Abstract
The yeast two-hybrid system has been utilized by many laboratories in the characterization of protein-protein interactions that occur in eukaryotic and prokaryotic cell systems. In addition, the two-hybrid system has been used to isolate and characterize novel interacting proteins to aid in the understanding of biochemical signaling pathways for various receptors and enzymes. The interacting components are fused to inert components of the transcriptional apparatus. One of the components is fused to a DNA-binding domain and is generally referred to as the “bait.” The second protein is fused to a transcriptional activation domain and is referred to as the “fish” or the “prey.” When there is an association between the “bait” and the “prey,” the DNA-binding domain and the transcriptional-activation domain are brought into close proximity to activate the transcription of the reporter gene.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Fields S. and Song O.-K. (1989) A novel genetic system to detect protein-protein interactions. Nature 340, 245–246.
Bai C. and Elledge S. J. (1997) Searching for interacting proteins with the two-hybrid system I, in The Yeast Two-Hybrid System (Bartel P. L. and Fields S., eds.), Oxford University Press, New York, pp. 11–28.
Brachmann R. K. and Boeke J. D. (1997) Tag games in yeast: the two-hybrid system and beyond. Curr. Opin. Biotechnol. 8, 561–568.
Hunter T. (1995) Protein kinases and phsophatases: the yin and yang of protein phosphorylation and signaling. Cell 80, 225–236.
Heldin C.-H. (1995). Dimerization of Cell Surface Receptors in Signal Transduction. Cell 80, 213–223.
Gartner A., Nsamyth K., and Ammerer G. (1992) Signal transduction in Saccha-romyces cerevisiae requires tyrosine and threonine phosphorylation of FUS3 and KSS1. Genes. Dev. 6, 1280–1292.
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, 21,155–21,164.
Pandey A., Lazar D. F., Saltiel A. R., and Dixit V. M. (1994) Activation of theEck receptor protein tyrosine kinase stimulates phosphatidylinositel 3-kinase activity. J. Biol. Chem. 269, 30,154–30,157.
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.
Gustafson T. A., He W., Craparo A., Schaub C. D., and Oneill 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.
Pandey A., Duan H., Di Fiore P. P., and Dixit V. M. (1995) The ret receptor protein tyrosine kinase associates with the SH2-containing adapter protein Grb10. J. Biol. Chem. 270, 21461–21463.
Xing Z., Chen H.-C., Nowlen J. K., Taylor S. J., Shalloway D., and Guan J.-L. (1994) Direct Interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain. Mol. Biol. Cell 5, 413–421.
Vasavada H. A., Ganguly S., Germino F. J., Wang Z. X., and Weissman S. M. (1991) A contingent replication assay for the detection of protein-protein interactions in animal cells. Proc. Natl. Acad. Sci. USA 88, 10,686–10,690.
Fearon E. R., Finkel T., Gillison M. L., Kennedy S. P., Casella J. F., Tomaselli G. F., et al. (1992) Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells. Proc. Natl. Acad. Sci. USA 89, 7958–7962.
Tsan J. T., Wang Z., Jin Y., Hwang L.-Y., Bash R. O., and Baer R. (1997) Mammalian cells as hosts for two-hybrid studies of protein-protein interaction, in The Yeast Two-Hybrid System (Bartel P. L. and Fields S., eds.), Oxford University Press, New York, pp. 217–232.
Osborne M. A., Zenner G., Lubinus M., Zhang X., Songyang Z., Cantley L. C., et al. (1996) The inositol 5′-phosphatase SHIP binds to immunoreceptor signaling motifs and responds to high affinity IgE receptor aggregation. J. Biol. Chem. 271, 29,271–29,278.
Osborne M. A., Dalton S., and Kochan J. P. (1995) The yeast tribrid system — genetic detection of trans-phosphorylated ITAM-SH2-interactions. Bio/Technology 13, 1474–1478.
Johnston M. (1987) A model fungal gene regulatory mechanism: the GAL genes of Saccharomyces cerevisiae. Microbiolog. Rev. 51:4, 458–476.
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.
Russo P. and Sherman F. (1989) Transcription terminates near the poly(A) site in the CYC1 gene of the yeast Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 86, 8348–8352.
Kalderon D., Roberts B. L., Richardson W. D., and Smith A. E. (1984) A short amino acid sequence able to specify nuclear location. Cell 39, 499–509.
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. Nucleic Acids Res. 13, 7865–7879.
Osborne M. A., Lubinus M., and Kochan J. P. (1997) Detection of protein-protein interactions dependent on post-translational modifications, in The Yeast Two-Hybrid System (Bartel P. L. and Fields S., eds.), Oxford University Press, New York, pp. 233–258.
Mizushima S. and Nagata S. (1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18, 5322.
Sherman F. (1991) Getting started with yeast. Meth. Enzymol. 194, 3–21.
Sambrook J., Fritsch E. F., and Maniatis T. (1989) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Towbin H., Staehelin T., and Gordon J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc. Natl. Acad. Sci. USA 76, 4350–4354.
Zhu L., Gunn D., Kuchibhatla S. (1997) Constructing an activation domain-fusion library, in The Yeast Two-Hybrid System (Bartel P. L. and Fields S., eds.), Oxford University Press, New York, pp. 73–96.
Gubler U. and Chua A. O. (1991) The establishment of cDNA libraries in lambda gt10, in Essential Molecular Biology. A Practical Approach, vol. II (Brown T. A., ed.), Oxford University Press, New York, pp. 39–56.
Cullen B. R. (1987) Use of eukaryotic expression technology in the functional analysis of cloned genes. Meth. Enzymol. 152, 684–704.
Kaiser C., Michaelis S., and Mitchell A. (1994) Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Wong C. and Naumovski L. (1997) Method to screen for relevant yeast two-hybrid-derived clones by coimmunoprecipitation and colocalization of epitope-tagged fragments — applications to Bcl-xL. Anal. Biochem. 252, 33–39.
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2000 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Volpers, C., Lubinus, M., Osborne, M.A., Kochan, J.P. (2000). cDNA Expression Cloning and Characterization of Phosphorylation Dependent Protein Interactors Using the Yeast Tribrid System. In: Reith, A.D. (eds) Protein Kinase Protocols. Methods in Molecular Biology™, vol 124. Humana Press. https://doi.org/10.1385/1-59259-059-4:271
Download citation
DOI: https://doi.org/10.1385/1-59259-059-4:271
Publisher Name: Humana Press
Print ISBN: 978-0-89603-700-7
Online ISBN: 978-1-59259-059-9
eBook Packages: Springer Protocols