Application of the PathHunter® Protein Interaction Assay to Receptor Tyrosine Kinases (RTKs): Developing a Non-Antibody One-Step Cell-Based Kinase Activity

Application of the PathHunter® Protein Interaction Assay to Receptor Tyrosine Kinases (RTKs): Developing a Non-Antibody One-Step Cell-Based Kinase Activity
Version:
REV1_0409

File Name/Number:
SBS 2009

Year:
2009

Kinase targets have been extensively studied in biochemical assays using purified protein fragments for the kinase and the substrate. However, there is an increasing need to understand how kinases function in the context of a whole cell assay. DiscoveRx has pioneered a protein interaction assay system using our established Enzyme Fragment Complementation (EFC) β-Galactosidase detection system. This is proving to be a generic assay system applicable to a wide range of protein interaction, with the key feature being the very low endogenous activity of the two fragment of our split β-Gal system, called ProLinkTM and EA (enzyme acceptor). In our approach to a functional, whole-cell kinase assay, we express a full-length human receptor tyrosine kinase (RTK) fused at its C-terminus to the small ProLink peptide. The EA component of our assay is fused to one of a series of SH2 or PTB domain constructs that we have generated. When co-expressed in a cell, we have shown that we can detect the protein interaction that occurs in the cells when the RTK is activated and recruits an SH2/PTB domain to phospho-tyrosine residues that occur during receptor activation. We have successfully applied this assay approach to the insulin receptor, the entire Trk family, IGF1R, PDGF and FGF receptor family members, and most recently FLT3. We will present data showing both agonist and antagonist assay results for these targets, as well as our findings from a focused set of kinase inhibitors comparing TrkA analyzed in a cell-based and biochemical assay format. Furthermore, we have also shown that we can create assay systems with RTK heterodimers, with our case study example being the ErbB3 and ErbB3 activated kinase. We have recently extended these studies to show the effect of P75 co-expression with each of the Trk Family RTKs. In conclusion, the PathHunter® protein interaction platform has proven to be a very useful tool in the development of a cell-based assay format for RTKs that does not require antibody or wash steps, and can be implemented in a simple one-addition HTS assay format.