FAK Cellular Phosphorylation Assay (intracellular kinase activity assay) for compound screening and profiling in intact cells
The focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that plays an important role in signal transduction pathways of integrin or growth factor receptor mediated cell adhesion. FAK has an important role in a large number of processes involved in the development and progression of cancer (survival, migration, proliferation and invasion). FAK overexpression/activity is found in a variety of human cancers making it an attractive target for anti-cancer therapy.
The cellular phosphorylation assay for FAK determines the autophosphorylation on Tyr397 of the FAK protein. Murine embryonal fibroblast cells deficient of the endogenous FAK express a human full-length FAK which is constantly phosphorylated in adherent cells. By adding Staurosporine phospho-FAK-Tyr397 levels are largely decreased and thus the dynamic behaviour to quantify inhibitory potentials of compounds can be obtained. The assay is validated based on known inhibitors of FAK kinase activity (see Fig. 1).
Substrate phosphorylation as a readout of intracellular kinase activity via ELISA
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Reference compound IC50 for FAK
Staurosporine is an inhibitor of the phospho-FAKTyr397 signal found in the described cells. The graph shows a representative result.