Knowledge-Based Design of a Biosensor to Quantify Localized ERK Activation in Living Cells
| Autor: | Christopher J. MacNevin, Bastian Zimmermann, Chia Wen Hsu, Onur Dagliyan, Klaus M. Hahn, Lutz Kummer, Andreas Plückthun, Melanie Kaufholz, Nikolay V. Dokholyan |
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| Přispěvatelé: | University of Zurich, Plückthun, Andreas |
| Jazyk: | angličtina |
| Předmět: |
MAPK/ERK pathway
1303 Biochemistry Nucleolus Clinical Biochemistry Muscle Proteins Plasma protein binding Biosensing Techniques 1308 Clinical Biochemistry 01 natural sciences Biochemistry Substrate Specificity Mice Drug Discovery Nuclear protein Phosphorylation Mitogen-Activated Protein Kinase 1 0303 health sciences Kinase 3002 Drug Discovery Nuclear Proteins General Medicine 3. Good health Cell biology 3004 Pharmacology DARPin Molecular Medicine Protein Binding Pyrimidinones macromolecular substances Biology 010402 general chemistry Article 03 medical and health sciences Nitriles 10019 Department of Biochemistry 1312 Molecular Biology Butadienes Animals Humans Molecular Biology 030304 developmental biology Pharmacology HEK 293 cells 0104 chemical sciences Enzyme Activation HEK293 Cells 1313 Molecular Medicine Mutagenesis Site-Directed NIH 3T3 Cells 570 Life sciences biology |
| Zdroj: | Chemistry & Biology Chemistry & Biology; Vol 20 |
| ISSN: | 1074-5521 |
| DOI: | 10.1016/j.chembiol.2013.04.016 |
| Popis: | Investigation of protein activation in living cells is fundamental to understand how proteins are influenced by the full complement of upstream regulators they experience. We describe here the generation of a biosensor based on the Designed Ankyrin Repeat Protein (DARPin) binding scaffold suited for intracellular applications. Combining selection and evolution from libraries, knowledge-based design and efficient and rapid testing of conjugate candidates, we created an ERK activity biosensor by derivatizing a DARPin specific for phosphorylated ERK (pERK) with a solvatochromic merocyanine dye (mero87), whose fluorescence increases upon pERK binding. The biosensor specifically responded to pERK2, recognized by its conformation, but not to non-phosphorylated ERK2 or other closely related mitogen-activated kinases tested. Activated endogenous ERK was visualized in mouse embryo fibroblasts incubated in 2% serum, revealing greater activation in the nucleus, perinuclear regions, and especially the nucleoli. Activity was greatly reduced by the MEK1/2 inhibitor U0126. The DARPin-based biosensor will serve as useful tool for studying biological functions of ERK in vitro and in vivo. |
| Databáze: | OpenAIRE |
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