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Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells


Interactions between proteins are at the heart of the cellular machinery. It is therefore not surprising that altered interaction profiles caused by aberrant protein expression patterns or by the presence of mutations can trigger cellular dysfunction, eventually leading to disease. Moreover, many viral and bacterial pathogens rely on protein-protein interactions to exert their damaging effects. Interfering with such interactions is an obvious pharmaceutical goal, but detailed insights into the protein binding properties as well as efficient screening platforms are needed. In this report, we describe a cytokine receptor–based assay with a positive readout to screen for disrupters of designated protein-protein interactions in intact mammalian cells and evaluate this concept using polypeptides as well as small organic molecules. These reverse mammalian protein-protein interaction trap (MAPPIT) screens were developed to monitor interactions between the erythropoietin receptor (EpoR) and suppressors of cytokine signaling (SOCS) proteins, between FKBP12 and ALK4, and between MDM2 and p53.

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Figure 1: Schematic view of the forward MAPPIT and reverse MAPPIT principles.
Figure 2: Inhibition of signaling via a chimeric SOCS3-CIS construct is lost upon coexpression of a competitor SOCS protein.
Figure 3: Analysis of the interaction between FKBP12 and ALK4.
Figure 4: Structure-function analysis using ReverseMAPPIT.
Figure 5: Dose-dependent disruption of the p53-MDM2 interaction by Nutlin-3 as evaluated by reverse MAPPIT.


  1. 1

    Zhang, Z.Y., Poorman, R.A., Maggiora, L.L., Heinrikson, R.L. & Kezdy, F.J. Dissociative inhibition of dimeric enzymes. Kinetic characterization of the inhibition of HIV-1 protease by its COOH-terminal tetrapeptide. J. Biol. Chem. 266, 15591–15594 (1991).

    CAS  PubMed  Google Scholar 

  2. 2

    Moss, N. et al. Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase with improved in vivo antiviral activity. J. Med. Chem. 39, 4173–4180 (1996).

    CAS  Article  Google Scholar 

  3. 3

    Adachi, T., Stafford, S., Sur, S. & Alam, R. A novel Lyn-binding peptide inhibitor blocks eosinophil differentiation, survival, and airway eosinophilic inflammation. J. Immunol. 163, 939–946 (1999).

    CAS  PubMed  Google Scholar 

  4. 4

    Degterev, A. et al. Identification of small-molecule inhibitors of interaction between the BH3 domain and Bcl-xL . Nat. Cell Biol. 3, 173–182 (2001).

    CAS  Article  Google Scholar 

  5. 5

    Hayashi, M. et al. Suppression of bone resorption by madindoline A, a novel nonpeptide antagonist to gp130. Proc. Natl. Acad. Sci. USA 99, 14728–14733 (2002).

    CAS  Article  Google Scholar 

  6. 6

    Hayashi, M. et al. Biological activity of a novel nonpeptide antagonist to the interleukin-6 receptor 20S,21-epoxy-resibufogenin-3-formate. J. Pharmacol. Exp. Ther. 303, 104–109 (2002).

    CAS  Article  Google Scholar 

  7. 7

    Weitz-Schmidt, G. et al. Statins selectively inhibit leukocyte function antigen-1 by binding to a novel regulatory integrin site. Nat. Med. 7, 687–692 (2001).

    CAS  Article  Google Scholar 

  8. 8

    Vassilev, L.T. et al. In vivo activation of the p53 pathway by small-molecule antagonists of MDM2. Science 303, 844–848 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Lepourcelet, M. et al. Small-molecule antagonists of the oncogenic Tcf/β-catenin protein complex. Cancer Cell 5, 91–102 (2004).

    CAS  Article  Google Scholar 

  10. 10

    Gadek, T.R. & Nicholas, J.B. Small molecule antagonists of proteins. Biochem. Pharmacol. 65, 1–8 (2003).

