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.     

Activation of phospholipase D1 by ADP-ribosylated RhoA

Clostridium botulinum exoenzyme C3 exclusively ADP-ribosylates RhoA, B, and C to inactivate them, resulting in disaggregation of the actin filaments in intact cells. The ADP-ribose resides at Asn-41 in the effector binding region, leading to the notion that ADP-ribosylation inactivates Rho by blocking coupling of Rho to its downstream effectors. In a recombinant system, however, ADP-ribosylated Rho bound to effector proteins such as phospholipase D-1 (PLD1), Rho-kinase (ROK), and rhotekin. The ADP-ribose rather mediated binding of Rho-GDP to PLD1. ADP-ribosylation of Rho-GDP followed by GTP-gamma-S loading resulted in binding but not in PLD activation. On the other hand, ADP-ribosylation of Rho previously activated by binding to GTP-gamma-S resulted in full PLD activation. This finding indicates that ADP-ribosylation seems to prevent GTP-induced change to the active conformation of switch I, the prerequisite of Rho-PLD interaction. In contrast to recombinant systems, ADP-ribosylation in intact cells results in functional inactivation of Rho, indicating other mechanisms of inactivation than blocking effector coupling.     

Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho.

Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation. We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation. In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h. Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h. The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA. Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology. Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min. The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA. The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.     

Effect of the Rho GTPase inhibitor-1 on the entry of dengue serotype 2 virus into EAhy926 cells

Dengue virus (DV) is the most rapidly spreading arbovirus in the world. Our previous studies indicated that Rac1, a kind of Rho GTPase, was related with the increased vascular permeability in DV infection. However, the molecular mechanisms that regulate the activity of the Rac1 pathway during DV infection is not fully understood yet. Recently, Rho-specific guanine nucleotide dissociated inhibitors (Rho GDIs), as a pivotal upstream regulator of Rho GTPase, attract our attention. To identify the role of GDI-1 in DV2 infection, the expression of GDI in Eahy926 cells was detected. Moreover, a GDI-1 down-regulated cell line was constructed to explore the correlation between GDI-1 and Rac1 and to further evaluate the function of GDI in DV life cycle. Our results indicated that DV2 infection could up-regulate GDI-1 expression, and down-regulation of GDI enhanced the activity of Rac1. In addition, down-regulated GDI-1 significantly inhibited all steps of DV2 replication cycle. GDI-1 plays an important role in DV2 infection via negatively regulating the activation of the Rac1-actin pathway. These results not only contribute to our further understanding of the pathogenesis of severe dengue but also provide further insight into the development of antiviral drugs.

     

Functional modification by cholera-toxin-catalyzed ADP-ribosylation of a guanine-nucleotide-binding regulatory protein serving as the substrate of pertussis toxin.

The alpha subunits of Gi (Gi alpha) and Gs (guanine-nucleotide-binding proteins involved in adenylate cyclase inhibition and stimulation, respectively) was ADP-ribosylated by cholera toxin in differentiated HL-60 cell membranes upon stimulation of chemotactic receptors by fMLF (fM, N-formylmethionine). The ADP-ribosylation site of Gi alpha modified by cholera toxin appeared to be different from that modified by pertussis toxin [Iiri, T., Tohkin, M., Morishima, N., Ohoka, Y., Ui, M. & Katada, T. (1989) J. Biol. Chem. 264, 21,394-21,400]. This allowed us to investigate how the two types of ADP-ribosylation influence the function of the signal-coupling protein. The major findings observed in HL-60 cell membranes, where the same Gi alpha molecule was ADP-ribosylated by treatment of the membranes with either toxin, are summarized as follows. (a) More fMLF bound with a high affinity to cholera-toxin-treated membranes than to the control membranes. The high-affinity binding was, however, not observed in pertussis-toxin-treated membranes. (b) Although fMLF stimulated guanine nucleotide binding and GTPase activity in control membranes, stimulation was almost completely abolished in pertussis-toxin-treated membranes. In contrast, fMLF-dependent stimulation of GTPase activity, but not that of guanine nucleotide binding was attenuated in cholera-toxin-treated membranes. (c) Gi alpha, once modified by cholera toxin, still served as a substrate of pertussis-toxin-catalyzed ADP-ribosylation; however, the ADP-ribosylation rate of modified Gi was much lower than that of intact Gi. These results suggested that Gi ADP-ribosylated by cholera toxin was effectively capable of coupling with fMLF receptors, resulting in formation of high-affinity fMLF receptors, and that hydrolysis of GTP bound to the alpha subunit was selectively impaired by its ADP-ribosylation by cholera toxin. Thus, unlike the ADP-ribosylation of Gi by pertussis toxin, cholera-toxin-induced modification would be of great advantage to the interaction of Gi with receptors and effectors that are regulated by the signal-coupling protein. This type of modification might also be a candidate for unidentified G proteins which were less sensitive to pertussis toxin and appeared to be involved in some signal-transduction systems.     

