Background: Fibroblast growth factor 14 (FGF14) binds to and regulates the voltage-gated Na ϩ (Nav) channel.
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.
Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.
G-proteins cycle between an inactive GDP-bound state and active GTP-bound state, serving as molecular switches that coordinate cellular signaling. We recently used phage-display to identify a series of peptides that bind Gα subunits in a nucleotide-dependent manner [Johnston, C. A., Willard, F. S., Jezyk, M. R., Fredericks, Z., Bodor, E. T., Jones, M. B., Blaesius, R., Watts, V. J., Harden, T. K., Sondek, J., Ramer, J. K., and Siderovski, D. P. (2005) Structure 13, 1069-1080]. Here we describe the structural features and functions of KB-1753, a peptide that binds selectively to GDP·AlF 4 − -and GTPγS-bound states of Gα i subunits. KB-1753 blocks interaction of Gα transducin with its effector, cGMP phosphodiesterase, and inhibits transducin-mediated activation of cGMP degradation. Additionally, KB-1753 interferes with RGS protein binding and resultant GAP activity. A fluorescent KB-1753 variant was found to act as a sensor for activated Gα in vitro. The crystal structure of KB-1753 bound to Gα i1 ·GDP·AlF 4 − reveals binding to a conserved hydrophobic groove between switch II and α3 helices, and, along with supporting biochemical data and previous structural analyses, supports the notion that this is the site of effector interactions for Gα i subunits.Heterotrimeric G-proteins serve as critical relays that transmit cues from extracellular stimuli as diverse as neurotransmitters, hormones, photons, and odorants/tastants to intracellular signaling cascades responsible for eliciting specific cellular effects (1,2). In the traditional model of G-protein signaling, cell surface G-protein coupled receptors (GPCRs), upon activation by the aforementioned stimuli, catalyze the exchange of GDP for GTP on the Gα *To whom correspondence should be addressed: UNC Pharmacology, 1106 M.E. Jones Bldg., Chapel Hill, NC 27599-7365. Telephone: 919-843-9363. Fax: 919-966-5640. E-mail: dsiderov@med.unc.edu. Current addresses: Entegrion, 5312 Farrington Rd., Chapel Hill, NC 27517 (J.K.R); Becton Dickinson, 21 Davis Dr., RTP, NC 27709 (R.B.); Amgen Inc., 1201 Amgen Court W., Seattle, WA 98119 (Z.F.). † This work was supported by NIH R01 GM074268 (D.P.S.) and EY12859 (V.Y.A.). C.A.J. was supported by an NIH postdoctoral fellowship (1 F32 GM076944). V.Y.A. is the recipient of the Senior Scientific Investigator Award from Research to Prevent Blindness Inc. Coordinates of the KB-1753/Gα i1 ·GDP·AlF 4 − complex were deposited in the Protein Data Bank (accession code 2G83). 1 Abbreviations: AlF 4 − , aluminum tetrafluoride; CFP, cyan fluorescent protein; cGMP, cyclic guanosine monophosphate; FRET, fluorescence resonance energy transfer; GAP, GTPase-accelerating protein; GDP, guanosine diphosphate; GEF, guanine nucleotide exchange factor; GMP, guanosine monophosphate; GPCR, G protein-coupled receptor; GTP, guanosine triphosphate; PDE, phosphodiesterase; RGS, regulator of G-protein signaling; ROS, rod outer segment; SPR, surface plasmon resonance; YFP, yellow fluorescent protein. subunit. This results in adoption of the active, GTP-bou...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.