Functional Whole-cell Assays for Tyrosine Phosphorylation Using PathHunter® Technology

Version:: MIPTEC1009_REV1
File Name/Number:: MIPTEC 2009
Year:: 2009

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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 detection system using well-established Enzyme Fragment Complementation (EFC) technology. We show here that this system can be used to report the phosphorylation status of proteins in whole cells by monitoring the interaction of target proteins with SH2 phosphotyrosine binding domains. We have successfully applied this approach to multiple receptor tyrosine kinases including; the insulin receptor family, the Trk family, PDGF and FGF receptor family members. This approach has been extended to receptors without kinase domains that couple to cytosolic kinases. Using the G-CSFR and Prolactin receptor we demonstrate that assays specific for a receptor target or assays specific for a cytosolic tyrosine kinase, such as Jak1 or Jak2, can be rapidly developed. In conclusion, the PathHunter^® protein interaction platform has proven to be a useful tool in the development of a cell-based assay format for tyrosine phosphorylation that does not require antibody or wash steps, and can be implemented in a simple one-addition HTS assay.