Stimulation of phospholipase C-beta2 by the Rho GTPases Cdc42Hs and Rac1.

Neutrophils contain a soluble guanine-nucleotidebinding protein, made up of two components with molecular masses of 23 and 26 kDa, that mediates stimulation of phospholipase C-beta2 (PLCbeta2). We have identified the two components of the stimulatory heterodimer by amino acid sequencing as a Rho GTPase and the Rho guanine nucleotide dissociation inhibitor LyGDI. Using recombinant Rho GTPases and LyGDI, we demonstrate that PLCbeta2 is stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-activated Cdc42HsxLyGDI, but not by RhoAxLyGDI. Stimulation of PLCbeta2, which was also observed for GTP[S]-activated recombinant Rac1, was independent of LyGDI, but required C-terminal processing of Cdc42Hs/Rac1. Cdc42Hs/Rac1 also stimulated PLCbeta2 in a system made up of purified recombinant proteins, suggesting that this function is mediated by direct protein-protein interaction. The Cdc42Hs mutants F37A and Y40C failed to stimulate PLCbeta2, indicating that the Cdc42Hs effector site is involved in this interaction. The results identify PLCbeta2 as a novel effector of the Rho GTPases Cdc42Hs and Rac1, and as the first mammalian effector directly regulated by both heterotrimeric and low-molecular-mass GTP-binding proteins.     

Recognition and activation of Rho GTPases by Vav1 and Vav2 guanine nucleotide exchange factors

Vav proteins are Rho GTPase-specific guanine nucleotide exchange factors (GEFs) that are distinguished by the tandem arrangement of Dbl homology (DH), Pleckstrin homology (PH), and cysteine rich domains (CRD). Whereas the tandem DH-PH arrangement is conserved among Rho GEFs, the presence of the CRD is unique to Vav family members and is required for efficient nucleotide exchange. We provide evidence that Vav2-mediated nucleotide exchange of Rho GTPases follows the Theorell-Chance mechanism in which the Vav2.Rho GTPase complex is the major species during the exchange process and the Vav2.GDP-Mg(2+).Rho GTPase ternary complex is present only transiently. The GTPase specificity for the DH-PH-CRD Vav2 in vitro follows this order: Rac1 > Cdc42 > RhoA. Results obtained from fluorescence anisotropy and NMR chemical shift mapping experiments indicate that the isolated Vav1 CRD is capable of directly associating with Rac1, and residues K116 and S83 that are in the proximity of the P-loop and the guanine base either are part of this binding interface or undergo a conformational change in response to CRD binding. The NMR studies are supported by kinetic measurements on Rac1 mutants S83A, K116A, and K116Q and Vav2 CRD mutant K533A in that these mutants affect both the initial binding event of Vav2 with Rac1 (k(on)) and the rate-limiting dissociation of Vav2 from the Vav2.Rac1 binary complex (thereby influencing the enzyme turnover number, k(cat)). The results suggest that the CRD domain in Vav proteins plays an active role, affecting both the k(on) and the k(cat) for Vav-mediated nucleotide exchange on Rho GTPases.     

Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling.

The small G proteins of the Ras family act as bimodal relays in the transfer of intracellular signals. This is a dynamic phenomenon involving a cascade of protein-protein interactions modulated by chemical modifications, structural rearrangements and intracellular relocalisations. Most of the small G proteins could be operationally defined as proteins having two conformational states, each of which interacts with different cellular partners. These two states are determined by the nature of the bound nucleotide, GDP or GTP. This capacity to cycle between a GDP-bound conformation and a GTP-bound conformation enables them to filter, to amplify or to temporise the upstream signals that they receive. Thus the control of this cycle is crucial. Membrane anchoring of the proteins in the Ras family is a prerequisite for their activity. Most of the proteins in the Rho/Rac and Rab subfamilies of Ras proteins cycle between cytosol and membranes. Then the control of membrane association/dissociation is an other important regulation level. This review will describe one family of crucial regulators acting on proteins in the Rho/Rac family-the Rho guanine nucleotide dissociation inhibitors, or RhoGDIs. As yet, only three RhoGDIs have been described: RhoGDI-1, RhoGDI-2 (or D4/Ly-GDI) and RhoGDI-3. RhoGDI 1 and 2 are cytosolic and participate in the regulation of both the GDP/GTP cycle and the membrane association/dissociation cycle of Rho/Rac proteins. The non-cytosolic RhoGDI-3 seems to act in a slightly different way.     

