Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Monitoring regulated protein-protein interactions using split TEV

Nature Methods volume 3, pages 985–993 (2006)Cite this article

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Principle of the split-TEV system.
Figure 2: Principles of 'transcription-coupled' and 'proteolysis-only' split-TEV reporters.
Figure 3: Functionality of split-TEV reporters.
Figure 4: Monitoring constitutive and rapamycin-regulated interactions of soluble and membrane model and full-length proteins at the membrane.
Figure 5: Monitoring of constitutive and rapamycin-regulated PPIs in the cytosol.
Figure 6: Monitoring Nrg-1-ErbB2/4 signaling using split TEV.

Similar content being viewed by others

References

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

    Article  CAS  Google Scholar 

  2. Papin, J.A., Hunter, T., Palsson, B.O. & Subramaniam, S. Reconstruction of cellular signalling networks and analysis of their properties. Nat. Rev. Mol. Cell Biol. 6, 99–111 (2005).

    Article  CAS  Google Scholar 

  3. Dumas, J., Smith, R.A. & Lowinger, T.B. Recent developments in the discovery of protein kinase inhibitors from the urea class. Curr. Opin. Drug Discov. Dev. 7, 600–616 (2004).

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Fry, D.C. & Vassilev, L.T. Targeting protein-protein interactions for cancer therapy. J. Mol. Med. 83, 955–963 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  12. Ghosh, I., Hamilton, A.D. & Regan, L. Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein. J. Am. Chem. Soc. 122, 5658–5659 (2000).

    Article  CAS  Google Scholar 

  13. Hu, C.D., Chinenov, Y. & Kerppola, T.K. Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation. Mol. Cell 9, 789–798 (2002).

    Article  CAS  Google Scholar 

  14. Rossi, F., Charlton, C.A. & Blau, H.M. Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation. Proc. Natl. Acad. Sci. USA 94, 8405–8410 (1997).

    Article  CAS  Google Scholar 

  15. Pelletier, J.N., Campbell-Valois, F.X. & Michnick, S.W. Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments. Proc. Natl. Acad. Sci. USA 95, 12141–12146 (1998).

    Article  CAS  Google Scholar 

  16. Wehrman, T., Kleaveland, B., Her, J.H., Balint, R.F. & Blau, H.M. Protein-protein interactions monitored in mammalian cells via complementation of beta-lactamase enzyme fragments. Proc. Natl. Acad. Sci. USA 99, 3469–3474 (2002).

    Article  CAS  Google Scholar 

  17. Galarneau, A., Primeau, M., Trudeau, L.E. & Michnick, S.W. Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions. Nat. Biotechnol. 20, 619–622 (2002).

    Article  CAS  Google Scholar 

  18. Paulmurugan, R., Umezawa, Y. & Gambhir, S.S. 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).

    Article  CAS  Google Scholar 

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

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  21. Stagljar, I., Korostensky, C., Johnsson, N. & te Heesen, S. 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).

    Article  CAS  Google Scholar 

  22. Kapust, R.B. & Waugh, D.S. Controlled intracellular processing of fusion proteins by TEV protease. Protein Expr. Purif. 19, 312–318 (2000).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  24. Indra, A.K. 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).

    Article  CAS  Google Scholar 

  25. Hynes, N.E. & Lane, H.A. ERBB receptors and cancer: the complexity of targeted inhibitors. Nat. Rev. Cancer 5, 341–354 (2005).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  29. Kanno, A., Ozawa, T. & Umezawa, Y. Intein-mediated reporter gene assay for detecting protein-protein interactions in living mammalian cells. Anal. Chem. 78, 556–560 (2006).

    Article  CAS  Google Scholar 

  30. Rojo-Niersbach, E., Morley, D., Heck, S. & Lehming, N. A new method for the selection of protein interactions in mammalian cells. Biochem. J. 348, 585–590 (2000).

    Article  CAS  Google Scholar 

Download references

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

  1. Michael C Wehr and Rico Laage: These authors contributed equally to this work.

Authors and Affiliations

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

    Michael C Wehr, Tobias M Fischer, Klaus-Armin Nave & Moritz J Rossner

  2. Axaron Bioscience AG, INF515, Heidelberg, D-69120, Germany

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

Authors
  1. Michael C Wehr
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rico Laage
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ulrike Bolz
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Tobias M Fischer
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sylvia Grünewald
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Sigrid Scheek
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alfred Bach
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Klaus-Armin Nave
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Moritz J Rossner
    View author publications

    You can also search for this author in PubMed Google Scholar

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.

Corresponding authors

Correspondence to Rico Laage or Moritz J Rossner.

Ethics declarations

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.

Supplementary information

Supplementary Fig. 1

Summary of principles and properties of all membrane reporters. (PDF 368 kb)

Supplementary Fig. 2

Comparsion of GV-ER and GV-2ER activation by full-length TEV. (PDF 35 kb)

Supplementary Fig. 3

Western blotting of model membrane Split-TEV fusion constructs. (PDF 180 kb)

Supplementary Fig. 4

Analysis of TM-Luc reporter activation by transmembrane and cytosolic GCN4/GBR1a/GBR2 cc domain fragment pairs. (PDF 49 kb)

Supplementary Fig. 5

Time-dependent analysis of the rapamycin-induced FKBP-FRB interactions monitored with LucER. (PDF 25 kb)

Supplementary Fig. 6

Specificity of PPIs in the cytosol measured with LucER in CHO cells. (PDF 22 kb)

Supplementary Fig. 7

Comparsion of TM-GV with GV-2ER and GV-ER activated by transmembrane and cytosolic GCN4cc-TEV fragment pairs. (PDF 73 kb)

Supplementary Fig. 8

Western blot of Split-TEV interaction pair dependent cleavage of GV-2ER. (PDF 133 kb)

Supplementary Fig. 9

Comparing the kinetics of the rapamycin-induced FKBP-FRB interactions monitored with the GV-2ER and LucER. (PDF 28 kb)

Supplementary Note 1

TEV protease fragments suited for transcomplementation. (PDF 142 kb)

Supplementary Note 2

A recombinase reporter system for permanent reporter activation. (PDF 540 kb)

Supplementary Note 3

Fluorescent 'Proteolysis-only' TEV-Reporters. (PDF 1998 kb)

Supplementary Methods (PDF 28 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Wehr, M., Laage, R., Bolz, U. et al. Monitoring regulated protein-protein interactions using split TEV. Nat Methods 3, 985–993 (2006). https://doi.org/10.1038/nmeth967

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nmeth967

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing