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

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.

SUBMITTER: Xu X

PROVIDER: S-EPMC7446724 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Modular bioengineered kinase sensors <i>via</i> scaffold protein-mediated split-luciferase complementation.

Xu Xiaolu X Lemmens Lenne J M LJM den Hamer Anniek A Merkx Maarten M Ottmann Christian C Brunsveld Luc L

Chemical science 20200512 21

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 exempl ...[more]

PMID: 32874496

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Project description:Kinases play fundamental roles in the brain. Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons. Changes induced by kinases on neuronal excitability, synaptic plasticity and brain connectivity are linked to complex brain disorders, but the molecular mechanisms underlying these cellular events remain for the most part elusive. To further our understanding of brain disease, new methods for rapidly surveying kinase pathways in the cellular context are needed. The bioluminescence-based luciferase complementation assay (LCA) is a powerful, versatile toolkit for the exploration of PPI. LCA relies on the complementation of two firefly luciferase protein fragments that are functionally reconstituted into the full luciferase enzyme by two interacting binding partners. Here, we applied LCA in live cells to assay 12 kinase pathways as regulators of the PPI complex formed by the voltage-gated sodium channel, Nav1.6, a transmembrane ion channel that elicits the action potential in neurons and mediates synaptic transmission, and its multivalent accessory protein, the fibroblast growth factor 14 (FGF14). Through extensive dose-dependent validations of structurally-diverse kinase inhibitors and hierarchical clustering, we identified the PI3K/Akt pathway, the cell-cycle regulator Wee1 kinase, and protein kinase C (PKC) as prospective regulatory nodes of neuronal excitability through modulation of the FGF14:Nav1.6 complex. Ingenuity Pathway Analysis shows convergence of these pathways on glycogen synthase kinase 3 (GSK3) and functional assays demonstrate that inhibition of GSK3 impairs excitability of hippocampal neurons. This combined approach provides a versatile toolkit for rapidly surveying PPI signaling, allowing the discovery of new modular pathways centered on GSK3 that might be the basis for functional alterations between the normal and diseased brain.

Project description:BackgroundIP3-induced Ca2+ release, mediated by IP3R, is one of the most momentous cellular signaling mechanisms that regulate in a wide variety of essential cellular functions. Involvement of disrupted IP3 signaling pathways in numerous pathophysiology conditions is implicated to find the best methods for its measurement. Hence, several different biosensors have developed to monitor temporal changes of IP3 by using the IP3-binding domain of IP3 receptors.ObjectivesBased on a previous study, we developed and characterized a series of bioluminescent biosensors using the human type-II IP3 receptor ligand binding domain (residues 1-604), named LAIRE (luminescent analyzer for IP3 receptor element) to study the effect of flexible and rigid linkers on the luminescence intensity of split luciferase. The effect of a mutation in IP3 binding residues and suppressor domain in the IP3 binding domain on luciferase complementary assay is also investigated.Materials and methodsIn the present study, first IP3-binding domain (residues 1-604) of IP3-receptor type 2 (LAIRE) was fused between complementary non-functional fragments of firefly luciferase and then the rigid linker sequence (LLRAIEAQQHLL), selected by ProDA database, introduced between Nluc and ligand binding domain and compared with that of the flexible linker ((GGGGS)2) in LAIRE chimera. The IP3-insensitive mutant of the biosensor was constructed using the Stratagene QuikChange® procedure. In order to the analysis of the dynamical movements of selected structures in the large-scale, coarse-graining method of molecular dynamics simulation (1µs) was applied.ResultsAs expected, the flexible linker brings two inactive fragments of luciferase together relative to the rigid linker and leads to complementation of luciferase activity, which is detected using luciferin. However, this conformational flexibility in linker increases background to noise ratio and attenuates fold induction.ConclusionsIt seems that the ligand binding properties of IP3 binding core make it more suitable for the design of biosensor than the ligand binding domain.

Dataset Information

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

Publications

Modular bioengineered kinase sensors <i>via</i> scaffold protein-mediated split-luciferase complementation.

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