Tyrosine phosphorylation-dependent yeast two-hybrid system for the identification of the SH2 domain-binding proteins.
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 allowed the efficient identification of SH2 domain-binding proteins. The general applicability of the system was tested using various combinations of SH2-kinase fusion bait and prey. The results indicate that the system specifically detected the previously reported in vivo interactions between the SH2 domains and their binding partners. In addition, using this system, we found the interaction between the adaptor protein, Lad, and the SH2 domain of Grb2 or PLC-gamma1. The binding of Lad to Grb2 was further confirmed in mammalian cells by a co-immunoprecipitation study. The conclusion is that the established tyrosine phosphorylation-dependent yeast two-hybrid system provides a novel and efficient way to define the SH2 domain-binding molecules.
Park
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Division of Molecular Life Science & the Center for Cell Signaling Research at Ewha Woman's University, Seoul, Korea.
Yun
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Y
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D051380:GRB2 Adaptor Protein; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D051966:Phospholipase C gamma; D010766:Phosphorylation; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D010738:Type C Phospholipases / Q000378:metabolism; D014443:Tyrosine; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D002352:Carrier Proteins; D051380:GRB2 Adaptor Protein; C495949:Grb2 protein, mouse; D007527:Isoenzymes; D010982:Platelet-Derived Growth Factor; D011506:Proteins; D011993:Recombinant Fusion Proteins; C402726:Sh2d2a protein, mouse; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D010738:Type C Phospholipases; D051966:Phospholipase C gamma