Kinases are message bearers of the cell, passing information between molecules by adding a phosphate group to target proteins. Understanding their regulatory roles calls for tools to follow activity in vivo. Most kinase reporters require two fluorophores to carry out fluorescence resonance energy transfer (FRET) and can be tricky to implement. Markus Covert and his team at Stanford University have recently come up with simple alternatives to report on kinase dynamics in single cells.

The idea was born from their studies of fluorescently labeled NF-κB movement to the nucleus during signaling. Postdoc Sergi Regot wondered whether nuclear fluorescence could serve as a readout for other regulatory events and focused on kinases. In the JNK kinase substrate c-Jun, he noticed a nuclear export signal sequence modified by two phosphorylation sites. Regot attached this sequence and the kinase docking site to a fluorescent protein and found that phosphorylation enhanced movement out of the nucleus.

The team engineered variations of the nuclear export sequence and added a weak nuclear localization signal, improving the signal-to-noise ratio and dynamic range of the resulting 'kinase translocation reporters'. Covert likes their simplicity. Unlike with FRET probes, researchers “can pop a kinase reporter right into what they're doing—it's just one color, so there's not a big overhead,” he says.

The reporters appear to work whether kinases are located in the nucleus or cytoplasm. “The misconception is thinking that this is a static system,” says Covert; reporters continually shuttle across the nuclear envelope, and reporter activation simply shifts the balance of this movement. However, the shuttling also means that reporters will not work normally when the nuclear envelope breaks down during cell division.

Kinases can change the localization of some endogenous targets, and reporters have been used before to visualize this movement in response to kinase activity. Covert's team has shown that this behavior can be exploited systematically in probe design. But effective designs do require “a pretty good read on the docking site,” says Covert. Some reporters are more difficult to make because specificity is determined by phosphorylation site context, so export and import signals need to be carefully 'folded' into the endogenous sequence, he says. This was the case for an AGC kinase reporter that they designed.

Kinases can have multiple targets, and multiple kinases can modify the same target. Determining the rules governing kinase specificity can be an attractive use of the reporters, as the ease of introducing mutations into a single construct will make it possible to test many hypotheses about specificity.

To convert reporter localization to a quantitative measure of activity, the researchers developed a model based on the four reporter states—active or inactive, nuclear or cytoplasmic—with different rate constants for shuttling and activation. They estimated these parameters by stimulating cells to trigger activation and by imaging JNK reporters bearing mutations that constantly either prevent phosphorylation or mimic it. “That was really exciting to me as a model-driven guy,” says Covert. “Now we can go from looking at a reporter to ... the active kinase concentration in an individual cell.”

These reporters have begun to shed light on cellular heterogeneity and dynamic activity in unstimulated cells and could be used to screen for kinase-modulating drugs. They can also be used to study how signaling elements are coordinated in the cell. In a potent demonstration, the researchers coexpressed different-colored reporters they created for ERK, p38 and JNK kinases, finding different activation dynamics when stimulating cells or adding kinase inhibitors. “We see some cross-talk,” says Covert. “It's happening in the same individual cells, and to me that is a beautiful result.”