    CAS  Article  Google Scholar 

  11. 11

    Bergendahl, V., Heyduk, T. & Burgess, R.R. Luminescence resonance energy transfer-based high-throughput screening assay for inhibitors of essential protein-protein interactions in bacterial RNA polymerase. Appl. Environ. Microbiol. 69, 1492–1498 (2003).

    CAS  Article  Google Scholar 

  12. 12

    Zhao, H.F. et al. A mammalian genetic system to screen for small molecules capable of disrupting protein-protein interactions. Anal. Chem. 76, 2922–2927 (2004).

    CAS  Article  Google Scholar 

  13. 13

    Kato-Stankiewicz, J. et al. Inhibitors of Ras/Raf-1 interaction identified by two-hybrid screening revert Ras-dependent transformation phenotypes in human cancer cells. Proc. Natl. Acad. Sci. USA 99, 14398–14403 (2002).

    CAS  Article  Google Scholar 

  14. 14

    Huang, J. & Schreiber, S.L. A yeast genetic system for selecting small molecule inhibitors of protein-protein interactions in nanodroplets. Proc. Natl. Acad. Sci. USA 94, 13396–13401 (1997).

    CAS  Article  Google Scholar 

  15. 15

    Vidal, M., Braun, P., Chen, E., Boeke, J.D. & Harlow, E. Genetic characterization of a mammalian protein-protein interaction domain by using a yeast reverse two-hybrid system. Proc. Natl. Acad. Sci. USA 93, 10321–10326 (1996).

    CAS  Article  Google Scholar 

  16. 16

    Park, S.H. & Raines, R.T. Genetic selection for dissociative inhibitors of designated protein-protein interactions. Nat. Biotechnol. 18, 847–851 (2000).

    CAS  Article  Google Scholar 

  17. 17

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

    CAS  Article  Google Scholar 

  18. 18

    Krebs, D.L. & Hilton, D.J. SOCS proteins: negative regulators of cytokine signaling. Stem Cells 19, 378–387 (2001).

    CAS  Article  Google Scholar 

  19. 19

    Yoshimura, A. et al. A novel cytokine-inducible gene CIS encodes an SH2-containing protein that binds to tyrosine-phosphorylated interleukin 3 and erythropoietin receptors. EMBO J. 14, 2816–2826 (1995).

    CAS  Article  Google Scholar 

  20. 20

    Baumann, H. et al. The full-length leptin receptor has signaling capabilities of interleukin 6-type cytokine receptors. Proc. Natl. Acad. Sci. USA 93, 8374–8378 (1996).

    CAS  Article  Google Scholar 

  21. 21

    Eyckerman, S., Broekaert, D., Verhee, A., Vandekerckhove, J. & Tavernier, J. Identification of the Y985 and Y1077 motifs as SOCS3 recruitment sites in the murine leptin receptor. FEBS Lett. 486, 33–37 (2000).

    CAS  Article  Google Scholar 

  22. 22

    Zhang, J.G. et al. The conserved SOCS box motif in suppressors of cytokine signaling binds to elongins B and C and may couple bound proteins to proteasomal degradation. Proc. Natl. Acad. Sci. USA 96, 2071–2076 (1999).

    CAS  Article  Google Scholar 

  23. 23

    Bjorbaek, C., Elmquist, J.K., Frantz, J.D., Shoelson, S.E. & Flier, J.S. Identification of SOCS-3 as a potential mediator of central leptin resistance. Mol. Cell 1, 619–625 (1998).

    CAS  Article  Google Scholar 

  24. 24

    Huse, M., Chen, Y.G., Massague, J. & Kuriyan, J. Crystal structure of the cytoplasmic domain of the type I TGF β receptor in complex with FKBP12. Cell 96, 425–436 (1999).

    CAS  Article  Google Scholar 

  25. 25

    Wang, T., Donahoe, P.K. & Zervos, A.S. Specific interaction of type I receptors of the TGF-beta family with the immunophilin FKBP-12. Science 265, 674–676 (1994).

