Science 355, 836–842 (2017)

Credit: AAAS

The Raf–MEK–ERK kinase pathway, which mediates cellular responses to a variety of extracellular signals, is regulated by positive and negative feedback mechanisms ranging from receptor degradation to transcriptional induction of inhibitory phosphatases. However, detecting these effects with temporal precision remains difficult. Zhou et al. engineered photodissociable dimeric variants of the Dronpa fluorescent protein (pdDronpa) that monomerize with cyan-light (500 nm) exposure and then attached pdDronpa domains to two locations on the Raf1 kinase domain. The interaction of the two pdDronpa domains under basal conditions prevents substrate access, while cyan-light exposure promotes dissociation, enabling kinase activity. This effect was reversible such that pdDronpa dimerization was restored with violet light (400 nm), inactivating kinase activity. Zhou et al. characterized a fast negative feedback mechanism on MEK, showing that a pulse of MEK activation by pdDronpa-modified Raf1 was followed by dephosphorylation attributable to ERK-mediated activation of protein phosphatases. Finally, the application of this technology to other kinases such as CDK5 provides additional support that pdDronpa may be a generally useful reagent for spatiotemporal control of kinase signaling.