Crosstalk among WEE1 Kinase, AKT, and GSK3 in Nav1.2 Channelosome Regulation

Abstract

The signaling complex around voltage-gated sodium (Nav) channels includes accessory proteins and kinases crucial for regulating neuronal firing. Previous studies showed that one such kinase, WEE1-critical to the cell cycle-selectively modulates Nav1.2 channel activity through the accessory protein fibroblast growth factor 14 (FGF14). Here, we tested whether WEE1 exhibits crosstalk with the AKT/GSK3 kinase pathway for coordinated regulation of FGF14/Nav1.2 channel complex assembly and function. Using the in-cell split luciferase complementation assay (LCA), we found that the WEE1 inhibitor II and GSK3 inhibitor XIII reduce the FGF14/Nav1.2 complex formation, while the AKT inhibitor triciribine increases it. However, combining WEE1 inhibitor II with either one of the other two inhibitors abolished its effect on the FGF14/Nav1.2 complex formation. Whole-cell voltage-clamp recordings of sodium currents (I_Na) in HEK293 cells co-expressing Nav1.2 channels and FGF14-GFP showed that WEE1 inhibitor II significantly suppresses peak I_Na density, both alone and in the presence of triciribine or GSK3 inhibitor XIII, despite the latter inhibitor's opposite effects on I_Na. Additionally, WEE1 inhibitor II slowed the tau of fast inactivation and caused depolarizing shifts in the voltage dependence of activation and inactivation. These phenotypes either prevailed or were additive when combined with triciribine but were outcompeted when both WEE1 inhibitor II and GSK3 inhibitor XIII were present. Concerted regulation by WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII was also observed in long-term inactivation and use dependency of Nav1.2 currents. Overall, these findings suggest a complex role for WEE1 kinase-in concert with the AKT/GSK3 pathway-in regulating the Nav1.2 channelosome.

Keywords: fibroblast growth factors; kinase inhibitors; split luciferase assay; voltage-gated sodium channels.

Figure 1

Evaluation of the effects of kinase inhibitors on the FGF14/Nav1.2 complex assembly using the in-cell split luciferase complementation assay (LCA). (A) Representative bar graph (top) and dose-response graph (bottom) of % luminescence reflecting the FGF14/Nav1.2 complex assembly in response to WEE1 inhibitor II (1–150 µM), (B) the AKT inhibitor (0.5–100 µM), and (C) GSK3 inhibitor XIII (1–150 µM). (D,E) are representative bar graphs (top) and dose responses (bottom) of % luminescence reflecting the FGF14/Nav1.2 complex assembly in response to WEE1 inhibitor II (15 µM) in combination with the indicated inhibitors. Percentage of luminescence (normalized to per plate control wells treated with 0.5% DMSO; n = 8 wells per plate) is plotted as a function of log concentration in the dose–response graphs at the bottom. Data are represented ± SEM. Half maximal inhibitory concentration (IC₅₀) and half maximal effective concentration (EC₅₀) were calculated using a sigmoidal dose–response fitting.

Figure 2

The interplay between WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII in regulating Nav1.2-mediated I_Na. (A) Representative traces of I_Na from HEK293-Nav1.2 cells expressing FGF14-GFP (HEK-Nav1.2/FGF14). Traces were recorded in response to depolarizing voltage steps in the presence of WEE1 inhibitor II (WEE1; 10 µM), triciribine (tri; 10 µM), GSK3 inhibitor XIII (30 µM), and/or DMSO (0.01%); the voltage-clamp stimulating protocol is depicted on the right. (B) Current-voltage (I-V) relationships derived from the experimental groups are described in Panel (A). (C) Bar graphs representing peak current densities and (D) the time constant (tau) of fast inactivation of Nav1.2 channels from the experimental groups are described in Panel (A). Data are presented as mean ± SEM. Statistical significance is indicated as follows: * p < 0.05, ** p < 0.001, *** p < 0.0001, ns = nonsignificant, determined by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6–10). In this figure, GSK3 inhibitor XIII is referred to as XIII.

Figure 3

Synergy and competition between WEE1 inhibitor II, triciribine, and GSK3 inhibitor XIII in regulating Na_v1.2 channel voltage dependence of activation and steady-state inactivation. (A) Normalized conductance plotted as a function of the voltage in HEK-Nav1.2/FGF14 cells treated with 0.1% DMSO or respective kinase inhibitors as shown with color-coded labels. The data were fitted with the Boltzmann function. (B) Bar graph summary of V_1/2 of activation. (C) Voltage dependence of steady-state inactivation. (D) Bar graph summary of V_1/2 of inactivation. Data are presented as mean ± SEM. Statistical significance is indicated as * p < 0.05, ** p < 0.001, *** p < 0.0001, ns = nonsignificant, determined by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6–10). In this figure, GSK3 inhibitor XIII is referred to as XIII.

Figure 4

Crosstalk among WEE1 inhibitor, triciribine, and GSK3 inhibitor XIII on modulation of Na_v1.2 channel long-term inactivation and cumulative inactivation properties. (A) Representative traces of I_Na elicited by HEK-Nav1.2/FGF14 cells from the indicated experimental groups in response to the depicted voltage-clamp protocol to induce long-term inactivation. (B) Long-term inactivation of Nav1.2 measured as channel availability versus depolarization. (C) Comparison of the relative I_Na amplitude at the 1st pulse to the 4th pulse for the indicated experimental groups. (D) Representative traces of I_Na elicited by HEK-Nav1.2/FGF14 cells from the indicated experimental groups in response to the depicted voltage-clamp protocol to induce use-dependent cumulative inactivation. (E) Characterization of cumulative inactivation of Nav1.2 channels induced by use dependency for the experimental groups described in (D). (F) Comparison of the relative I_Na amplitude at the 1st pulse to the 20th pulse for the indicated experimental groups. Data are presented as mean ± SEM. Statistical significance is indicated as * p < 0.05, ** p < 0.001, *** p < 0.0001, ns = nonsignificant, determined by one-way ANOVA followed by Tukey’s multiple comparisons test (n = 6–10). In this figure, GSK3 inhibitor XIII is referred to as XIII.

Figure 5

Putative crosstalk between WEE1 kinase, AKT, and GSK3 in regulating the Nav1.2/FGF14 signalosome. WEE1 kinase and GSK3β have been shown to directly regulate the FGF14/Nav1.2 complex assembly and its functional activity via phosphorylation of FGF14Y158 (1) and FGF14S226 (2), respectively. Additionally, GSK3β directly phosphorylates the Nav1.2 C-terminal tail at T1966 (3). Phosphorylation of FGF14Y158 by WEE1 kinase may increase its assembly with Nav1.2. Similarly, phosphorylation of FGF14S226 or Nav1.2T1966 by GSK3 may enhance the assembly of the FGF14/Nav1.2 complex. Moreover, GSK3 has been shown to degrade WEE1 kinase via ubiquitination (4), leading to a reduction in WEE1 kinase levels. There are no reports of direct phosphorylation of FGF14 or Nav1.2 by AKT. Therefore, AKT may influence the FGF14/Nav1.2 complex assembly and its functional activity indirectly through the suppression of GSK3β via inhibitory phosphorylation (5) or through a synergistic effect with WEE1 kinase (6).