The role of Mg2+ cofactor in the guanine nucleotide exchange and GTP hydrolysis reactions of Rho family GTP-binding proteins

The biological activities of Rho family GTPases are controlled by their guanine nucleotide binding states in cells. Here we have investigated the role of Mg(2+) cofactor in the guanine nucleotide binding and hydrolysis processes of the Rho family members, Cdc42, Rac1, and RhoA. Differing from Ras and Rab proteins, which require Mg(2+) for GDP and GTP binding, the Rho GTPases bind the nucleotides in the presence or absence of Mg(2+) similarly, with dissociation constants in the submicromolar concentration. The presence of Mg(2+), however, resulted in a marked decrease in the intrinsic dissociation rates of the nucleotides. The catalytic activity of the guanine nucleotide exchange factors (GEFs) appeared to be negatively regulated by free Mg(2+), and GEF binding to Rho GTPase resulted in a 10-fold decrease in affinity for Mg(2+), suggesting that one role of GEF is to displace bound Mg(2+) from the Rho proteins. The GDP dissociation rates of the GTPases could be further stimulated by GEF upon removal of bound Mg(2+), indicating that the GEF-catalyzed nucleotide exchange involves a Mg(2+)-independent as well as a Mg(2+)-dependent mechanism. Although Mg(2+) is not absolutely required for GTP hydrolysis by the Rho GTPases, the divalent ion apparently participates in the GTPase reaction, since the intrinsic GTP hydrolysis rates were enhanced 4-10-fold upon binding to Mg(2+), and k(cat) values of the Rho GTPase-activating protein (RhoGAP)-catalyzed reactions were significantly increased when Mg(2+) was present. Furthermore, the p50RhoGAP specificity for Cdc42 was lost in the absence of Mg(2+) cofactor. These studies directly demonstrate a role of Mg(2+) in regulating the kinetics of nucleotide binding and hydrolysis and in the GEF- and GAP-catalyzed reactions of Rho family GTPases. The results suggest that GEF facilitates nucleotide exchange by destabilizing both bound nucleotide and Mg(2+), whereas RhoGAP utilizes the Mg(2+) cofactor to achieve high catalytic efficiency and specificity.     

Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1

BACKGROUND: Members of the Rho family of small GTP-binding proteins, such as Rho, Rac and Cdc42, have a role in a wide range of cell responses. These proteins function as molecular switches by virtue of a conformational change between the GTP-bound (active) and GDP-bound (inactive) forms. In addition, most members of the Rho and Rac subfamilies cycle between the cytosol and membrane. The cytosolic guanine nucleotide dissociation inhibitors, RhoGDIs, regulate both the GDP/GTP exchange cycle and the membrane association/dissociation cycle. RESULTS: We have used NMR spectroscopy and site-directed mutagenesis to identify the regions of human RhoGDI-1 that are involved in binding Rac-1. The results emphasise the importance of the flexible regions of both proteins in the interaction. At least one specific region (residues 46-57) of the flexible N-terminal domain of RhoGDI, which has a tendency to form an amphipathic helix in the free protein, makes a major contribution to the binding energy of the complex. In addition, the primary site of Rac-1 binding on the folded domain of RhoGDI involves the beta4-beta5 and beta6-beta7 loops, with a slight movement of the 3(10) helix accompanying the interaction. This binding site is on the same face of the protein as the binding site for the isoprenyl group of post-translationally modified Rac-1, but is distinct from this site. CONCLUSIONS: Isoprenylated Rac-1 appears to interact with three distinct sites on RhoGDI. The isoprenyl group attached to the C terminus of Rac-1 binds in a pocket in the folded domain of RhoGDI. This is distinct from the major site on this domain occupied by Rac-1 itself, which involves two loops at the opposite end to the isoprenyl-binding site. It is probable that the flexible C-terminal region of Rac-1 extends from the site at which Rac-1 contacts the folded domain of RhoGDI to allow the isoprenyl group to bind in the pocket at the other end of the RhoGDI molecule. Finally, the flexible N terminus of RhoGDI-1, and particularly residues 48-58, makes a specific interaction with Rac-1 which contributes substantially to the binding affinity.     

