ The University of Tokyo.
2516 Analytical Chemistry, Vol. 73, No. 11, June 1, 2001
biological processes, several techniques have been developed for examining the interactions between proteins, such as coimmunoprecipitation, and cross-linking proteins and thereafter cofractionation by chromatography. In contrast to such biochemical techniques, the yeast twohybrid system has been extremely useful for detecting and identifying protein-protein interactions in vivo,1,2 where a library of proteins is screened for interaction with a “bait” protein. The two-hybrid method, a standard functional assay, facilitates the identification of potential protein-protein interactions and has been proposed as a method for the generation of protein interaction maps.3-5 This approach allows the rapid detection of protein binding partners, including the relevant interacting domains, and immediately provides the gene that encodes the identified interacting proteins; there are, however, cases where this is not applicable. For example, the set of detectable protein interactions is those occurring in the nucleus, in proximity to the reporter gene, so the method is not useful for the study of membrane proteins. Moreover, if one of the proteins is a transcriptional activator, it may itself induce transcription of the reporter gene. Conceptually, the biological information generated by two-hybrid analysis is often questioned because of the inherent artificial nature of the assay. Nonetheless, numerous previously unknown protein interactions have been identified using this yeast two-hybrid system. To overcome the limitation in the yeast two-hybrid assay, various permutations of the two-hybrid method have been described, including the split-ubiquitin system (USPS),6-8 SOSrecruitment system,9-11 dihydrofolate reductase complementa(1) Chien, C. T.; Bartel, P. L.; Sternglanz, R.; Fields, S. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 9578-9582. (2) Fields, S.; Song, O. Nature 1989, 340, 245-246. (3) Flores, A.; Briand, J. F.; Gadal, O.; Andrau, J. C.; Rubbi, L.; 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. (4) 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. 1999, 97, 11431147. (5) Walhout, A. J. M.; Sordella, R.; Lu, X.; Hartley, J. L.; Temple, G. F.; Brasch, M. A.; Thierry-Mieg, N.; Vidal, M. Science 2000, 287, 116-122. (6) Dunnwald, M.; Varshavsky, A.; Johnsson, N. Mol. Biol. Cell 1999, 10, 329344. (7) Johnsson, N.; Varshavsky, A. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 1034010344. (8) Stagljar, I.; Korostensky, C.; Johnsson, N.; Heesen, S. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 5187-5192. 10.1021/ac0013296 CCC: $20.00
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tion,12,13 β-galactosidase complementation,14 and G-protein fusion system.15 These systems are well suited for assaying interactions between cytoplasm and membrane-proximal proteins, but they can be utilized only in appropriately engineered cells and/or are prone to false positive signals. Fluorescent ratio imaging has also been used to study protein interactions in living cells.16 The detection mechanisms are based on the fluorescence resonance energy transfer (FRET) that occurs when donor and acceptor fluorophores are in sufficient proximity (
