Whereas binding of activated RhoA to the PH domains

Whereas binding of activated RhoA to the PH domains of the RhoGEFs is proposed to mediate positive feedback regulation (Chen et al., 2010b, Medina et al., 2013), the sequestration of p190RhoGEF by activated Rac1 reveals a novel putative physiological role, that is to utilize Rac1 to directly drive regulation of RhoA. The low affinity for Rac1 may then suggest that this putative regulation requires either very robust action by Rac1 prior to initiating regulation of RhoA, or the action of a partner that facilitates the ability of Rac1 to localize p190RhoGEF. The relatively weak interaction of p190-PH for phosphoinositides (Miller et al., 2013) may offer a mechanism for the latter facilitory role. Prior studies on Dbs, a RhoGEF that activates RhoA, support this hypothesis. The PH domain of this GEF can bind to phosphoinositides (Snyder et al., 2001) and activated Rac1 (Cheng et al., 2004); further, coexpression of activated Rac1 and Dbs in NIH3T3 LDC1267 sale enhanced the ability of overexpressed Dbs to stimulate activation of RhoA and cause foci formation (Cheng et al., 2004). What is the physiological role of Rac binding to p190RhoGEF? Many RhoGEFs play roles in various aspects of cellular motility, such as adhesion, contractility, and tail retraction (Chikumi et al., 2002, Dubash et al., 2007, Francis et al., 2006, Iwanicki et al., 2008, Lim et al., 2008, Miller et al., 2012, Zhai et al., 2003). An interesting feature of p190RhoGEF is its potential contribution to cellular matrix attachment and localization to the focal adhesions involved (Miller et al., 2012). A current model for adhesion predicts a major role for activation of Rac in the formation process with an increasing contribution of RhoA in maturation and strengthening of the focal adhesion (Guilluy et al., 2011, Guo et al., 2006). Binding of p190RhoGEF to focal adhesion kinase (FAK) has been proposed as a mechanism for localization of the GEF to foci (Zhai et al., 2003). The data reported herein suggest that activated Rac could be a recruitment mechanism or synergize with the binding to FAK to keep p190RhoGEF at the focal adhesion and drive effective catalysis of RhoA at the same site. This would then coordinate a switch to the stabilizing action of RhoA, which could then enhance its own function through its positive feedback mechanism. An interesting test of this hypothesis would be the use of p190RhoGEF mutants, where the binding to Rac1 and RhoA were selectively dissected; unfortunately, the residues on the PH domain used for interaction with the GTPases are essentially the same and mutation of core residues eliminates binding to both GTPases (Fig. 1, Fig. 7). Similarly, attempts to exploit differences between residues in the GTPases produced only modest changes (Fig. S8). The physiological importance of the interaction of activated Rac1 and the p190 PH domain may be indicated from an examination of the effect of Rac1 on other Lbc RhoGEFs. The seven members of this family can be split into two groups characterized by their relative homology among the DHPH domains and the absence (p190RhoGEF, p114RhoGEF, AKAP-Lbc, GEFH1) or presence of an RH domain (p115RhoGEF, LARG, PRG) (Medina et al., 2013, Sternweis et al., 2007). All five of the Lbc RhoGEFs that were tested for activation of RhoA in the vesicle assay showed an increased activity in the presence of membrane associated Rac1•GTPγS (Fig. 8). While the extent of activation increases with similarity to p190RhoGEF, the latter clearly stands out, suggesting that this is a specialized function of the RhoGEF. A second indication of specialized function is that the order of potency for binding of RhoA to the same site on Lbc RhoGEFs is completely different; indeed, p190RhoGEF ranks second to PRG in potency for binding activated RhoA (see Fig. 1 in Chen et al. (Submitted for publication)). The RhoGEFs with an RH domain and specialized responsiveness to signaling via the heterotrimeric G12/13 proteins show the least effect of Rac1; this emphasizes the divergence in function of these homologous RhoGEFs. The relatively smaller effects of Rac1 on related Lbc RhoGEFs relative to RhoA raises the question of whether this merely reflects the residual interaction of proteins with highly conserved interacting surfaces, RhoA versus Rac1, with the conserved hydrophobic region of Lbc PH domains. If this is largely true, the ability of Rac1 to interact more effectively with p190RhoGEF may have evolved from this motif to serve a specialized crosstalk function.