The Phosphatidylinositol (3,4,5)-Trisphosphate-dependent Rac Exchanger 1·Ras-related C3 Botulinum Toxin Substrate 1 (P-Rex1·Rac1) Complex Reveals the Basis of Rac1 Activation in Breast Cancer Cells

The P-Rex (phosphatidylinositol (3,4,5)-trisphosphate (PIP₃)-dependent Rac exchanger) family (P-Rex1 and P-Rex2) of the Rho guanine nucleotide exchange factors (Rho GEFs) activate Rac GTPases to regulate cell migration, invasion, and metastasis in several human cancers. The family is unique among Rho GEFs, as their activity is regulated by the synergistic binding of PIP₃ and Gβγ at the plasma membrane. However, the molecular mechanism of this family of multi-domain proteins remains unclear. We report the 1.95 Å crystal structure of the catalytic P-Rex1 DH-PH tandem domain in complex with its cognate GTPase, Rac1 (Ras-related C3 botulinum toxin substrate-1). Mutations in the P-Rex1·Rac1 interface revealed a critical role for this complex in signaling downstream of receptor tyrosine kinases and G protein-coupled receptors. The structural data indicated that the PIP₃/Gβγ binding sites are on the opposite surface and markedly removed from the Rac1 interface, supporting a model whereby P-Rex1 binding to PIP₃ and/or Gβγ releases inhibitory C-terminal domains to expose the Rac1 binding site.     

RhoGDIs revisited: novel roles in Rho regulation

Small GTP-binding proteins of the Rho/Rac/Cdc42 family combine their GDP/GTP cycle, regulated by guanine nucleotide-exchange factors and GTPase-activating proteins, to a cytosol/membrane cycle, regulated by guanine nucleotide dissociation inhibitors (rhoGDIs). RhoGDIs are endowed with dual functions in the cytosol where they form soluble complexes with geranylgeranylated GDP-bound Rho proteins and at membrane interfaces where they monitor the delivery and extraction of Rho proteins to/from their site of action. They have little diversity compared with other Rho protein regulators and therefore have been regarded mostly as housekeeping regulators that distribute Rho proteins equally to any membranes. Recently, acquired data show that rhoGDIs, by interacting with candidate receptors/displacement factors or by phosphorylation, may in fact have active contributions to targeting Rho proteins to specific subcellular membranes and signaling pathways. In addition, the GDP/GTP and membrane/cytosol cycles can be uncoupled in certain cases, with Rho proteins either escaping the membrane/cytosol cycle or being regulated by rhoGDIs in their GTP-bound form. Here, we survey recent structure-function relationships and cellular studies on rhoGDIs and revisit their classical housekeeping role into novel and more specific functions. We also review their involvement in diseases.     

Nuclear translocation of cleaved LyGDI dissociated from Rho and Rac during Trp53-dependent ionizing radiation-induced apoptosis of thymus cells in vitro

LyGDI inhibits the dissociation of GDP from Rho family GTPases and is found in abundance in hematopoietic cells. Here we report truncation of LyGDI after irradiation in mouse 3SB thymus cells. A 21-kDa fragment of LyGDI, resulting from activated caspase 3-induced cleavage at an N-terminal consensus site following the Asp(18) residue, accumulated at peak quantities between 5 and 12 h after irradiation. Cleavage of LyGDI was inhibited by the caspase inhibitor benzoyloxycarbonyl-Val-Asp-fluoromethylketone. Subcellular fractionation and immunofluorescence revealed the truncated 21-kDa fragment of LyGDI within the nuclear fraction of irradiated 3SB cells, whereas full-length LyGDI was found only in the cytoplasmic fraction. Truncated LyGDI within the nucleus had no association with the Rho family proteins RhoA and Rac1, since these proteins were observed only in the cytoplasmic fractions. These data demonstrate that regulation of Rho family GTPases by LyGDI is disrupted during apoptosis, suggesting that fragmentation of LyGDI implicates the transmission of a signal from the cytoplasm to the nucleus during Trp53-dependent apoptosis of thymus cells after irradiation.     

