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
  • Published:

The Yeast Tribrid System—Genetic Detection of trans-phosphorylated ITAM-SH2-Interactions

Abstract

Protein-protein interactions are often dependent on the post-translational modification of one component of a complex. To facilitate the study of these interactions in signal transduction, we have developed the yeast tribrid system, a modification of the yeast two-hybrid system. We demonstrate that the interactions are dependent upon the presence of a tyrosine kinase, an SH2 domain and a tyrosine containing substrate. Using the gamma subunit of the high-affinity IgE receptor, FcεRI, this approach has been used to isolate a novel SH2-containing family member. The mRNA encoding this novel protein is differentially expressed in rat tissues. The yeast tribrid system can be readily adapted for the characterization of novel tyrosine kinases or substrates, as well as the study of protein-protein interactions which involve other post-translational modifications.

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

Access options

Buy this article

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

Similar content being viewed by others

References

  1. Cambier, J.C. 1995. New nomenclature for the Reth motif (or ARH1/TAM/ARAM/YXXL). Imm. Tod. 16: 110.

    Article  CAS  Google Scholar 

  2. Beaven, M.A. and Metzger, H. 1993. Signal transduction by Fc receptors, the FceRI case. Imm. Tod. 14: 222–226.

    Article  CAS  Google Scholar 

  3. Rivera, V.M. and Brugge, J.S. 1995. Clustering of Syk is sufficient to induce tyrosine phosphorylation and release of allergic mediators from rat basophilic leukemia cells. Mol. Cell. Biol. 15: 1582–1590.

    Article  CAS  Google Scholar 

  4. Taylor, J.A., Karas, J.L., Ram, M.K., Green, O.M. and Seidel-Dugan, C. 1995. Activation of the high-affinity immunoglobulin E receptor FceRI in RBL-2H3 cells is inhibited by Syk SH2 domains. Mol. Cell. Biol. 15: 4149–4157.

    Article  CAS  Google Scholar 

  5. Kolanus, W., Romeo, C. and Seed, B. 1993. T cell activation by clustered tyrosine kinases. Cell 74: 171–183.

    Article  CAS  Google Scholar 

  6. Fields, S. and Sternglanz, R. 1994. The two-hybrid system: an assay for protein-protein interactions. Trends Genet. 10: 286–292.

    Article  CAS  Google Scholar 

  7. Castellanos, R.M.P. and Mazon, M.J. 1985. Identification of phosphotyrosine in yeast proteins and of a protein tyrosine kinase associated with the plasma membrane. J. Biol. Chem. 260: 8240–8242.

    CAS  PubMed  Google Scholar 

  8. Gartner, A., Nsamyth, K. and Ammerer, G. 1992. Signal transduction in Saccharomyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1. Genes. Dev. 6: 1280–1292.

    Article  CAS  Google Scholar 

  9. Lim, M.-Y., Dailey, D., Martin, G.S. and Thorner, J. 1993. Yeast MCK1 protein kinase autophosphorylates at tyrosine and serine but phosphorylates exogenous substrates at serine and threonine. J. Biol. Chem. 268: 21155–21164.

    CAS  PubMed  Google Scholar 

  10. Navon, A., Schwarz, Y., Hazan, B., Kassir, Y. and Nir, U. 1994. Meiosis-dependent tyrosine phosphorylation of a yeast protein related to the mouse p51ferT. Mol. Gen. Gen. 244: 160–167.

    CAS  Google Scholar 

  11. Pandey, A., Lazar, D.F., Saltiel, A.R. and Dixit, V.M. 1994. Activation of the Eck receptor protein tyrosine kinase stimulates phosphatidylinositel 3-kinase activity. J. Biol. Chem. 269: 30154–30157.

    CAS  PubMed  Google Scholar 

  12. O'Neill, T.J., Craparo, A. and Gustafson, T.A. 1995. Characterization of an interaction between insulin receptor substrate 1 and the insulin receptor by using the two-hybrid system. Mol. Cell. Biol. 14: 6433–6442.

    Article  Google Scholar 

  13. Gustafson, T.A., He, W., Craparo, A., Schaub, C.D. and O'Neill, T.J. 1995. Phosphotyrosine-dependent interaction of SHC and insulin receptor substrate 1 with the NPEY motif of the insulin receptor via a novel non-SH2 domain. Mol. Cell. Biol. 15: 2500–2508.

    Article  CAS  Google Scholar 

  14. Kornbluth, S., Jove, R. and Hanafusa, H. 1987. Characterizaton of avian and viral p60src proteins expressed in yeast. Proc. Natl. Acad. Sci. USA 84: 4455–4459.

    Article  CAS  Google Scholar 

  15. Kuster, H., Thompson, H. and Kinet, J.P. 1990. Characterizaton and expression of the gene for the human Fc receptor γ subunit. J. Biol. Chem. 265: 6448–6452.

    CAS  PubMed  Google Scholar 

  16. Law, C., Sidorenko, S., Chadran, K., Draves, K.E., Chan, A.C., Weiss, A. et al. 1994. Molecular cloning of human syk. J. Biol. Chem. 269: 12310–12319.

