The electron transfer sensor allows the researcher to measure activities of kinases, phosphatases, proteases, and phosphodiesterases with one assay platform, enabling the establishment of signaling-pathway signatures within lysates of cells that have undergone perturbations.
As an example, the cyclic AMP dependant activation of PKA was measured with a phosphodiesterase assay and a kinase activity assay in one experiment and quantified with a single detection reagent (Figure 3, left). cAMP is an important cellular second messenger that is regulated by GPCRs. The levels of cAMP are balanced by phosphodiesterases, which hydrolyze the 3´ cyclic bond.
cAMP mediates various signaling events by binding to the regulatory domain of PKA, one of its downstream targets. Upon binding, the regulatory domain disassociates from the catalytic domain and the enzyme becomes active. With increasing concentrations of the generic cAMP phosphodiesterase inhibitor IBMX, endogenous phosphodiesterase mediated cleavage of a fluorescein-labeled cAMP was decreased in lysates of rat brain (Figure 3, left).
The resulting increase in cAMP levels correlated with increase in PKA activity that was monitored using a Chromeo 642-labeled substrate (LRRASLG). Connecting GPCR signaling through measurement of phosphodiesterase-mediated cAMP hydrolysis with downstream protein kinase activity using one assay system illustrates the viability of the sensor as a tool to generate drug-induced signaling signatures.