Biosensors of protein kinase action: from in vitro assays to living cells.

Protein kinases, and the signal transduction pathways in which they participate, are now recognized to be medicinally attractive targets of opportunity. Inhibitors of the protein kinase family not only hold great promise as therapeutic agents, but are also of profound utility in the characterization of signaling pathways. The direct visualization of protein kinase activity in living cells provides a genuine assessment of the efficacy and selectivity of these inhibitors in a physiological setting. In addition, the ability to visualize the activity of a protein kinase in real time furnishes a direct measurement of the activation of specific signaling pathways in response to extracellular stimuli. We have developed two series of fluorescent substrates for protein kinase C (PKC) using a strategy that positions the reporter-group directly on the residue undergoing phosphorylation. The first series of PKC substrates is based, in part, on the Ca(+2) indicators developed by Tsien and his collaborators during the 1980s. In this case, phosphorylation of the substrate creates a divalent metal ion binding site. Upon metal ion coordination, a fluorescence change transpires via a mechanism analogous to that described for the Ca(+2) indicators. The second series of PKC sensors was identified via the preparation and subsequent screen of a library of fluorescently-labeled PKC peptide substrates. The lead derivative displays a phosphorylation-induced fluorescence change that allows the visualization of real-time PKC activity in both cell lysates and living cells. Furthermore, immunodepletion experiments demonstrate that the fluorescently-tagged peptide is selectively, if not exclusively, phosphorylated by the conventional PKCs. Both of the protein kinase biosensor strategies take advantage of the ease with which peptides can be modified to create libraries of structurally altered analogs. However, the inherent synthetic mutability of peptides is not just limited to library construction. For example, it may ultimately be possible to simultaneously monitor multiple protein kinases by affixing fluorophores with distinct photophysical properties to appropriately designed active site-directed peptides.