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.