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

Hsu WC, Nenov MN, Shavkunov A, Panova N, Zhan M, Laezza F - PLoS ONE (2015)

Bottom Line: Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons.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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

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Dose-response studies of identified partial agonists of FGF14:Nav1.6 regulatory pathways.Fitting, pIC50/EC50 calculation, axes as in Fig. 3. Partial agonists were defined as compounds that act as agonists (promote FGF14:Nav1.6 complementation with increasing dose).
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pone.0117246.g005: Dose-response studies of identified partial agonists of FGF14:Nav1.6 regulatory pathways.Fitting, pIC50/EC50 calculation, axes as in Fig. 3. Partial agonists were defined as compounds that act as agonists (promote FGF14:Nav1.6 complementation with increasing dose).

Mentions: Investigating the dose-response characteristics of inhibitors can reveal novel insights into pharmacological mechanisms. Recent investigations into the clinical possibilities of kinase inhibitors and continuing efforts towards drug discovery of better inhibitors underlines modern thinking of kinase inhibitors as both agonists and antagonists of complex cellular pathways [29]. Towards this aim, we have adapted the concept of efficacy, a pharmacological parameter quantifying receptor occupancy by drug molecules [30], as a quantitative way to determine the ability of kinase inhibitors to target the FGF14:Nav complex at various dose concentrations. Additionally, we define an “agonist” of the FGF14:Nav system as a compound that increases the interaction of FGF14 with Nav as a function of increasing dosage, and an “inverse agonist” as a compound that similarly decreases the FGF14:Nav interaction. Dose-dependent experiments were performed with n = 6 biological replicates for up to 7 working concentrations (50 uM, 40 uM, 25 uM, 10 uM, 4 uM, 2.5 uM, 1 uM), and runs for all experiments were combined to produce a dose-response curve for each inhibitor. The dose-response data were subsequently normalized with respect to the maximum peak intensity observed for each compound to determine the degree of agonism/inverse agonism. The dose-response data hint at the heterogeneity of kinase inhibition, and the need to establish effective, working concentrations for further study. The inhibitors Cdk1 inhibitor, NH125, TX-1918, BAY 11–7082 and BAY 11–7085, Syk inhibitor III, and Wee1 inhibitor I produced a dose-response consistent with pharmacological full inverse agonists, with FGF14:Nav interaction almost absent at saturating concentrations and high (e>1) efficacy values (Fig. 3), making them most suitable for studying the dose-dependent inhibition of the FGF14:Nav complex. Conversely, inhibitors such as Ro 3306 and Go 6983 can be classified as partial inverse agonists, with substantial residual FGF14:Nav response at saturating concentrations and low (e<1) efficacy (Fig. 4). This is probably explained through activation of competing or compensatory pathways that may increase FGF14:Nav binding, while other inhibitors such as triciribine and NSC 625987 produce responses consistent with partial agonism of the FGF14:Nav1.6 complex, useful in studying the phenotype of increased FGF14:Nav binding (Fig. 5). In addition, the observed effective IC50 values for each of the inhibitors differs markedly from in vitro reported values (S1 Table), many of which are in the low nanomolar range. These results demonstrate the hazard of relying on in vitro IC50 values to gauge effectiveness in actual cells and tissues, as well as the need to conduct dose-response screenings independent of manufacturer-provided data for a particular biological system of choice.


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

Hsu WC, Nenov MN, Shavkunov A, Panova N, Zhan M, Laezza F - PLoS ONE (2015)

Dose-response studies of identified partial agonists of FGF14:Nav1.6 regulatory pathways.Fitting, pIC50/EC50 calculation, axes as in Fig. 3. Partial agonists were defined as compounds that act as agonists (promote FGF14:Nav1.6 complementation with increasing dose).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4319734&req=5

pone.0117246.g005: Dose-response studies of identified partial agonists of FGF14:Nav1.6 regulatory pathways.Fitting, pIC50/EC50 calculation, axes as in Fig. 3. Partial agonists were defined as compounds that act as agonists (promote FGF14:Nav1.6 complementation with increasing dose).
Mentions: Investigating the dose-response characteristics of inhibitors can reveal novel insights into pharmacological mechanisms. Recent investigations into the clinical possibilities of kinase inhibitors and continuing efforts towards drug discovery of better inhibitors underlines modern thinking of kinase inhibitors as both agonists and antagonists of complex cellular pathways [29]. Towards this aim, we have adapted the concept of efficacy, a pharmacological parameter quantifying receptor occupancy by drug molecules [30], as a quantitative way to determine the ability of kinase inhibitors to target the FGF14:Nav complex at various dose concentrations. Additionally, we define an “agonist” of the FGF14:Nav system as a compound that increases the interaction of FGF14 with Nav as a function of increasing dosage, and an “inverse agonist” as a compound that similarly decreases the FGF14:Nav interaction. Dose-dependent experiments were performed with n = 6 biological replicates for up to 7 working concentrations (50 uM, 40 uM, 25 uM, 10 uM, 4 uM, 2.5 uM, 1 uM), and runs for all experiments were combined to produce a dose-response curve for each inhibitor. The dose-response data were subsequently normalized with respect to the maximum peak intensity observed for each compound to determine the degree of agonism/inverse agonism. The dose-response data hint at the heterogeneity of kinase inhibition, and the need to establish effective, working concentrations for further study. The inhibitors Cdk1 inhibitor, NH125, TX-1918, BAY 11–7082 and BAY 11–7085, Syk inhibitor III, and Wee1 inhibitor I produced a dose-response consistent with pharmacological full inverse agonists, with FGF14:Nav interaction almost absent at saturating concentrations and high (e>1) efficacy values (Fig. 3), making them most suitable for studying the dose-dependent inhibition of the FGF14:Nav complex. Conversely, inhibitors such as Ro 3306 and Go 6983 can be classified as partial inverse agonists, with substantial residual FGF14:Nav response at saturating concentrations and low (e<1) efficacy (Fig. 4). This is probably explained through activation of competing or compensatory pathways that may increase FGF14:Nav binding, while other inhibitors such as triciribine and NSC 625987 produce responses consistent with partial agonism of the FGF14:Nav1.6 complex, useful in studying the phenotype of increased FGF14:Nav binding (Fig. 5). In addition, the observed effective IC50 values for each of the inhibitors differs markedly from in vitro reported values (S1 Table), many of which are in the low nanomolar range. These results demonstrate the hazard of relying on in vitro IC50 values to gauge effectiveness in actual cells and tissues, as well as the need to conduct dose-response screenings independent of manufacturer-provided data for a particular biological system of choice.

Bottom Line: Through complex signaling pathways, kinases regulate the strength of protein:protein interactions (PPI) influencing cell cycle, signal transduction, and electrical activity of neurons.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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

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.

Show MeSH
Related in: MedlinePlus