Oncogenic Dbl, Cdc42, and p21-activated kinase form a ternary signaling intermediate through the minimum interactive domains

Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.     

Pseudomonas aeruginosa ExoS ADP-ribosyltransferase inhibits ERM phosphorylation

Pseudomonas aeruginosa causes life-threatening infections in compromised and cystic fibrosis patients. Pathogenesis stems from a number of virulence factors, including four type III translocated cytotoxins: ExoS, ExoT, ExoY and ExoU. ExoS is a bifunctional toxin: the N terminus (amino acids 96-219) encodes a Rho GTPase Activating Protein (GAP) domain. The C terminus (amino acids 234-453) encodes a 14-3-3-dependent ADP-ribosyltransferase domain which transfers ADP-ribose from NAD onto substrates such as the Ras GTPases and vimentin. Ezrin/radixin/moesin (ERM) proteins have recently been identified as high-affinity substrates for ADP-ribosylation by ExoS. Expression of ExoS in HeLa cells led to a loss of phosphorylation of ERM proteins that was dependent upon the expression of ADP-ribosyltransferase activity. MALDI-MS and site-directed mutagenesis studies determined that ExoS ADP-ribosylated moesin at three C-terminal arginines (Arg553, Arg560 and Arg563), which cluster Thr558, the site of phosphorylation by protein kinase C and Rho kinase. ADP-ribosylated-moesin was a poor target for phosphorylation by protein kinase C and Rho kinase, which showed that ADP-ribosylation directly inhibited ERM phosphorylation. Expression of dominant active-moesin inhibited cell rounding elicited by ExoS, indicating that moesin is a physiological target in cultured cells. This is the first demonstration that a bacterial toxin inhibits the phosphorylation of a mammalian protein through ADP-ribosylation. These data explain how the expression of the ADP-ribosylation of ExoS modifies the actin cytoskeleton and indicate that ExoS possesses redundant enzymatic activities to depolymerize the actin cytoskeleton.     

The N-terminal region of GAP regulates cytoskeletal structure and cell adhesion.

Ras GTPase activating protein (GAP) possesses a C-terminal domain that interacts with GTP-bound Ras, and an N-terminal region containing two SH2 domains and an SH3 domain. In addition to its association with Ras, GAP binds stably to autophosphorylated beta PDGF receptors, and to two cytoplasmic phosphoproteins: p62, an RNA binding protein, and p190, which possesses GAP activity towards small guanine nucleotide binding proteins in the Rho/Rac family. To define the region of GAP that mediates these interactions with cellular phosphoproteins, and to investigate the biological significance of these complexes, a truncated GAP polypeptide (GAP-N) containing residues 1-445 was stably expressed in Rat-2 fibroblasts. GAP-N contains the SH2 and SH3 domains, but lacks the Ras GTPase activating domain. Stimulation of cells expressing GAP-N with PDGF induced association of GAP-N with the beta PDGF receptor, and phosphorylation of GAP-N on tyrosine, consistent with the notion that GAP SH2 domains direct binding to the autophosphorylated beta PDGF receptor in vivo. GAP-N bound constitutively to p190 in both serum-deprived and growth factor-stimulated cells. This GAP-N-p190 complex had Rho GAP activity in vitro. The expression of GAP-N in Rat-2 cells correlated with changes in the cytoskeleton and in cell adhesion, typified by the disruption of action stress fibres, a reduction in focal contacts, and an impaired ability to adhere to fibronectin. These results suggest that the N-terminal domain of GAP can direct interactions with cellular phosphoproteins in vivo, and thereby exert an effector function which modulates the cytoskeleton and cell adhesion.(ABSTRACT TRUNCATED AT 250 WORDS)     