    CAS  Article  Google Scholar 

  26. 26

    Cook, W.S. & Unger, R.H. Protein tyrosine phosphatase 1B: a potential leptin resistance factor of obesity. Dev. Cell 2, 385–387 (2002).

    CAS  Article  Google Scholar 

  27. 27

    ten Hoeve, J. et al. Identification of a nuclear Stat1 protein tyrosine phosphatase. Mol. Cell. Biol. 22, 5662–5668 (2002).

    CAS  Article  Google Scholar 

  28. 28

    Chung, C.D. et al. Specific inhibition of Stat3 signal transduction by PIAS3. Science 278, 1803–1805 (1997).

    CAS  Article  Google Scholar 

  29. 29

    Chen, Y.G., Liu, F. & Massague, J. Mechanism of TGFbeta receptor inhibition by FKBP12. EMBO J. 16, 3866–3876 (1997).

    CAS  Article  Google Scholar 

  30. 30

    Charng, M.J., Kinnunen, P., Hawker, J., Brand, T. & Schneider, M.D. FKBP-12 recognition is dispensable for signal generation by type I transforming growth factor-βreceptors. J. Biol. Chem. 271, 22941–22944 (1996).

    CAS  Article  Google Scholar 

  31. 31

    Bogan, A.A. & Thorn, K.S. Anatomy of hot spots in protein interfaces. J. Mol. Biol. 280, 1–9 (1998).

    CAS  Article  Google Scholar 

  32. 32

    Archakov, A.I. et al. Protein-protein interactions as a target for drugs in proteomics. Proteomics 3, 380–391 (2003).

    CAS  Article  Google Scholar 

  33. 33

    Arkin, M.R. et al. Binding of small molecules to an adaptive protein-protein interface. Proc. Natl. Acad. Sci. USA 100, 1603–1608 (2003).

    CAS  Article  Google Scholar 

  34. 34

    Vidal, M. & Endoh, H. Prospects for drug screening using the reverse two-hybrid system. Trends Biotechnol. 17, 374–381 (1999).

    CAS  Article  Google Scholar 

  35. 35

    Gaber, R.F., Copple, D.M., Kennedy, B.K., Vidal, M. & Bard, M. The yeast gene ERG6 is required for normal membrane function but is not essential for biosynthesis of the cell-cycle-sparking sterol. Mol. Cell. Biol. 9, 3447–3456 (1989).

    CAS  Article  Google Scholar 

  36. 36

    Eyckerman, S. et al. Analysis of Tyr to Phe and fa/fa leptin receptor mutations in the PC12 cell line. Eur. Cytokine Netw. 10, 549–556 (1999).

    CAS  PubMed  Google Scholar 

  37. 37

    Lemmens, I. et al. Heteromeric MAPPIT: a novel strategy to study modification-dependent protein-protein interactions in mammalian cells. Nucleic Acids Res. 31, e75 (2003).

    Article  Google Scholar 

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We wish to acknowledge R. Devos for the pGEX-PTP-1B vector; K. Kas for critical reading of the manuscript, M. Goethals for peptide synthesis and D. Defeau for technical assistance. This project was supported by grants from the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT-Vlaanderen; GBOU 010090), Ghent University (UGent; GOA 12051401) and the valorisation fund of the Flanders Interuniversity Institute for Biotechnology (VIB). S.E. is a Postdoctoral Fellow of the Fund for Scientific Research-Flanders (FWO-V).

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Correspondence to Jan Tavernier.

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Supplementary information

Supplementary Fig. 1

Dose-dependent disruption of the p53/MDM2 interaction: comparison of different configurations. (PDF 975 kb)

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Eyckerman, S., Lemmens, I., Catteeuw, D. et al. Reverse MAPPIT: screening for protein-protein interaction modifiers in mammalian cells. Nat Methods 2, 427–433 (2005).

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