Positive regulation of Rho GTPase activity by RhoGDIs as a result of their direct interaction with GAPs

Abstract

Background: Rho GTPases function as molecular switches in many different signaling pathways and control a wide range of cellular processes. Rho GDP-dissociation inhibitors (RhoGDIs) regulate Rho GTPase signaling and can function as both negative and positive regulators. The role of RhoGDIs as negative regulators of Rho GTPase signaling has been extensively investigated; however, little is known about how RhoGDIs act as positive regulators. Furthermore, it is unclear how this opposing role of GDIs influences the Rho GTPase cycle. We constructed ordinary differential equation models of the Rho GTPase cycle in which RhoGDIs inhibit the regulatory activities of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) by interacting with them directly as well as by sequestering the Rho GTPases. Using this model, we analyzed the role of RhoGDIs in Rho GTPase signaling.

Results: The model constructed in this study showed that the functions of GEFs and GAPs are integrated into Rho GTPase signaling through the interactions of these regulators with GDIs, and that the negative role of GDIs is to suppress the overall Rho activity by inhibiting GEFs. Furthermore, the positive role of GDIs is to sustain Rho activation by inhibiting GAPs under certain conditions. The interconversion between transient and sustained Rho activation occurs mainly through changes in the affinities of GDIs to GAPs and the concentrations of GAPs.

Conclusions: RhoGDIs positively regulate Rho GTPase signaling primarily by interacting with GAPs and may participate in the switching between transient and sustained signals of the Rho GTPases. These findings enhance our understanding of the physiological roles of RhoGDIs and Rho GTPase signaling.

Figure 1

Representation of the models of Rho GTPase cycle regulation (left) and simulations of their Rho activation dynamics (right). The activation levels of GTPases were defined as the concentration of the GTP-Rho/Effector complex. A) The canonical model of the Rho GTPase cycle in which GDIs inhibit the activities of GEFs and GAPs by sequestering GTPase. B) The GDI-integrated model of the Rho GTPase cycle in which GDIs inhibit the activities of GEFs and GAPs not only by sequestering GTPase but also by interacting with GEFs and GAPs. C) GDI/GEF interaction was removed from the GDI-integrated model. D) GDI/GAP interaction was removed from the GDI-integrated model. All parameters and reactions in the models are shown in Additional file 1: Tables S1 and S2. Reaction numbers (re#) correspond to the reaction numbers in Additional file 1: Table S2.

Figure 2

Free (non-GTPase-complexed) GDI concentration affects the prolongation of Rho activation in the GDI-integrated model. Rho activation dynamics were simulated at various concentration of free GDI. A) 600 min after stimulation in the canonical model. B) 600 min after stimulation in the GDI-integrated model. C) 1,800 min after stimulation in the GDI-integrated model. The activation levels of GTPases were expressed as the concentration of GTP-Rho/Effector complex.

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.

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.