GDI-1 phosphorylation switch at serine 96 induces RhoA activation and increased endothelial permeability

We identified the GDI-1-regulated mechanism of RhoA activation from the Rho-GDI-1 complex and its role in mediating increased endothelial permeability. Thrombin stimulation failed to induce RhoA activation and actin stress fiber formation in human pulmonary arterial endothelial cells transduced with full-length GDI-1. Expression of a GDI-1 mutant form (C-GDI) containing the C terminus (aa 69 to 204) also prevented RhoA activation, whereas further deletions failed to alter RhoA activation. We observed that protein kinase Calpha-mediated phosphorylation of the C terminus of GDI-1 at Ser96 reduced the affinity of GDI-1 for RhoA and thereby enabled RhoA activation. Rendering GDI-1 phosphodefective with a Ser96 --> Ala substitution rescued the inhibitory activity of GDI-1 toward RhoA but did not alter the thrombin-induced activation of other Rho GTPases, i.e., Rac1 and Cdc42. Phosphodefective mutant GDI-1 also suppressed myosin light chain phosphorylation, actin stress fiber formation, and the increased endothelial permeability induced by thrombin. In contrast, expressing the phospho-mimicking mutant S96D-GDI-1 protein induced RhoA activity and increased endothelial permeability independently of thrombin stimulation. These results demonstrate the crucial role of the phosphorylation of the C terminus of GDI-1 at S96 in selectively activating RhoA. Inhibiting GDI-1 phosphorylation at S96 is a potential therapeutic target for modulating RhoA activity and thus preventing the increase in endothelial permeability associated with vascular inflammation.     

Differential expression of Rho1GTPase and Rho3GTPase during isotropic and polarized growth of Mucor circinelloides

Evidence has been obtained that indicates the presence of small 22 kDa GTP-binding Rho proteins through ADP-ribosylation by Clostridium botulinum C3 exotoxin in Mucor circinelloides. Rho protein was detected at all stages of growth studied. During polarized growth, both under aerobic conditions and during the yeast-mycelia transition, the radiolabeling of the [32P]ADP-ribosylated protein increased when tube formation occurred and decreased as the hyphae branched. However, when Mucor grew isotropically, the Rho protein band was thick and its intensity did not vary significantly even after bud formation and separation of daughter cells. Crude extracts of yeast and mycelial cells exhibited a broad 22 kDa band of the [32P]ADP-ribosylated Rho protein that was resolved into a protein with a pI of 6.0, after two-dimensional electrophoresis, corresponding to the Rho1p homolog. Furthermore, [32P]ADP-ribosylated Rho protein from soluble and particulate extracts of multipolarized mycelial cells obtained from the yeast-mycelia transition was separated into two proteins with pI of 6.0 and 6.4, respectively, after two-dimensional electrophoresis. These correspond to the Rho1p and Rho3p homologs, respectively. Therefore, our results show that an increase in Rho accumulation is associated with polarized growth.     

Interaction of the Rho-ADP-ribosylating C3 exoenzyme with RalA

RhoA, -B, and -C are ADP-ribosylated and biologically inactivated by Clostridium botulinum C3 exoenzyme and related C3-like transferases. We report that RalA GTPase, which is not ADP-ribosylated by C3, inhibits ADP-ribosylation of RhoA by C3 from C. botulinum (C3bot), Clostridium limosum (C3lim), and Bacillus cereus (C3cer) but not from Staphylococcus aureus (C3stau) in human platelet membranes and rat brain lysate. Inhibition by RalA occurs with the GDP- and guanosine 5'-3-O-(thio)triphosphate-bound forms of RalA and is overcome by increasing concentrations of C3. A direct interaction of RalA with C3 was verified by precipitation of the transferase with GST-RalA-Sepharose. The affinity constant (K(d)) of the binding of RalA to C3lim was 12 nm as determined by fluorescence titration. RalA increased the NAD glycohydrolase activity of C3bot by about 5-fold. Although RalA had no effect on glucosylation of Rho GTPases by Clostridium difficile toxin B, C3bot and C3lim inhibited glucosylation of RalA by Clostridium sordellii lethal toxin. Furthermore, C3bot decreased activation of phospholipase D by RalA. The data indicate that several C3 exoenzymes directly interact with RalA without ADP-ribosylating the GTPase. The interaction is of high affinity and interferes with essential functions of C3 and RalA.     