Liposome Reconstitution and Modulation of Recombinant Prenylated Human Rac1 by GEFs,GDI1 and Pak1

Small Rho GTPases are well known to regulate a variety of cellular processes by acting as molecular switches. The regulatory function of Rho GTPases is critically dependent on their posttranslational modification at the carboxyl terminus by isoprenylation and association with proper cellular membranes. Despite numerous studies, the mechanisms of recycling and functional integration of Rho GTPases at the biological membranes are largely unclear. In this study, prenylated human Rac1, a prominent member of the Rho family, was purified in large amount from baculovirus-infected Spodoptera frugiperda insect cells using a systematic detergent screening. In contrast to non-prenylated human Rac1 purified from Escherichia coli, prenylated Rac1 from insect cells was able to associate with synthetic liposomes and to bind Rho-specific guanine nucleotide dissociation inhibitor 1 (GDI1). Subsequent liposome reconstitution experiments revealed that GDI1 efficiently extracts Rac1 from liposomes preferentially in the inactive GDP-bound state. The extraction was prevented when Rac1 was activated to its GTP-bound state by Rac-specific guanine nucleotide exchange factors (GEFs), such as Vav2, Dbl, Tiam1, P-Rex1 and TrioN, and bound by the downstream effector Pak1. We found that dissociation of Rac1-GDP from its complex with GDI1 strongly correlated with two distinct activities of especially Dbl and Tiam1, including liposome association and the GDP/GTP exchange. Taken together, our results provided first detailed insights into the advantages of the in vitro liposome-based reconstitution system to study both the integration of the signal transducing protein complexes and the mechanisms of regulation and signaling of small GTPases at biological membranes.     

Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1

The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 Å resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1's ability to contact a range of Rho GTPases using a novel protein-protein interaction network.     

A crystallographic view of interactions between Dbs and Cdc42: PH domain-assisted guanine nucleotide exchange

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho guanosine triphosphatases (GTPases) and invariably possess tandem Dbl (DH) and pleckstrin homology (PH) domains. While it is known that the DH domain is the principal catalytic subunit, recent biochemical data indicate that for some Dbl-family proteins, such as Dbs and Trio, PH domains may cooperate with their associated DH domains in promoting guanine nucleotide exchange of Rho GTPases. In order to gain an understanding of the involvement of these PH domains in guanine nucleotide exchange, we have determined the crystal structure of a DH/PH fragment from Dbs in complex with Cdc42. The complex features the PH domain in a unique conformation distinct from the PH domains in the related structures of Sos1 and Tiam1.Rac1. Consequently, the Dbs PH domain participates with the DH domain in binding Cdc42, primarily through a set of interactions involving switch 2 of the GTPase. Comparative sequence analysis suggests that a subset of Dbl-family proteins will utilize their PH domains similarly to Dbs.     

Cholecystokinin-Mediated RhoGDI Phosphorylation via PKCα Promotes both RhoA and Rac1 Signaling

RhoA and Rac1 have been implicated in the mechanism of CCK-induced amylase secretion from pancreatic acini. In all cell types studied to date, inactive Rho GTPases are present in the cytosol bound to the guanine nucleotide dissociation inhibitor RhoGDI. Here, we identified the switch mechanism regulating RhoGDI1-Rho GTPase dissociation and RhoA translocation upon CCK stimulation in pancreatic acini. We found that both Gα13 and PKC, independently, regulate CCK-induced RhoA translocation and that the PKC isoform involved is PKCα. Both RhoGDI1 and RhoGDI3, but not RhoGDI2, are expressed in pancreatic acini. Cytosolic RhoA and Rac1 are associated with RhoGDI1, and CCK-stimulated PKCα activation releases the complex. Overexpression of RhoGDI1, by binding RhoA, inhibits its activation, and thereby, CCK-induced apical amylase secretion. RhoA translocation is also inhibited by RhoGDI1. Inactive Rac1 influences CCK-induced RhoA activation by preventing RhoGDI1 from binding RhoA. By mutational analysis we found that CCK-induced PKCα phosphorylation on RhoGDI1 at Ser96 releases RhoA and Rac1 from RhoGDI1 to facilitate Rho GTPases signaling.     

Structural basis for the reversible activation of a Rho protein by the bacterial toxin SopE

The bacterial enteropathogen Salmonella typhimurium employs a type III secretion system to inject bacterial toxins into the host cell cytosol. These toxins transiently activate Rho family GTP-binding protein-dependent signaling cascades to induce cytoskeletal rearrangements. One of these translocated Salmonella toxins, SopE, can activate Cdc42 in a Dbl-like fashion despite its lack of sequence similarity to Dbl-like proteins, the Rho-specific eukaryotic guanine nucleotide exchange factors. To elucidate the mechanism of SopE-mediated guanine nucleotide exchange, we have analyzed the structure of the complex between a catalytic fragment of SopE and Cdc42. SopE binds to and locks the switch I and switch II regions of Cdc42 in a conformation that promotes guanine nucleotide release. This conformation is strikingly similar to that of Rac1 in complex with the eukaryotic Dbl-like exchange factor Tiam1. However, the catalytic domain of SopE has an entirely different architecture from that of Tiam1 and interacts with the switch regions via different amino acids. Therefore, SopE represents the first example of a non-Dbl-like protein capable of inducing guanine nucleotide exchange in Rho family proteins.     

