Knowledge-based design of a biosensor to quantify localized ERK activation in living cells

Lutz Kummer, Chia Wen Hsu, Onur Dagliyan, Christopher MacNevin, Melanie Kaufholz, Bastian Zimmermann, Nikolay V. Dokholyan, Klaus M. Hahn, Andreas Plückthun

Research output: Contribution to journalArticle

37 Scopus citations

Abstract

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.

Original languageEnglish (US)
Pages (from-to)847-856
Number of pages10
JournalChemistry and Biology
Volume20
Issue number6
DOIs
StatePublished - Jun 20 2013

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Medicine
  • Molecular Biology
  • Pharmacology
  • Drug Discovery
  • Clinical Biochemistry

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    Kummer, L., Hsu, C. W., Dagliyan, O., MacNevin, C., Kaufholz, M., Zimmermann, B., Dokholyan, N. V., Hahn, K. M., & Plückthun, A. (2013). Knowledge-based design of a biosensor to quantify localized ERK activation in living cells. Chemistry and Biology, 20(6), 847-856. https://doi.org/10.1016/j.chembiol.2013.04.016