Identifying a kinase network regulating FGF14: Nav1.6 complex assembly using split-luciferase complementation

Wei Chun Hsu, Miroslav N. Nenov, Alexander Shavkunov, Neli Panova, Ming Zhan, Fernanda Laezza

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Article numbere0117246
JournalPLoS One
Volume10
Issue number2
DOIs
StatePublished - Feb 6 2015

Fingerprint

fibroblast growth factors
luciferase
Luciferases
phosphotransferases (kinases)
Phosphotransferases
protein-protein interactions
Assays
Brain
Proteins
Glycogen Synthase Kinase 3
assays
brain
neurons
Brain Diseases
Neurons
cell cycle
Surveying
NAV1.6 Voltage-Gated Sodium Channel
central nervous system diseases
synaptic transmission

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Medicine(all)

Cite this

Identifying a kinase network regulating FGF14 : Nav1.6 complex assembly using split-luciferase complementation. / Hsu, Wei Chun; Nenov, Miroslav N.; Shavkunov, Alexander; Panova, Neli; Zhan, Ming; Laezza, Fernanda.

In: PLoS One, Vol. 10, No. 2, e0117246, 06.02.2015.

Research output: Contribution to journalArticle

Hsu, Wei Chun ; Nenov, Miroslav N. ; Shavkunov, Alexander ; Panova, Neli ; Zhan, Ming ; Laezza, Fernanda. / Identifying a kinase network regulating FGF14 : Nav1.6 complex assembly using split-luciferase complementation. In: PLoS One. 2015 ; Vol. 10, No. 2.
@article{0d9a13fa08d94ec596f47b38c8d196c0,
title = "Identifying a kinase network regulating FGF14: Nav1.6 complex assembly using split-luciferase complementation",
abstract = "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.",
author = "Hsu, {Wei Chun} and Nenov, {Miroslav N.} and Alexander Shavkunov and Neli Panova and Ming Zhan and Fernanda Laezza",
year = "2015",
month = "2",
day = "6",
doi = "10.1371/journal.pone.0117246",
language = "English (US)",
volume = "10",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "2",

}

TY - JOUR

T1 - Identifying a kinase network regulating FGF14

T2 - Nav1.6 complex assembly using split-luciferase complementation

AU - Hsu, Wei Chun

AU - Nenov, Miroslav N.

AU - Shavkunov, Alexander

AU - Panova, Neli

AU - Zhan, Ming

AU - Laezza, Fernanda

PY - 2015/2/6

Y1 - 2015/2/6

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=84922572433&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84922572433&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0117246

DO - 10.1371/journal.pone.0117246

M3 - Article

C2 - 25659151

AN - SCOPUS:84922572433

VL - 10

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 2

M1 - e0117246

ER -