u-0126 and merocyanine-dye

Investigation of protein activation in living cells is fundamental to understanding how proteins are influenced by the full complement of upstream regulators they experience. Here, we describe the generation of a biosensor based on the DARPin binding scaffold suited for intracellular applications. Combining library selection and knowledge-based design, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK with a solvatochromatic merocyanine dye, whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. The DARPin-based biosensor will serve as a useful tool for studying biological functions of ERK in vitro and in vivo.

Topics: Animals; Biosensing Techniques; Butadienes; Enzyme Activation; HEK293 Cells; Humans; Mice; Mitogen-Activated Protein Kinase 1; Muscle Proteins; Mutagenesis, Site-Directed; NIH 3T3 Cells; Nitriles; Nuclear Proteins; Phosphorylation; Protein Binding; Pyrimidinones; Substrate Specificity