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.
authors: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America; M.D./Ph.D. Combined Degree Program, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors: Wei-Chun
authors: WC
authors: Hsu
authors: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors: Miroslav N
authors: MN
authors: Nenov
authors: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors: Alexander
authors: A
authors: Shavkunov
authors: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors: Neli
authors: N
authors: Panova
authors: Department of Systems Medicine and Bioengineering, Weill Cornell Medical College, Methodist Hospital Research Institute, Houston, Texas, United States of America.
authors: Ming
authors: M
authors: Zhan
authors: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, Texas, United States of America; Mitchell Center for Neurodegenerative Diseases, University of Texas Medical Branch, Galveston, Texas, United States of America; Center for Biomedical Engineering, University of Texas Medical Branch, Galveston, Texas, United States of America; Center for Addiction Research, University of Texas Medical Branch, Galveston, Texas, United States of America.
authors: Fernanda
authors: F
authors: Laezza
mesh_terms: D000818:Animals; D005346:Fibroblast Growth Factors / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test / Q000379:methods; D057809:HEK293 Cells; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D062557:NAV1.6 Voltage-Gated Sodium Channel / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*
chemical_list: D062557:NAV1.6 Voltage-Gated Sodium Channel; D047428:Protein Kinase Inhibitors; C100681:SCN8A protein, human; C507811:Scn8a protein, rat; C112971:fibroblast growth factor 14; D005346:Fibroblast Growth Factors; D008156:Luciferases; D011494:Protein Kinases