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.

  • Research Article
  • Published:

Mapping signal transduction pathways by phage display

Nature Biotechnology volume 17, pages 1193–1198 (1999)Cite this article

Abstract

Rapid identification of proteins that interact with a novel gene product is an important element of functional genomics. Here we describe a phage display–based technique for interaction screening of complex cDNA libraries using proteins or synthetic peptides as baits. Starting with the epidermal growth factor receptor (EGFR) cytoplasmic tail, we identified known protein interactions that link EGFR to the Ras/MAP kinase signal transduction cascade and several novel interactions. This approach can be used as a rapid and efficient tool for elucidating protein networks and mapping intracellular signal transduction pathways.

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
Figure 2: (A) Binding of Shc phage to immobilized tyrosine phosphorylated (pY) and nonphosphorylated (Y) TrkA-Y490 and Shc-Y317 peptides.
Figure 3
Figure 4: The protein sequences encoded by some phage display clones binding to GST-Grb2 protein bait.
Figure 5: Association of endogenous Grb2 with GST fusions encoding fragments of Gab1, Gab2, AMSH, and Fyn proteins selected in the phage display screens.

Similar content being viewed by others

References

  1. Smith, G.P. Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315 –1317 (1985).

    Article  CAS  Google Scholar 

  2. Kay, B.K., Winter, J. & McCaferty, J. Phage display of peptides and proteins. A laboratory manual (Academic Press, San Diego, CA; 1996).

    Google Scholar 

  3. Griffiths, A.D. & Duncan, A.R. Strategies for selection of antibodies by phage display. Curr. Opin. Biotechnol. 9, 102–108 ( 1998).

    Article  CAS  Google Scholar 

  4. Maruyama, I.N., Maruyama, H.I. & Brenner, S. λ foo: A λ phage vector for the expression of foreign proteins. Proc. Natl. Acad. Sci. USA 91, 8273–8277 (1994).

    Article  CAS  Google Scholar 

  5. Sternberg, N. & Hoess, R.H. Display of peptides and proteins on the surface on the surface of bacteriophage λ. Proc. Natl. Acad. Sci. USA 92, 1609–1613 (1995).

    Article  CAS  Google Scholar 

  6. Dunn, I.S. Assembly of functional bacteriophage lambda virions incorporating C-terminal peptide or protein fusions with the major tail protein. J. Mol. Biol. 248, 497–506 ( 1995).

    Article  CAS  Google Scholar 

  7. Santini, C. et al. Efficient display of an HCV cDNA expression library as C-terminal fusion to the capsid protein D bacteriophage lambda. J. Mol. Biol. 282, 125–135 (1998).

    Article  CAS  Google Scholar 

  8. Efimov, V.P., Nepluev, I.V. & Mesyanzhinov, V.V. Bacteriophage T4 as a surface display vector. Virus Genes 10, 172–177 (1995).

    Article  Google Scholar 

  9. Ren, Z.J. et al. Phage display of intact domains at high copy number: a system based on SOC, the smaller outer capsid protein of bacteriophage T4. Protein Sci. 5, 1833–1843 ( 1996).

    Article  CAS  Google Scholar 

  10. Rosenberg, A. et al. T7Select phage display system: a powerful new protein display system based on bacteriophage T7. inNovations 6, 1–6 (1996).

    Google Scholar 

  11. Crameri, R. & Suter, M. Display of biologically active proteins on the surface of filamentous phages: a cDNA cloning system for selection of functional gene products linked to the genetic information responsible for their production. Gene 137, 69– 75 (1993).

    Article  CAS  Google Scholar 

  12. Crameri, R., Jaussi, R., Menz, G. & Blaser, K. Display of expression products of cDNA libraries on phage surfaces. A versatile screening system for selective isolation of genes by specific gene-product/ligand interaction. Eur. J. Biochem, 226, 53– 58 (1994).

    Article  CAS  Google Scholar 

  13. Kuriyan, J. & Cowburn, D. Modular peptide recognition domains in eukaryotic signaling. Annu. Rev. Biophys. Biomol. Struct. 26, 259–288 (1997).

    Article  CAS  Google Scholar 

  14. Cohen, G.B., Ren, R. & Baltimore, D. Modular binding domains in signal transduction proteins. Cell 80, 237–248 (1995).

    Article  CAS  Google Scholar 

  15. Pawson, T. Protein modules and signaling networks. Nature 373, 573–580 (1995).

    Article  CAS  Google Scholar 

  16. Mandiyan, V. et al. Thermodynamic studies of Shc phosphotyrosine interaction domain recognition of the NPXpY motif. J. Biol. Chem. 271, 4770–4775 (1996).

