Journal
CHEMISTRY & BIOLOGY
Volume 20, Issue 6, Pages 847-856Publisher
CELL PRESS
DOI: 10.1016/j.chembiol.2013.04.016
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Funding
- Ernst Schering Foundation
- Schweizerische Nationalfonds grant [3100A0B-128671]
- Swiss National Center of Competence in Research in Structural Biology
- PhosphoNetX Project in SystemsX
- European Union FP7 Collaborative Project AffinityProteome [222635]
- National Institutes of Health [GM090317]
- Swiss National Science Foundation (SNF) [3100A0B-128671] Funding Source: Swiss National Science Foundation (SNF)
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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.
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