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

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Researchers used a novel assay to identify key kinase pathways regulating neuronal excitability. They found that PI3K/Akt, Wee1 kinase, and PKC modulate the FGF14:Nav1.6 complex, converging on GSK3, which impacts brain function.

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Area of Science:

  • Neuroscience
  • Molecular Biology
  • Biochemistry

Background:

  • Kinases are crucial for brain function, regulating protein:protein interactions (PPI) that influence neuronal excitability and connectivity.
  • Dysregulation of kinase activity is implicated in brain disorders, but the underlying molecular mechanisms are often unclear.
  • New methods are needed to rapidly survey kinase pathways in cellular contexts for better understanding of brain disease.

Purpose of the Study:

  • To apply the luciferase complementation assay (LCA) to investigate kinase pathway regulation of the FGF14:Nav1.6 protein complex in live cells.
  • To identify specific kinase pathways that modulate the interaction between FGF14 and Nav1.6, a key complex in neuronal excitability.
  • To uncover novel signaling nodes, particularly GSK3, involved in regulating neuronal function and potentially implicated in brain disease.

Main Methods:

  • Utilized a bioluminescence-based luciferase complementation assay (LCA) in live cells to study 12 kinase pathways.
  • Assayed kinase regulation of the protein:protein interaction (PPI) complex formed by fibroblast growth factor 14 (FGF14) and voltage-gated sodium channel Nav1.6.
  • Employed dose-dependent validation with kinase inhibitors, hierarchical clustering, and Ingenuity Pathway Analysis.

Main Results:

  • Identified the PI3K/Akt pathway, Wee1 kinase, and protein kinase C (PKC) as key regulators of the FGF14:Nav1.6 complex.
  • Demonstrated that these pathways converge on glycogen synthase kinase 3 (GSK3).
  • Showed that inhibition of GSK3 significantly impairs excitability in hippocampal neurons.

Conclusions:

  • The LCA provides a versatile toolkit for rapid surveying of PPI signaling pathways in the brain.
  • Discovered novel modular pathways centered on GSK3 that regulate neuronal excitability through modulation of the FGF14:Nav1.6 complex.
  • These findings offer new insights into molecular mechanisms underlying normal and diseased brain function, highlighting GSK3 as a potential therapeutic target.