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Modular bioengineered kinase sensors via scaffold protein-mediated split-luciferase complementation

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Abstract

Phosphorylation is a key regulation event in cellular signaling. Sensing the underlying kinase activity is of crucial importance for its fundamental understanding and for drug development. For this, modular kinase activity sensing concepts are urgently needed. We engineered modular serine kinase sensors based on complementation of split NanoBiT luciferase on protein assembly platforms generated from the scaffold protein 14-3-3. The bioengineered platforms are modular and easy adaptable as exemplary shown using novel sensors for the kinases PKA, PKB, and CHK1. Two designs were conceptualized, both relying on binding of defined mono- or bivalent kinase recognition motifs to the 14-3-3 platform upon phosphorylation, resulting in reconstitution of active split-luciferase. Especially the design based on double phosphorylation and bivalent 14-3-3 binding exhibits high efficiency for signal amplification (>1000-fold) and sensitivity to specific kinases, including in cellular lysates.

Graphical abstract: Modular bioengineered kinase sensors via scaffold protein-mediated split-luciferase complementation

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Supplementary files

Article information


Submitted
06 Jan 2020
Accepted
11 May 2020
First published
12 May 2020

This article is Open Access
All publication charges for this article have been paid for by the Royal Society of Chemistry

Chem. Sci., 2020, Advance Article
Article type
Edge Article

Modular bioengineered kinase sensors via scaffold protein-mediated split-luciferase complementation

X. Xu, L. J. M. Lemmens, A. den Hamer, M. Merkx, C. Ottmann and L. Brunsveld, Chem. Sci., 2020, Advance Article , DOI: 10.1039/D0SC00074D

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