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Monitoring regulated protein-protein interactions using split TEV

Nature Methods volume 3, pages 985993 (2006) | Download Citation

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Abstract

Signaling cascades integrate extracellular stimuli primarily through regulated protein-protein interactions (PPIs). Intracellular signal transduction strictly depends on PPIs occurring at the membrane and in the cytosol. To monitor constitutive and regulated protein interactions within living mammalian cells, we have developed a biological assay termed split TEV. We engineered inactive fragments of the NIa protease from the tobacco etch virus (TEV protease) that regain activity only when coexpressed as fusion constructs with interacting proteins. Functional reconstitution of TEV protease fragments can be monitored with 'proteolysis-only' reporters, which can be previously silent fluorescent and luminescent reporter proteins. Additionally, proteolytically cleavable inactive transcription factors can be combined with any downstream reporter gene of choice to yield 'transcription-coupled' reporter systems. Thus, split TEV combines the advantages of split enzyme– and reporter gene–mediated assays, and provides full flexibility with regard to the final readout. In a first biological application, we monitored neuregulin-induced ErbB2/ErbB4 receptor tyrosine kinase heterodimerization.

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References

  1. 1.

    Specificity in signal transduction: from phosphotyrosine-SH2 domain interactions to complex cellular systems. Cell 116, 191–203 (2004).

  2. 2.

    , , & Reconstruction of cellular signalling networks and analysis of their properties. Nat. Rev. Mol. Cell Biol. 6, 99–111 (2005).

  3. 3.

    , & Recent developments in the discovery of protein kinase inhibitors from the urea class. Curr. Opin. Drug Discov. Dev. 7, 600–616 (2004).

  4. 4.

    Making protein interactions druggable: targeting PDZ domains. Nat. Rev. Drug Discov. 3, 1047–1056 (2004).

  5. 5.

    & Targeting protein-protein interactions for cancer therapy. J. Mol. Med. 83, 955–963 (2005).

  6. 6.

    et al. Proteome survey reveals modularity of the yeast cell machinery. Nature 440, 631–636 (2006).

  7. 7.

    & A novel genetic system to detect protein-protein interactions. Nature 340, 245–246 (1989).

  8. 8.

    et al. A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403, 623–627 (2000).

  9. 9.

    Protein fragment complementation strategies for biochemical network mapping. Curr. Opin. Biotechnol. 14, 610–617 (2003).

  10. 10.

    Visualization of molecular interactions by fluorescence complementation. Nat. Rev. Mol. Cell. Biol. 7, 449–456 (2006).

  11. 11.

    , & The use of resonance energy transfer in high-throughput screening: BRET versus FRET. Trends Pharmacol. Sci. 23, 351–354 (2002).

  12. 12.

    , & Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658–5659 (2000).

  13. 13.

    , & Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9, 789–798 (2002).

  14. 14.

    , & Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc. Natl. Acad. Sci. USA 94, 8405–8410 (1997).

  15. 15.

    , & Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. USA 95, 12141–12146 (1998).

  16. 16.

    , , , & Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments. Proc. Natl. Acad. Sci. USA 99, 3469–3474 (2002).

  17. 17.

    , , & Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat. Biotechnol. 20, 619–622 (2002).

  18. 18.

    , & Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies. Proc. Natl. Acad. Sci. USA 99, 15608–15613 (2002).

  19. 19.

    , & Transforming a (beta/alpha)8–barrel enzyme into a split-protein sensor through directed evolution. Chem. Biol. 11, 681–689 (2004).

  20. 20.

    & Split ubiquitin as a sensor of protein interactions in vivo. Proc. Natl. Acad. Sci. USA 91, 10340–10344 (1994).

  21. 21.

    , , & A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Proc. Natl. Acad. Sci. USA 95, 5187–5192 (1998).

  22. 22.

    & Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr. Purif. 19, 312–318 (2000).

  23. 23.

    et al. Importance of the γ-aminobutyric acid(B) receptor C termini for G-protein coupling. Mol. Pharmacol. 61, 1070–1080 (2002).

  24. 24.

    et al. Temporally-controlled site-specific mutagenesis in the basal layer of the epidermis: comparison of the recombinase activity of the tamoxifen-inducible Cre-ER(T) and Cre-ER(T2) recombinases. Nucleic Acids Res. 27, 4324–4327 (1999).

  25. 25.

