We have used a modification of the very useful yeast two-hybrid system that allows the identification of protein-protein interactions requiring tyrosine phosphorylation. Yeast do not contain tyrosine kinases, and we therefore introduced tyrosine kinases (TK) from the PDGF receptor, Src, or the Fms receptor. The modified, or TK two-hybrid method permits identification of either PTB, SH2 domains or other interactions requiring tyrosine phosphorylation. Binding partners for these domains also may be detected. The properties and uses of the system will be described below. The system is based on the following two publications:
Keegan and Cooper, Oncogene 12, 1537-1544 (1996).
Lioubin et al., Genes & Development 10:1084-1095 (1996).
1) Components of the modified, or TK two-hybrid system
Bait, in our case the bait consisted of the LexA DNA-binding domain fused to the Shc PTB domain. Either WW or SH2 domains could be substituted for the PTB domain; or,sequences expressing known tyrosine phosphorylation sites could be used to fish-out binding domains from a library.
Tysosine Kinase, we used the TK domain of the PDGF receptor expressed from the same plasmid as the LexA-PTB fusion protein (see below). Plasmids are also available with the Src or Fms TK domains, and a plasmid with the Flt3 TK domain is under development.
Target, The target for the screen is the VP16 transactivation domain fused to a Library of sequences. It is very important to have a positive control in these experiments before starting any library screen, and we used the VP16 protein fused to the C-terminal tail sequences of the EGF receptor. This contains known PTB binding sites and positive results will help assure success with the library screen.
2) Plasmids expressing various TK domains
3) Tyrosine kinase activity of the plasmids expressed in yeast
The results to the right demonstrate the relative tyrosine kinase activities of the three plasmids (above) expressed in yeast. The total yeast proteins were run on a gel and after transfer, blotted with anti-phosphotyrosine antibodies. In this example, the BTM-Fms plasmid did not phosphorylate host proteins much above background (BTM lane). Other Fms plasmids are now available that do tyrosine phosphorylate but the activity is less than with either BTM-Src, or BTM-PDGFR. Both Src and PDGFR tyrosine phosphorylate well, with overlapping substrates as might be expected. Some are the same, but many are different between the two kinases. We also have demonstrated this same specificity in the two-hybrid system by comparing the different kinases with the same bait (Kristen Carlberg, Mario Lioubin, unpublished results).
4) Yeast Two-Hybrid Protocols
See: Vojtek, AB and Hollenberg, SM, Ras-Raf interactions: two-hybrid analysys. Methods in Enzymology. 255:331-342, 1995.
Lioubin, MN, Algate, PA, Tsai, S, Carlberg, K, Aebersold, R and Rohrschneider, LR. p150ship, a signal transduction molecule with inositol polyphosphate 5-phosphatase activity. Genes & Development. 10:1084-1095, 1996.
