title: Knowledge-based design of a biosensor to quantify localized ERK activation in living cells.
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.
authors.affiliation: Department of Biochemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland.
authors.forename: Lutz
authors.initials: L
authors.lastname: Kummer
authors.forename: Chia-Wen
authors.initials: CW
authors.lastname: Hsu
authors.forename: Onur
authors.initials: O
authors.lastname: Dagliyan
authors.forename: Christopher
authors.initials: C
authors.lastname: MacNevin
authors.forename: Melanie
authors.initials: M
authors.lastname: Kaufholz
authors.forename: Bastian
authors.initials: B
authors.lastname: Zimmermann
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Dokholyan
authors.forename: Klaus M
authors.initials: KM
authors.lastname: Hahn
authors.forename: Andreas
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002070:Butadienes / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D019950:Mitogen-Activated Protein Kinase 1 / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000378:metabolism; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D041681:NIH 3T3 Cells; D009570:Nitriles / Q000494:pharmacology; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011744:Pyrimidinones / Q000737:chemistry; D013379:Substrate Specificity
chemical_list: C472289:ANKRD23 protein, human; D002070:Butadienes; D009124:Muscle Proteins; D009570:Nitriles; D009687:Nuclear Proteins; D011744:Pyrimidinones; C113580:U 0126; C003954:merocyanine dye; D019950:Mitogen-Activated Protein Kinase 1