Interaction of GDI with GAP sustains Rho activation
A Rho GTPase switch can be regulated by three classes of regulators: GEFs, GAPs, and GDIs. In the canonical model of the Rho GTPase cycle (Figure 1A, left), GEFs promote GDP/GTP exchange, thereby activating Rho GTPases. In contrast, GAPs promote GTP hydrolysis, thereby inactivating Rho GTPases. GDIs sequester GDP-bound GTPases from GEFs and keep them inactive; however, GDIs can also sequester GTP-bound GTPases from GAPs and keep them active. In this model, the Rho GTPase cycle functions as a simple ON/OFF switch and Rho activation is transiently elevated upon stimulation (Figure 1A, right).
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.
The majority of Rho GTPases exist in biologically inactive cytosolic complexes with GDIs, and the dissociation of GTPases from GDIs is hypothesized to be a prerequisite for activation by GEFs. However, it has been suggested that GDI and Rho GTPase can simultaneously bind GEF or GAP and form a ternary complex (GEF/GDI/Rho GTPase or GAP/GDI/Rho GTPase) [25-27]. According to these observations, we constructed a model of the Rho GTPase cycle (Figure 1B, left) in which GDIs inhibit the activities of GEFs and GAPs by physically interacting with them as well as by sequestering Rho GTPases (see Methods). We designated this model the ‘GDI-integrated model’ because the activation dynamics and ultimate output of GEFs and GAPs are integrated by GDIs to regulate Rho activity. Rho activation is sustained for a longer period of time in this model (Figure 1B, right), compared with the canonical model (Figure 1A, right).
To clarify which interaction of GDIs with GEFs or GAPs participates in this sustained Rho activation, we further modified our GDI-integrated model. When the interaction of GDIs with GEFs was removed (Figure 1C, left), similar Rho activation dynamics, with a two-fold increase in the overall level, were obtained (Figure 1C, right). In contrast, when the interaction of GDIs with GAPs was removed (Figure 1D, left), Rho activation level decreased and was not sustained (Figure 1D, right). These results therefore suggest that GDIs sustain Rho activation through interaction with GAPs.