    CAS  PubMed  Google Scholar 

  17. Paolini, R., Renard, V., Vivier, E., Ochiai, K., Jouvin, M.H., Malissen, B., et al. 1995. Different roles for the Fc epsilon RI gamma chain as a function of the receptor context. J. Exp. Med. 181: 247–255.

    Article  CAS  Google Scholar 

  18. Kihara, H. and Siraganian, R.P. 1994. Src homology 2 domains of Syk and Lyn bind to tyrosine-phosphorylated subunits of the high affinity IgE receptor. J. Biol. Chem. 269: 22427–22432.

    CAS  PubMed  Google Scholar 

  19. Shiue, L., Green, J., Karas, J.L., Morgenstern, J.P., Ram, M.K., Taylor, M.K., et al 1995. Interaction of p72syk with the γ and β subunits of the high-affinity receptor for immunoglobulin E FceRI. Mol. Cell. Biol. 15:1: 272–281.

    Article  Google Scholar 

  20. Shiue, L., Zoller, M.J. and Brugge, J.S. 1995. Syk is activated by phosphotyrosine-containing peptides representing the tyrosine-based activation motifs of the high affinity receptor for IgE. J. Biol. Chem. 270: 10498–10502.

    Article  CAS  Google Scholar 

  21. Dalrymple, M.A., Mcgeoch, D.J., Davison, A.J. and Preston, C.M. 1985. DNA sequence of the herpes simplex virus type 1 gene whose product is responsible for transcriptional activation of immediate early promoters. Nucl. Acids Res. 13: 7865–7879.

    Article  CAS  Google Scholar 

  22. Perlmutter, R.M., Marth, J.D., Lewis, D.B., Peet, R., Ziegler, S.F. and Wilson, C.B. 1988. Structure and expression of Ick transcripts in human lymphoid cells. J. Cell Biochem. 38: 117–126.

    Article  CAS  Google Scholar 

  23. Yamanashi, Y., Fukushige, S., Semba, K., Sukegawa, J., Miyajima, N., Matsubara, K., et al 1987. The yes-related cellular gene lyn encodes a possible tyrosine kinase similar to p56lck. Mol. Cell. Biol. 7:1: 237–243.

    Article  Google Scholar 

  24. Pawson, T. 1995. Protein modules and signalling networks. Nature (Lond.) 373: 573–580.

    Article  CAS  Google Scholar 

  25. Turner, H., Reif, K., Rivera, J. and Cantrell, D. 1995. Regulation of the adapter molecule Grb2 by the FceRI in the mast cell line RBL2H3. J. Biol. Chem. 270: 9500–9506.

    Article  CAS  Google Scholar 

  26. Rose, M.D., Winston, F., and Heiter, P. (1990). Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

    Google Scholar 

  27. Guthrie, C. and Fink, G.R. 1991. Guide to yeast genetics and molecular biology. Meth. Enz. 194 1–933.

    Google Scholar 

  28. Schiestl, R.H. and Gietz, R.D. 1989. High efficiency transformation of intact yeast cells using single stranded nucleic acids as a carrier. Curr. Genet. 16: 339–346.

    Article  CAS  Google Scholar 

  29. Dalton, S. and Treisman, R. 1992. Characterization of SAP-1 a protein recruited by serum response factor to the c-fos serum response element. Cell 68: 597–612.

    Article  CAS  Google Scholar 

  30. Kalderon, D., Roberts, B.L., Richardson, W.D. and Smith, A.E. 1984. A short amino acid sequence able to specify nuclear locaton. Cell 39: 499–509.

    Article  CAS  Google Scholar 

  31. Evan, G.I., Lewis, G.K., Ramsay, G. and Bishop, J. M. 1985. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol. Cell. Biol. 5: 3610–3616.

    Article  CAS  Google Scholar 

  32. Kinet, J.P., Blank, U., Ra, C., White, K., Metzger, H. and Kochan, J. 1988. Isolation and characterization of cDNAs coding for the β subunit of the high-affinity receptor for immunoglobulin E. Proc. Natl. Acad. Sci. USA 85: 6483–6487.

    Article  CAS  Google Scholar 

  33. Franzusoff, A., Rothblatt, J. and Schekman, R. 1991. Analysis of polypeptide transit through yeast secretory pathway. Meth. Enz. 194: 662–682.

    Article  CAS  Google Scholar 

  34. Pirozzi, G., Terry, R.W., Epstein, D. and Labow, M.A. 1995. Identification and characterization of a novel surface antigen gene induced in mast cells activated through the high affinity IgE receptor. J. Immunol. In press.

Download references

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Jarema P. Kochan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Osborne, M., Dalton, S. & Kochan, J. The Yeast Tribrid System—Genetic Detection of trans-phosphorylated ITAM-SH2-Interactions. Nat Biotechnol 13, 1474–1478 (1995). https://doi.org/10.1038/nbt1295-1474

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt1295-1474

This article is cited by

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