    & ERBB receptors and cancer: the complexity of targeted inhibitors. Nat. Rev. Cancer 5, 341–354 (2005).

  26. 26.

    et al. Heregulin induces tyrosine phosphorylation of HER4/p180erbB4. Nature 366, 473–475 (1993).

  27. 27.

    Neuregulins: functions, forms, and signaling strategies. Exp. Cell Res. 284, 14–30 (2003).

  28. 28.

    et al. Design and application of a cytokine-receptor-based interaction trap. Nat. Cell Biol. 3, 1114–1119 (2001).

  29. 29.

    , & Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells. Anal. Chem. 78, 556–560 (2006).

  30. 30.

    , , & A new method for the selection of protein interactions in mammalian cells. Biochem. J. 348, 585–590 (2000).

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Acknowledgements

We acknowledge the excellent technical assistance from F. Herzog with cell cultures, and H. Böhli and J. Ohsam for cloning expression constructs. The GST-Nrg-1β fusion constructs were a kind gift of C. Lai (The Scripps Institute). Her2 and mouse ErbB4 templates were kindly provided by A. Ullrich (Max Planck Institute for Biochemistry) and C. Lai.

Author information

Author notes

    • Michael C Wehr
    •  & Rico Laage

    These authors contributed equally to this work.

Affiliations

  1. Max Planck Institute of Experimental Medicine, Hermann Rein Str. 3, D-37075 Göttingen, Germany.

    • Michael C Wehr
    • , Tobias M Fischer
    • , Klaus-Armin Nave
    •  & Moritz J Rossner
  2. Axaron Bioscience AG, INF515, D-69120 Heidelberg, Germany.

    • Rico Laage
    • , Ulrike Bolz
    • , Sylvia Grünewald
    • , Sigrid Scheek
    • , Alfred Bach
    •  & Moritz J Rossner

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Contributions

M.C.W. cloned and western blot–verified TEV fragment constructs and reporters and conducted the final experiments except the TEV fragment screen; R.L. cloned TEV fragment constructs and the TM-GV reporter, and perfomed the TEV fragment screen and proof-of-principle experiments monitoring interactions at the membrane including rapamycin regulation; U.B. was involved in cloning of reporters and interaction constructs; T.M.F. cloned the Nrg1 constructs and performed the Nrg1 western blot; S.G. and S.S. contributed to initial data monitoring GPCR interactions; A.B. and K.-A.N. supported the project and contributed conceptually; M.J.R. developed the concept, cloned initial reporter constructs and supervised the project. M.C.W. and M.J.R. wrote the manuscript.

Competing interests

R.L., U.B. and A.B. are employees of Axaron Bioscience AG, Germany, which holds the patent for the described technology. S.G., S.S. and M.J.R. were temporarily employees of Axaron Bioscience AG during the initial phase of the project.

Corresponding authors

Correspondence to Rico Laage or Moritz J Rossner.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Summary of principles and properties of all membrane reporters.

  2. 2.

    Supplementary Fig. 2

    Comparsion of GV-ER and GV-2ER activation by full-length TEV.

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    Supplementary Fig. 3

    Western blotting of model membrane Split-TEV fusion constructs.

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    Supplementary Fig. 4

    Analysis of TM-Luc reporter activation by transmembrane and cytosolic GCN4/GBR1a/GBR2 cc domain fragment pairs.

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    Supplementary Fig. 5

    Time-dependent analysis of the rapamycin-induced FKBP-FRB interactions monitored with LucER.

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    Supplementary Fig. 6

    Specificity of PPIs in the cytosol measured with LucER in CHO cells.

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    Supplementary Fig. 7

    Comparsion of TM-GV with GV-2ER and GV-ER activated by transmembrane and cytosolic GCN4cc-TEV fragment pairs.

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    Supplementary Fig. 8

    Western blot of Split-TEV interaction pair dependent cleavage of GV-2ER.

  9. 9.

    Supplementary Fig. 9

    Comparing the kinetics of the rapamycin-induced FKBP-FRB interactions monitored with the GV-2ER and LucER.

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    Supplementary Note 1

    TEV protease fragments suited for transcomplementation.

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    Supplementary Note 2

    A recombinase reporter system for permanent reporter activation.

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    Supplementary Note 3

    Fluorescent 'Proteolysis-only' TEV-Reporters.

  13. 13.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth967

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