A novel role for RhoGDI as an inhibitor of GAP proteins.

RhoGDI inhibits guanine nucleotide dissociation from post-translationally processed Rho and Rac proteins but its biochemical role in vivo is unknown. We show here that N-terminal effector site mutations in the Rac protein do not compromise its interaction with RhoGDI and that, whilst geranylgeranylation and -AAX proteolysis of the C-terminal CAAX motif of Rac1 and RhoA are required for efficient interaction with RhoGDI, methylesterification of the C-terminal cysteine residue is not required. In vitro, RhoGDI can form stable complexes with Rho and Rac proteins in both the GTP and GDP bound states. Furthermore the Rac-GTP--RhoGDI complex is resistent to the action of recombinant RhoGAP and recombinant BCR. Thus GDI, by complexing with Rac-GTP and preventing GAP stimulated GTP hydrolysis, may allow transit of the activated form of the Rac protein between physically separated activator and effector proteins in the cell.     

Thermodynamics of Ras/effector and Cdc42/effector interactions probed by isothermal titration calorimetry

Proliferation, differentiation, and morphology of eucaryotic cells is regulated by a large network of signaling molecules. Among the major players are members of the Ras and Rho/Rac subfamilies of small GTPases that bind to different sets of effector proteins. Recognition of multiple effectors is important for communicating signals into different pathways, leading to the question of how an individual GTPase achieves tight binding to diverse targets. To understand the observed specificity, detailed information about binding energetics is expected to complement the information gained from the three-dimensional structures of GTPase/effector protein complexes. Here, the thermodynamics of the interaction of four closely related members of the Ras subfamily with four different effectors and, additionally, the more distantly related Cdc42/WASP couple were quantified by means of isothermal titration calorimetry. The heat capacity changes upon complex formation were rationalized in light of the GTPase/effector complex structures. Changes in enthalpy, entropy, and heat capacity of association with various Ras proteins are similar for the same effector. In contrast, although the structures of the Ras-binding domains are similar, the thermodynamics of the Ras/Raf and Ras/Ral guanine nucleotide dissociation stimulator interactions are quite different. The energy profile of the Cdc42/WASP interaction is similar to Ras/Ral guanine nucleotide dissociation stimulator, despite largely different structures and interface areas of the complexes. Water molecules in the interface cannot fully account for the observed discrepancy but may explain the large range of Ras/effector binding specificity. The differences in the thermodynamic parameters, particularly the entropy changes, could help in the design of effector-specific inhibitors that selectively block a single pathway.     

Dissociation of GDP dissociation inhibitor and membrane translocation are required for efficient activation of Rac by the Dbl homology-pleckstrin homology region of Tiam

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.     

RHO GTPase Signaling for Axon Extension: Is Prenylation Important?

Many lines of evidence indicate the importance of the Rho family guanine nucleotide triphosphatases (GTPases) in directing axon extension and guidance. The signaling networks that involve these proteins regulate actin cytoskeletal dynamics in navigating neuronal growth cones. However, the intricate patterns that regulate Rho GTPase activation and signaling are not yet fully defined. Activity and subcellular localization of the Rho GTPases are regulated by post-translational modification. The addition of a geranylgeranyl group to the carboxy (C-) terminus targets Rho GTPases to the plasma membrane and promotes their activation by facilitating interaction with guanine nucleotide exchange factors and allowing sequestering by association with guanine dissociation inhibitors. However, it is unclear how these modifications affect neurite extension or how subcellular localization alters signaling from the classical Rho GTPases (RhoA, Rac1, and Cdc42). Here, we review recent data addressing this issue and propose that Rho GTPase geranylgeranylation regulates outgrowth.     

