Protein kinases control a broad range of cellular processes. Dysregulation of kinase activity causes many diseases including cancer. Furthermore, temporal dynamics of kinase signaling can elicit distinct cellular responses. To image dynamics of kinase activity in living animals, genetically encoded fluorescent reporters are ideal because they require no exogenous molecule and are non-invasive. Although green fluorescent protein (GFP)-based Frster resonance energy transfer (FRET) reporters can be used to image dynamics of kinase signaling in cultured cells where they can achieve subcellular resolution and compartmentalized kinase signaling, in vivo use of the FRET-based kinase reporters is difficult because of small magnitude of the fluorescence changes of the fluorophores. We seek to design and demonstrate a new class of GFP-based reporters that, upon kinase activation, phase separate and form highly concentrated droplets via multivalent protein-protein interactions. Our approach is inspired by recent work showing that multivalent interactions drive protein phase separation to form protein droplets that concentrate the protein up to 100 fold. The phase separation-based kinase reporter has both large dynamic range and high brightness because fluorophores concentrate in discrete punctae. This punctal signal pattern is distinctive and easily detectable in whole animals and robust for in vivo detection of kinase activity. The reporter also achieves fast kinetics at the second-to-minute timescale and is reversible, with no apparent toxicity in transgenic animals. To further demonstrate the new design principle, we aim to design and apply phase separation-based kinase reporters to visualize dynamics of several key kinases during specific cellular processes in vivo.
Public Heath Relevance/Narrative Protein kinases play critical roles in animal development and homeostasis. Perturbation of kinase activity can lead to many human diseases including cancer. The proposed new class of fluorescent kinase reporters will have large dynamic range, high brightness and fast kinetics, and will be ideal for imaging dynamics of kinase activity in animals, which will advance mechanistic understanding of animal development and disease.