    Article  CAS  Google Scholar 

  17. Batzer, A.G., Rotin, D., Urena, J.M., Skolnik, E.Y. & Schlessinger, J. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol. Cell. Biol. 14, 5192–5201 (1994).

    Article  CAS  Google Scholar 

  18. Sakaguchi, K. et al. Shc phosphotyrosine-binding domain dominantly interacts with epidermal growth factor receptors and mediates Ras activation in intact cells. Mol. Endocrinol. 12, 536–543 (1998).

    Article  CAS  Google Scholar 

  19. Rozakis-Adcock, M. et al. Association of the Shc and Grb2/Sem5 SH2-containing proteins is implicated in activation of the Ras pathway by tyrosine kinases. Science 360, 689–692 ( 1992).

    CAS  Google Scholar 

  20. Van der Geer, P., Wiley, S., Gish, G.D. & Pawson, T. The Shc adapter protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein-protein interactions. Curr. Biol. 6, 1435–1444 (1996).

    Article  CAS  Google Scholar 

  21. Gotoh, N., Toyoda, M. & Shibuya, M. Tyrosine phosphorylation sites at amino acids 239 and 240 of Shc are involved in epidermal growth factor-induced mitogenic signaling that is distinct from Ras/mitogen-activated protein kinase activation. Mol. Cell. Biol. 17, 1824–1831 (1997).

    Article  CAS  Google Scholar 

  22. Li, N. et al. Guanine-nucleotide-releasing factor hSos1 binds to Grb2 and links receptor tyrosine kinases to Ras signalling. Nature 363, 85–88 (1993).

    Article  CAS  Google Scholar 

  23. >Rozakis-Adcock M., Fernley R., Wade J., Pawson T. & Bowtell D. The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1. Nature 363 , 83–85 (1993).

    Article  Google Scholar 

  24. Chardin, P. et al. Human Sos1: a guanine nucleotide exchange factor for Ras that binds to GRB2. Science 260, 1338– 1343 (1993).

    Article  CAS  Google Scholar 

  25. Dikic, I. et al. Shc binding to nerve growth factor receptor is mediated by the phosphotyrosine interaction domain. J. Biol. Chem. 270, 15125–15129 (1995).

    Article  CAS  Google Scholar 

  26. Bernard, D.J. et al. Effect of epidermal growth factor in HLA class I and class II transcription and protein expression in human breast adenocarcinoma cell lines. Br. J. Cancer 66, 88–92 (1992).

    Article  CAS  Google Scholar 

  27. Nutt, J.E. & Lunec, J. Induction of metalloproteinase (MMP1) expression by epidermal growth factor (EGF) receptor stimulation and serum deprivation in human breast tumour cells. Eur. J. Cancer 32A, 2127–2135 (1996).

    Article  CAS  Google Scholar 

  28. Soler, C., Felipe, A. & Carpenter, G. Epidermal growth factor increases protein and messenger RNA expression levels of Ras GTPase activating protein. Cell Growth & Differ. 5, 519–526 (1994).

    CAS  Google Scholar 

  29. Pronk, G.J. et al. Involvement of Shc in insulin- and epidermal growth factor-induced activation of p21ras. Mol. Cell. Biol. 14, 1575–1581 (1994).

    Article  CAS  Google Scholar 

  30. Tanaka, S. et al. A novel variant of human Grb7 associated with invasive esophageal carcinoma. J. Clin. Invest. 102, 821– 827 (1998).

    Article  CAS  Google Scholar 

  31. Kishi, T. et al. Molecular cloning of human GRB-7 co-amplified with CAB1 and c-ERBB-2 in primary gastric cancer. Biochem. Biophys. Res. Commun. 232, 5–9 (1997).

    Article  CAS  Google Scholar 

  32. Downward, J. The GRB2/Sem-5 adapter protein. FEBS Lett. 338, 113–117 (1994).

    Article  CAS  Google Scholar 

  33. Tanaka, N. et al. Possible involvement of a novel STAM-associated molecule "AMSH" in intracellular signal transduction mediated by cytokines. J. Biol. Chem. 274, 19129–19135 (1999).

    Article  CAS  Google Scholar 

  34. Takeshita, T. et al. Cloning of a novel signal-transducing adapter molecule containing an SH3 domain and ITAM. Biochem. Biophys. Res. Commun. 225, 1035–1039 (1996).

    Article  CAS  Google Scholar 

  35. Takeshita, T. et al. STAM, signal transducing adapter molecule, is associated with Janus kinases and involved in signaling for cell growth and c-myc induction. Immunity 6, 449–457 ( 1997).