Disruption of RhoGDI and RhoA regulation by a Rac1 specificity switch mutant

Rho family GTPases are important regulators of the actin cytoskeleton. Activation of these proteins can be promoted by guanine nucleotide exchange factors containing Dbl and Pleckstrin homology domains resulting in membrane insertion of a Rho family member, whereas the inactive GDP-bound form is sequestered primarily in the cytoplasm, bound to the guanosine dissociation inhibitor RhoGDI. Dominant interfering variants of Rac1, but not Cdc42, inhibit beta1 integrin-promoted uptake of Yersinia pseudotuberculosis. Unexpectedly, we found that the Rac1(W56F) guanine nucleotide exchange factors specificity switch mutant blocked invasin-promoted uptake as well as Cdc42-dependent uptake of enteropathogenic Escherichia coli. Fluorescence resonance energy transfer experiments demonstrated that Rac1(W56F) retained the ability to be loaded with GTP, bind a downstream effector, and interact with RhoGDI. Mutational analyses of intragenic suppressors and coexpression studies demonstrated that binding of the Rac1(W56F) mutant to RhoGDI appeared to play a role in the inhibition of uptake. As RhoGDI inhibits RhoA, overactivation of RhoA may account for the uptake interference caused by Rac1(W56F). Consistent with this model, a dominant interfering form of RhoA restored significant uptake in the presence of the Rac1(W56F) mutant but had no effect on another interfering Rac1 form. Furthermore, the cellular GTP-RhoA level was elevated by the presence of Rac1(W56F) mutant protein. These data are consistent with the proposition that Rac1(W56F) blocks invasin-promoted uptake by preventing RhoGDI from inactivating RhoA. We conclude that RhoGDI allows cross-talk between Rho family members that promote potentially antagonistic processes, and disruption of this cross-talk can interfere with invasin-promoted uptake.     

Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling.

The small GTPases Rho, Rac, and Cdc42 are monoglucosylated at effector domain amino acid threonine 37/35 by Clostridium difficile toxins A and B. Glucosylation renders the Rho proteins inactive by inhibiting effector coupling. To understand the functional consequences, effects of glucosylation on subcellular distribution and cycling of Rho GTPases between cytosol and membranes were analyzed. In intact cells and in cell lysates, glucosylation leads to a translocation of the majority of RhoA GTPase to the membranes whereas a minor fraction is monomeric in the cytosol without being complexed with the guanine nucleotide dissociation inhibitor (GDI-1). Rho complexed with GDI-1 is not substrate for glucosylation, and modified Rho does not bind to GDI-1. However, a membranous factor inducing release of Rho from the GDI complex makes cytosolic Rho available as a substrate for glucosylation. The binding of glucosylated RhoA to the plasma membranes is saturable, competable with unmodified Rho-GTPgammaS guanosine 5'-O-(3-thiotriphosphate), and takes place at a membrane protein with a molecular mass of about 70 kDa. Membrane-bound glucosylated Rho is not extractable by GDI-1 as unmodified Rho is, leading to accumulation of modified Rho at membranous binding sites. Thus, in addition to effector coupling inhibition, glucosylation also inhibits Rho cycling between cytosol and membranes, a prerequisite for Rho activation.     

Mechanism of nucleotide release from Rho by the GDP dissociation stimulator protein

Guanine nucleotide dissociation stimulator (GDS) promotes the release of tightly bound GDP from various Ras superfamily proteins, including RhoA, Rac1, K-Ras, Rap1A, and Rap1B. It displays no significant sequence homology to other known exchange factors for small G-proteins. Studies are reported here of the mechanism of GDS-mediated nucleotide release from RhoA using a combination of equilibrium and stopped-flow kinetic measurements, employing fluorescent N-methylanthraniloyl (mant) derivatives of GDP and 2'-deoxyGDP. It is proposed that GDS operates by an associative displacement mechanism where stimulated nucleotide release from the Rho.mantGDP complex occurs via a transiently populated ternary complex (Rho.GDS.mantGDP). In kinetic experiments where excess GDS was mixed with the Rho.mantGDP complex, stimulated mantGDP dissociation rates of 1 s(-)(1) were measured during a single turnover, representing a 5000-fold enhancement over the intrinsic rate of mantGDP dissociation from Rho. The stable, nucleotide-free binary complex Rho.GDS was isolated. When the Rho.GDS complex was mixed with excess mantGDP, a biphasic increase in fluorescence occurred, the observed rate constants of which both reached saturating values at high mantGDP concentrations. This is compelling evidence that an isomerization of the Rho.GDS.mantGDP ternary complex is an important feature of the mechanism of nucleotide release.