Influence of GDI affinity for GEF and GAP and the concentration of GEF and GAP on Rho activation dynamics
Phosphorylation affects the affinity of GDIs for various Rho GTPases [29-33] and affects the function of GEFs [34,35] and GAPs [36-38]. Therefore, phosphorylation may modify the regulation of Rho signaling by GDIs, GEFs and GAPs. To examine how the affinity of GDIs for GEFs (KmGEF/GDI) and GAPs (KmGAP/GDI) affects the ability of GDIs to sustain Rho activation, we simulated the Rho activation dynamics at 0.01, 0.1, and 1.0 μM of KmGEF/GDI and KmGAP/GDI in our model. The decrease of KmGEF/GDI resulted in overall decrease of Rho activation at all the tested concentrations of KmGAP/GDI (Figure 3A). The Rho activation was markedly sustained at 0.01 and 0.1 μM of KmGAP/GDI and decreasing KmGEF/GDI did not negate the sustained Rho activation (Figure 3A). Conversely, as the KmGAP/GDI value became smaller, the Rho activation was sustained to a greater degree at all KmGEF/GDI (Figure 3B). These results indicate that the sustained Rho activation can primarily be attributed to the interaction between GAPs and GDIs, and the higher affinity of GDIs for GAPs promotes sustained Rho activation.
Figure 3  Prolongation of Rho activation in the GDI-integrated model is dependent on K mGAP/GDI and the GAP concentration. Rho activation dynamics were simulated at various K mGEF/GDI values (A), K mGAP/GDI values (B), GEF concentration (C), and GAP concentrations (D) in the GDI-integrated model. The activation levels of GTPases were expressed as the concentration of GTP-Rho/Effector complex.
It was also suggested that the local concentration of GEFs and GAPs defined the modes of Rho GTPase signaling [22]. We examined how the concentration of GEFs and GAPs affected the ability of GDIs to sustain Rho activation. We simulated the Rho activation dynamics at 0.1, 0.3, and 0.9 μM concentrations of GEFs and GAPs in our model. The decrease of GEF concentration resulted in overall decrease of Rho activation at all of the tested GAP concentrations (Figure 3C). The sustained Rho activation was apparent only at 0.1 μM of GAP and the decrease of GEF concentration did not negate this sustained Rho activation (Figure 3C). However, at all of the tested GEF concentrations, as the GAP concentrations became smaller, Rho activation was sustained to a higher degree, and increasing GAP concentration negated this sustained Rho activation (Figure 3D). These results indicate that the sustained Rho activation is dependent on the concentration of GAPs, and a lower GAP concentration sustains Rho activation. Finally, we compared the Rho activation dynamics at 0.01, 0.1, and 1.0 μM KmGAP/GDI under various concentrations of free GDI. A decrease in the KmGAP/GDI value enhanced the prolongation of Rho activation regardless of free GDI concentration (Figure 4A). Surprisingly, at 0.01 μM KmGAP/GDI, Rho activation was sustained for a significant period of time, longer than 12,000 min (8.3 days), after stimulation in the presence of free GDI (Figure 4B). However, the overall levels of Rho activation markedly decreased in association with an increase in free GDI. These results suggest that GDIs enable extremely long-term retention of the activated state of the Rho GTPases.
Figure 4  GDI enables extremely long-term retention of the activation state of Rho GTPases. Simulation of Rho activation dynamics at K mGAP/GDI = 0.01, 01, and 1.0 μM in the presence of various free GDI concentrations (0–2.4 μM) in the GDI-integrated model. A) 600 min after stimulation. B) 12,000 min after stimulation. The activation levels of GTPases were expressed as the concentration of GTP-Rho/Effector complex.