In this paper, we established a modified yeast two-hybrid system, which is specialized for the detection of SH2 domain-binding proteins. The employment of the SH2 domain-tyrosine kinase fusion protein as bait al-lowed the efficient identification of SH2 domain-binding proteins. The general applicability of the sys-tem was tested using various combinations of SH2-kinase fusion bait and prey. The results indicate that the system specifically detected the previously re-ported in vivo interactions between the SH2 domains and their binding partners. In addition, using this sys-tem, we found the interaction between the adaptor protein, Lad, and the SH2 domain of Grb2 or PLC-g1. The binding of Lad to Grb2 was further confirmed in mammalian cells by a co-immunoprecipitation study. The conclusion is that the established tyrosine phos-phorylation-dependent yeast two-hybrid system pro-vides a novel and efficient way to define the SH2 do-main-binding molecules.

Keywords: SH2 Domain, Grb2, Lad, Tyrosine Phosphorylation-de