Catching a kinase in the act

Using a cleverly designed set of molecular probes, Violin et al., reporting on page 899, provide the first real-time analysis of phosphorylation and dephosphorylation downstream of protein kinase C (PKC). The results provide a striking demonstration of the importance of phosphatases in PKC signaling. When second messenger concentrations decrease, PKC interaction with the membrane loosens, PKC activity is rapidly lost, and the activity probe is quickly dephosphorylated.The authors designed a reporter protein that immediately indicates changes in PKC activity. Phosphorylation or dephosphorylation of the reporter causes a change in fluorescence resonance energy transfer. When tethered to the membranes of appropriately stimulated cells, the reporter is phosphorylated and dephosphorylated in sustained oscillations, in step with waves of calcium release. Similar reporter proteins, designed to monitor other signaling parameters, demonstrate that the oscillations can be driven...