Leading the Way in Life Science Technologies

GEN Exclusives

More »


More »
November 01, 2009 (Vol. 29, No. 19)

Cellular-Signaling Pathway Signatures

Measuring Various Post-Translational Modifications with One Detection Reagent

  • Intramolecular Quenching

    Click Image To Enlarge +
    Figure 2. Various types of post-translational modifications can be detected with one sensor.

    The mechanism of fluorescence quench by the sensor is electron transfer, which involves the physical exchange of an electron between the donor and acceptor molecule. The efficiency of photo-induced electron transfer is highest with ~20 Å distance between donor and acceptor fluorophores, which corresponds to approximately 13 amino acids.

    In order to overcome this limitation, a strategy was used to form a bridge between the site of a posttranslational modification and the fluorophore (Figure 1, left). This was accomplished by employing a Chromeo 642 fluorophore (Active Motif) containing a phosphonate group that acts as a chelator for the metal ion. Upon chelation, the metal ion forms a ternary complex consisting of [fluor phosphonate (P1)]-[metal ion (M)]-[substrate phosphate (P2)]. Formation of this intramolecular ternary complex is thought to reduce the distance between donor and acceptor molecules to an extent that results in marked increase in sensitivity.

    Assays using Chromeo 642 phosphonated fluorophores resulted in 35-fold higher sensitivity of detection of protein kinase A (PKA) enzyme activity than could be achieved using a nonphosphonated TAMRA peptide of the same sequence (LRRASLG) (Figure 1). In cellular lysates, endogenous PKA activity was detectable in as little as 260 ng total rat brain lysate using Chromeo 642-labeled substrates, whereas no activity was detected using the TAMRA-labeled peptide.

  • Activity Assays

    Click Image To Enlarge +
    Figure 3. Sensor performance in cellular lysates

    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.

  • Multiplexing

    In contrast to FRET, electron transfer does not require spectral overlap between the donor and acceptor molecules. Thus, the fluorescence of fluorophores of any spectral properties can be quenched with electron transfer sensors, providing a multiplexed readout of several substrates labeled with various fluorophores in a reaction mixture.

    As an example, phosphodiesterase-mediated cleavage of fluorescein-labeled cAMP was simultaneously monitored with hydrolysis of a TAMRA-labeled cGMP in one sample of 2 µg whole brain lysate (Figure 3).

    The sensor promises to be a cost-effective new tool for studying chemical compounds that offer potential disease intervention strategies, bringing drug discovery closer to a more relevant systems biology approach and a better understanding of a drug’s action on catalytic events within interconnected cellular pathways. 

Related content