Inhibition of the GTPase activity of transducin by an NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes.

The bacterial toxins, choleragen and pertussis toxin, inhibit the light-stimulated GTPase activity of bovine retinal rod outer segments by catalysing the ADP-ribosylation of the alpha-subunit (T alpha) of transducin [Abood, Hurley, Pappone, Bourne & Stryer (1982) J. Biol. Chem. 257, 10540-10543; Van Dop, Yamanaka, Steinberg, Sekura, Manclark, Stryer & Bourne (1984) J. Biol. Chem. 259, 23-26]. Incubation of retinal rod outer segments with NAD+ and a purified NAD+:arginine ADP-ribosyltransferase from turkey erythrocytes resulted in approx. 60% inhibition of GTPase activity. Inhibition was dependent on both enzyme and NAD+, and was potentiated by the non-hydrolysable GTP analogues guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) and guanosine 5'-[beta gamma-methylene]triphosphate (p[CH2]ppG). The transferase ADP-ribosylated both the T alpha and T beta subunits of purified transducin. T alpha (39 kDa), after ADP-ribosylation, migrated as two distinct peptides with molecular masses of 42 kDa and 46 kDa on SDS/polyacrylamide-gel electrophoresis. T beta (36 kDa), after ADP-ribosylation, migrated as a 38 kDa peptide. With purified transducin subunits, it was observed that the GTPase activity of ADP-ribosylated T alpha, reconstituted with unmodified T beta gamma and photolysed rhodopsin, was decreased by 80%; conversely, reconstitution of T alpha with ADP-ribosyl-T beta gamma resulted in only a 19% inhibition of GTPase. Thus ADP-ribosylation of T alpha, the transducin subunit that contains the guanine nucleotide-binding site, has more dramatic effects on GTPase activity than does modification of the critical 'helper subunits' T beta gamma. To elucidate the mechanism of GTPase inhibition by transferase, we studied the effect of ADP-ribosylation on p[NH]pp[3H]G binding to transducin. It was shown previously that modification of transducin by choleragen, which like transferase ADP-ribosylates arginine residues, did not affect guanine nucleotide binding. ADP-ribosylation by the transferase, however, decreased p[NH]pp[3H]G binding, consistent with the hypothesis that choleragen and transferase inhibit GTPase by different mechanisms.     

Structure of Salmonella effector protein SopB N-terminal domain in complex with host Rho GTPase Cdc42

SopB is a type III secreted Salmonella effector protein with phosphoinositide phosphatase activity and a distinct GTPase binding domain. The latter interacts with host Cdc42, an essential Rho GTPase that regulates critical events in eukaryotic cytoskeleton organization and membrane trafficking. Structural and biochemical analysis of the SopB GTPase binding domain in complex with Cdc42 shows for the first time that SopB structurally and functionally mimics a host guanine nucleotide dissociation inhibitor (GDI) by contacting key residues in the regulatory switch regions of Cdc42 and slowing Cdc42 nucleotide exchange.     

Ezrin/radixin/moesin proteins are high affinity targets for ADP-ribosylation by Pseudomonas aeruginosa ExoS

