Use of selective tyrosine kinase blockers to monitor growth factor receptor dephosphorylation in intact cells.

A novel assay was developed which allows measuring the activity of protein tyrosine phosphatases (PTPases) downregulating the signaling activity of the receptors for platelet-derived growth factor (PDGF) or epidermal growth factor (EGF) in intact Swiss 3T3 cells and nerve growth factor (TrkA) in TrkA-overexpressing PC12 cells. The assay is based on the inhibition of the receptor tyrosine kinases by specific inhibitors which enter the cells rapidly and do not affect the activity of PTPases. Thereafter, the decay of phosphotyrosine in the autophosphorylated receptors is monitored by immunoblotting with anti-phosphotyrosine antibodies. The dephosphorylation kinetics of EGF receptors and PDGF receptors in Swiss 3T3 cells as measured with this assay were found to be strikingly different. EGF receptors are almost completely dephosphorylated after 2 min at room temperature, whereas PDGF receptors are dephosphorylated only by 50% after 5 min. These data agree with previous findings about receptor dephosphorylation kinetics in isolated Swiss 3T3 cell membranes employing conventional dephosphorylation assays. The novel assay will facilitate characterization of the hitherto not identified receptor-directed PTPases for PDGF receptors, EGF receptors, and TrkA. The assay principle is general and should be applicable to any PTPases and their substrates, provided specific inhibitors for the respective kinases are available. Furthermore, it can be applied to screen for regulator molecules of specific PTPases in their physiological environment.