    Article  CAS  Google Scholar 

  36. Toda M. et al. Molecular cloning of cDNA encoding human drebrin E and chromosomal mapping of its gene. Biochem. Biophys. Res. Commun. 196, 468–472 (1993).

    Article  CAS  Google Scholar 

  37. Holgado-Madruga, M., Emlet, D.R., Moscatello, D.K., Godwin, A.K. & Wong, A.J. A Grb2-associated docking protein in EGF- and insulin-receptor signaling. Nature 379, 560–564 (1996).

    Article  CAS  Google Scholar 

  38. Rocchi, S. et al. Determination of Gab1 (Grb2-associated binder-1) interaction with insulin receptor-signaling molecules. Mol. Endocrinol. 12, 914–923 (1998).

    Article  CAS  Google Scholar 

  39. Takahashi-Tezuka, M. et al. Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol. Cell Biol. 18, 4109–4117 ( 1998).

    Article  CAS  Google Scholar 

  40. Bardelli, A., Longati, P., Gramaglia, D., Stella, M.C. & Comoglio, P.M. Gab1 coupling to the HGF/Met receptor multifunctional docking site requires binding of Grb2 and correlates with the transforming potential. Oncogene 15, 3103–3111 (1997).

    Article  CAS  Google Scholar 

  41. Weidner, K.M. et al. Interaction between Gab1 and the c-Met receptor tyrosine kinase is responsible for epithelial morphogenesis. Nature 384 , 173–176 (1996).

    Article  CAS  Google Scholar 

  42. Gu, H., Pratt, J.G., Burakoff, S.J. & Neel, B.G. Cloning of p97/Gab2, the major SHP2-binding protein in hematopoietic cells, reveals a novel pathway for cytokine-induced gene activation. Mol. Cell 2, 729–740 ( 1998).

    Article  CAS  Google Scholar 

  43. Nagase, T. et al. Prediction of the coding sequences of unidentified human genes. IX. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro. DNA Res. 5, 31– 39 (1998).

    Article  CAS  Google Scholar 

  44. Zhao C., Yu, D.H., Shen, R. & Feng, G.S. Gab2, a new pleckstrin homology domain-containing adapter protein, acts to uncouple signaling from ERK kinase to Elk-1. J. Biol. Chem. 274, 19649–19654 (1999).

    Article  CAS  Google Scholar 

  45. Lin, J.W. et al. Yotiao, a novel protein of neuromuscular junction and brain that interacts with specific splice variants of NMDA receptor subunit NR1. J. Neurosci. 18, 2017–2027 (1998).

    Article  CAS  Google Scholar 

  46. Satoh, T., Kato, J., Nishida, K. & Kaziro, Y. Tyrosine phosphorylation of ACK in response to temperature shift-down, hyperosmotic shock, and epidermal growth factor stimulation. FEBS Lett. 386, 230–234 (1996).

    Article  CAS  Google Scholar 

  47. Lowenstein, E.J. et al. The SH2 and SH3 domain-containing protein GRB2 links receptor tyrosine kinases to ras signaling. Cell 70, 431–442 (1992).

    Article  CAS  Google Scholar 

  48. Soule, H.D., Vazguez, J., Long, A., Albert, S. & Brennan, M. A human cell line from a pleural effusion derived from a breast carcinoma. J. Natl. Cancer Inst. 51, 1409–1416 (1973).

    Article  CAS  Google Scholar 

  49. Jainchill, J.L., Aaronson, S.A. & Todaro, G.J. Murine sarcoma and leukemia viruses: assay using clonal lines of contact-inhibited mouse cells. J. Virol. 4 , 549–553 (1969).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Novagen. T7Select System Manual TB178 (Novagen, Madison, WI; 1999).

    Google Scholar 

  51. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 ( 1990).

    Article  CAS  Google Scholar 

  52. Gish, W. & States, D.J. Identification of protein coding regions by database similarity search. Nat. Genet. 3, 266–272 (1993).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We are grateful to Tom Yu for peptide synthesis

Author information

Authors and Affiliations

  1. Sugen, Inc., 230 East Grand Ave., South San Francisco, CA, 04080

    Sergey Zozulya, Mario Lioubin, Ronald J. Hill, Clare Abram & Mikhail L. Gishizky

Authors
  1. Sergey Zozulya
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mario Lioubin
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ronald J. Hill
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Clare Abram
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Mikhail L. Gishizky
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Sergey Zozulya or Mikhail L. Gishizky.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Zozulya, S., Lioubin, M., Hill, R. et al. Mapping signal transduction pathways by phage display. Nat Biotechnol 17, 1193–1198 (1999). https://doi.org/10.1038/70736

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/70736

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