Pseudomonas aeruginosa ExoS is a bifunctional type III-secreted cytotoxin. The N terminus (amino acids 96-233) encodes a GTPase-activating protein activity, whereas the C terminus (amino acids 234-453) encodes a factor-activating ExoS-dependent ADP-ribosyltransferase activity. The GTPase-activating protein activity inactivates the Rho GTPases Rho, Rac, and Cdc42 in cultured cells and in vitro, whereas the ADP-ribosylation by ExoS is poly-substrate-specific and includes Ras as an early target for ADP-ribosylation. Infection of HeLa cells with P. aeruginosa producing a GTPase-activating protein-deficient form of ExoS rounded cells, indicating the ADP-ribosyltransferase domain alone is sufficient to elicit cytoskeletal changes. Examination of substrates modified by type III-delivered ExoS identified a 70-kDa protein as an early and predominant target for ADP-ribosylation. Matrix-assisted laser desorption ionization mass spectroscopy identified this protein as moesin, a member of the ezrin/radixin/moesin (ERM) family of proteins. ExoS ADP-ribosylated recombinant moesin at a linear velocity that was 5-fold faster and with a K(m) that was 2 orders of magnitude lower than Ras. Moesin homologs ezrin and radixin were also ADP-ribosylated, indicating the ERMs collectively represent high affinity targets of ExoS. Type III delivered ExoS ADP-ribosylated moesin and ezrin (and/or radixin) in cultured HeLa cells. The ERM proteins contribute to cytoskeleton dynamics, and the ability of ExoS to ADP-ribosylate the ERM proteins links ADP-ribosylation with the cytoskeletal changes associated with ExoS intoxication.     

Role of brefeldin A-dependent ADP-ribosylation in the control of intracellular membrane transport

The fungal toxin brefeldin A (BFA) dissociates coat proteins from Golgi membranes, causes the rapid disassembly of the Golgi complex and potently stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kDa. These proteins have been identified as the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and a novel guanine nucleotide binding protein (BARS-50), respectively. The role of ADP-ribosylation in mediating the effects of BFA on the structure and function of the Golgi complex was analyzed by several approaches including the use of selective pharmacological blockers of the reaction and the use of ADP-ribosylated cytosol and/or enriched preparations of the BFA-induced ADP-ribosylation substrates, GAPDH and BARS-50.A series of blockers of the BFA-dependent ADP-ribosylation reaction identified in our laboratory inhibited the effects of BFA on Golgi morphology and, with similar potency, the ADP-ribosylation of BARS-50 and GAPDH. In permeabilized RBL cells, the BFA-dependent disassembly of the Golgi complex required NAD+ and cytosol. Cytosol that had been previously ADP-ribosylated (namely, it contained ADP-ribosylated GAPDH and BARS-50), was instead sufficient to sustain the Golgi disassembly induced by BFA.Taken together, these results indicate that an ADP-ribosylation reaction is part of the mechanism of action of BFA and it might intervene in the control of the structure and function of the Golgi complex.     

Rho GTPase Recognition by C3 Exoenzyme Based on C3-RhoA Complex Structure

C3 exoenzyme is a mono-ADP-ribosyltransferase (ART) that catalyzes transfer of an ADP-ribose moiety from NAD⁺ to Rho GTPases. C3 has long been used to study the diverse regulatory functions of Rho GTPases. How C3 recognizes its substrate and how ADP-ribosylation proceeds are still poorly understood. Crystal structures of C3-RhoA complex reveal that C3 recognizes RhoA via the switch I, switch II, and interswitch regions. In C3-RhoA(GTP) and C3-RhoA(GDP), switch I and II adopt the GDP and GTP conformations, respectively, which explains why C3 can ADP-ribosylate both nucleotide forms. Based on structural information, we successfully changed Cdc42 to an active substrate with combined mutations in the C3-Rho GTPase interface. Moreover, the structure reflects the close relationship among Gln-183 in the QXE motif (C3), a modified Asn-41 residue (RhoA) and NC1 of NAD(H), which suggests that C3 is the prototype ART. These structures show directly for the first time that the ARTT loop is the key to target protein recognition, and they also serve to bridge the gaps among independent studies of Rho GTPases and C3.     

Identification of rho as a substrate for botulinum toxin C3-catalyzed ADP-ribosylation

Recombinant Aplysia rho and a GTP-binding protein purified from human neutrophil membranes (G22K) were ADP-ribosylated by botulinum toxin C3 with stoichiometries of 0.8 and 0.6, respectively. Rho and G22K appeared to be different proteins since (i) rho migrated faster on polyacrylamide gels, (ii) unlike G22K, rho did not require the presence of cytosol to be ADP-ribosylated, (iii) G22K was not recognized by an anti-rho antiserum, and (iv) antibody 142-24E05 recognized G22K effectively but only poorly cross reacted with rho. ADP-ribosylation had no effect on the ability of rho to bind or hydrolyse GTP. Therefore, it appears that there are multiple botulinum toxin C3 substrates and that the toxin exerts its effects on cell function by a mechanism other than modulating the GTPase activity of rho.     

