Dissociation of GDP Dissociation Inhibitor and Membrane Translocation Are Required for Efficient Activation of Rac by the Dbl Homology-Pleckstrin Homology Region of Tiam

Abstract

Small G proteins of the Rho/Rac/Cdc42 family are associated with lipid membranes through their prenylated C termini. Alternatively, these proteins form soluble complexes with GDI proteins. To assess how this membrane partitioning influences the activation of Rac by guanine nucleotide exchange factors, GDP-to-GTP exchange reactions were performed in the presence of liposomes using different forms of Rac-GDP. We show that both non-prenylated Rac-GDP and the soluble complex between prenylated Rac-GDP and GDI are poorly activated by the Dbl homology-pleckstrin homology (DH-PH) domain of the exchange factor Tiam1, whereas prenylated Rac-GDP bound to liposomes is activated about 10 times more rapidly. Sedimentation experiments with liposomes reveal that the DH-PH region of Tiam1 forms, with nucleotide-free prenylated Rac, a membrane-bound complex from which GDI is excluded. Taken together, these experiments demonstrate that the dissociation of Rac-GDP from GDI and its translocation to membrane lipids favor DH-PH-catalyzed nucleotide exchange because the steric hindrance caused by GDI is relieved and because the membrane environment favors functional interaction between the DH-PH domain and the small G protein.Small G proteins of the Rho family, including Rho, Rac, and Cdc42p, undergo two interdependent cycles. First, they cycle between inactive (GDP) and active (GTP) conformations through the catalytic action of guanine exchange factors (GEF) 1 and GTPase-activating proteins. Second, they cycle between cytosolic and membrane-associated forms through the action of GDI proteins (1-3). How these two cycles are coupled is critical for the proper interaction of Rho proteins with their targets. It is generally assumed that Rho proteins must be in the GTP conformation and associated to membranes to trigger a cellular response.Dbl homology (DH) domains catalyze the exchange of guanine nucleotides on small G proteins of the Rho family (4, 5). The DH domain is an all-␣-helix fold that is systematically followed by a pleckstrin homology (PH) domain. The DH-PH tandem is thus the hallmark of Rho GEFs, which otherwise display variable domain composition and organization. Recent structural and mutagenesis studies on DH-PH domains, either isolated or in complex with Rho proteins, have given insights into the mechanism by which these domains promote the release of the bound nucleotide (6 -10). One of the best-studied examples is Tiam, a GEF for Rac. The DH domain of Tiam makes extensive contacts with the switch I and II regions of Rac and modifies the magnesium, sugar, and guanine basebinding regions to destabilize the bound nucleotide (9). In addition, structure-based mutagenesis studies show that the specificity between functional GEF/Rho pairs relies on a few residues at the DH/switch II interface (11-13). Binding to switch I and II regions is a general property of GEFs for small G proteins (14).Despite these spectacular progresses, one aspect of the activation of Rho proteins by DH domains remains obscure: how does the nucleotide ...

In Vivo Dynamics of Rac-Membrane Interactions

“…However, Dbl-catalyzed nucleotide exchange is inhibited by RhoGDI in the absence of membranes (Yaku et al, 1994). Additionally, membrane-bound Rac is a better substrate for Tiam1 than soluble Rac (Robbe et al, 2003). These results suggest that partial or complete dissociation of the Rho GTPase/ RhoGDI complex precedes and is required for activation by GEFs.…”

S6K1 and mTOR regulate Rac1-driven platelet activation and aggregation

“…Current models of TIAM function hypothesize that TIAM1 recruits and activates Rac1 at the cytosolic surface of the plasma membrane. 33 As platelet activation increases the association of TIAM1 with Rac1 and phosphorylated S6K1 ( Figure 2D), TIAM1 may recruit a Rac1-S6K1 complex to the plasma membrane to help drive lamellipodia formation. In support of this model, confocal microscopy revealed that TIAM1 colocalized with Rac1 at the lamellipodial edge of activated platelets ( Figure 2E).…”

“…As seen in Figure 2C, Rac1-GST captured S6K1 from platelet lysates while GST alone failed to interact with S6K1. Previous work has established that Rac1 activity in tumor cells is regulated in part by TIAM1, 33 a Rac guanine nucleotide exchange factor (GEF) that is known to interact with S6K1. 34 To determine whether S6K1 and Rac1 are associated with TIAM1 in platelets, we next immunoprecipitated TIAM1 from platelet lysates with sc-872, an antibody that specifically captures TIAM1 from platelet lysates (supplemental Figure 3).…”

cAMP-Binding Protein Epac Induces Cardiomyocyte Hypertrophy

“…RhoGDI retains Rac into the cytoplasm and must dissociate to allow Rac to encounter its GEFs. 32,33 Recently, Price et al 34 have shown that Ca 2ϩ induces a disruption of the RacRhoGDI complex leading to the translocation and activation of Rac in PC3 cells. Thus, one could speculate that such a mechanism might occur in cardiomyocytes and contribute to Epac-induced Ca 2ϩ -dependent Rac activation We report for the first time to our knowledge that Epac is implicated in the activation of NFAT in cardiac myocytes.…”