A screening method of SH2 domain ligands and blockers using a solid phase binding.
We have developed a high throughput screening method for SH2 domain binding ligands and blockers. This method measures directly the binding of a 3H-labeled phosphopeptide derived from the sequence around tyrosine317 in the human Shc (SpYVNVK) to the SH2 domain of Grb2, which is precoated as glutathione S-transferase fusion proteins on solid phase. The optimum concentration for the fusion protein coating was 300 ng/100 microl/well for SH2 domain binding. Although an 8-h incubation at 4 degrees C for the coating of fusion protein was required to reach a maximum binding, even a 2-h coating produced 84% of the maximum binding. Saturation of ligand peptide binding in our assay system was observed at 10 pmol/well for the SH2 domain. However, 2 pmol/well showed consistent and reproducible results for the binding when the incubations were performed for 8 h at 4 degrees C. Competitive binding inhibition studies with various unlabeled phosphopeptides imply that the binding assay is highly specific to peptide sequences and able to screen possible ligands or blockers of signal transduction pathway mediated by Grb2 SH2 binding. In conclusion, our new method for SH2 domain binding is easy, rapid, and most of all inexpensive. These advantages over existing assay methods make this method especially suitable for a high throughput application, such as the screening for anticancer drug candidates.
Koh
W S
WS
Immune Regulation Research Unit, Korea Research Institute of Bioscience and Biotechnology, Yusung, Taejon, South Korea.
Yoon
S Y
SY
Lee
E K
EK
Lee
E K
EK
Kwon
B M
BM
Kim
J W
JW
Han
M Y
MY
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D001667:Binding, Competitive; D002474:Cell-Free System; D051380:GRB2 Adaptor Protein; D006801:Humans; D008024:Ligands; D008722:Methods; D010748:Phosphopeptides / Q000378:metabolism; D011506:Proteins / Q000737:chemistry*; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013997:Time Factors; D018909:src Homology Domains
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051380:GRB2 Adaptor Protein; C495948:GRB2 protein, human; D008024:Ligands; D010748:Phosphopeptides; D011506:Proteins; C528024:SHC1 protein, human; D055765:Shc Signaling Adaptor Proteins; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1