Recognition of RhoA by Clostridium botulinum C3 exoenzyme

The C3-like ADP-ribosyltransferases exhibit a very confined substrate specificity compared with other Rho-modifying bacterial toxins; they selectively modify the RhoA, -B, and -C isoforms but not other members of the Rho or Ras subfamilies. In this study, the amino acid residues involved in the RhoA substrate recognition by C3 from Clostridium botulinum are identified by applying mutational analyses of the nonsubstrate Rac. First, the minimum domain responsible for the recognition by C3 was identified as the N-terminal 90 residues. Second, the combination of the N-terminal basic amino acids ((Rho)Arg(5)-Lys(6)), the acid residues (Rho)Glu(47) and (Rho)Glu(54) only slightly increases ADP-ribosylation but fully restores the binding of the respective mutant Rac to C3. Third, the residues (Rho)Glu(40) and (Rho)Val(43) also participate in binding to C3 but they are mainly involved in the correct formation of the ternary complex between Rho, C3, and NAD(+). Thus, these six residues (Arg(5), Lys(6), Glu(40), Val(43), Glu(47), and Glu(54)) distributed over the N-terminal part of Rho are involved in the correct binding of Rho to C3. Mutant Rac harboring these residues shows a kinetic property with regard to ADP-ribosylation, which is identical with that of RhoA. Differences in the conformation of Rho given by the nucleotide occupancy have only minor effects on ADP-ribosylation.     

Physical association of the small GTPase Rho with a 68-kDa phosphatidylinositol 4-phosphate 5-kinase in Swiss 3T3 cells.

Our previous work showed that post-translationally modified Rho in its GTP-bound state stimulated phosphatidylinositol 4-phosphate 5-kinase (PIP5K) activity in mouse fibroblast lysates. To investigate whether Rho physically interacts with PIP5K, we incubated immobilized Rho-GST with Swiss 3T3 cell lysates and tested for retained PIP5K activity. Rho-GST, but not Ras-GST or GST alone, bound significant PIP5K activity. The binding of PIP5K was independent of whether Rho was in a GTP- or GDP-bound state. An antibody against a 68-kDa human erythrocyte type I PIP5K recognized a single 68-kDa protein eluted from Rho-GST column. The Rho-associated PIP5K responded to phosphatidic acid differentially from the erythrocyte type I PIP5K, suggesting that it could be a distinct isoform not reported previously. Rho co-immunoprecipitated with the 68-kDa PIP5K from Swiss 3T3 lysates, demonstrating that endogenous Rho also interacts with PIP5K. ADP-ribosylation of Rho with C3 exoenzyme enhanced PIP5K binding by approximately eightfold, consistent with the ADP-ribosylated Rho functioning as a dominant negative inhibitor. These results demonstrate that Rho physically interacts with a 68-kDa PIP5K, although whether the association is direct or indirect is unknown.     

Rap1 Spatially Controls ArhGAP29 To Inhibit Rho Signaling during Endothelial Barrier Regulation

The small GTPase Rap1 controls the actin cytoskeleton by regulating Rho GTPase signaling. We recently established that the Rap1 effectors Radil and Rasip1, together with the Rho GTPase activating protein ArhGAP29, mediate Rap1-induced inhibition of Rho signaling in the processes of epithelial cell spreading and endothelial barrier function. Here, we show that Rap1 induces the independent translocations of Rasip1 and a Radil-ArhGAP29 complex to the plasma membrane. This results in the formation of a multimeric protein complex required for Rap1-induced inhibition of Rho signaling and increased endothelial barrier function. Together with the previously reported spatiotemporal control of the Rap guanine nucleotide exchange factor Epac1, these findings elucidate a signaling pathway for spatiotemporal control of Rho signaling that operates by successive protein translocations to and complex formation at the plasma membrane.