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Originally published In Press as doi:10.1074/jbc.M201072200 on February 14, 2002

J. Biol. Chem., Vol. 277, Issue 17, 14771-14776, April 26, 2002
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Interaction of the Rho-ADP-ribosylating C3 Exoenzyme with RalA*

Christian Wilde, Holger Barth, Peter SehrDagger, Li Han§, Martina Schmidt§, Ingo Just, and Klaus Aktories||

From the Institut für Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg, Otto-Krayer-Haus, Albertstrasse 25, D-79104 Freiburg, the  Institut für Toxikologie, Medizinische Hochschule Hannover, D-30625 Hannover, and § Institut für Pharmakologie, Universitätsklinikum Essen, D-45122 Essen, Germany

Received for publication, February 1, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 (Kd) 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.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Rho GTPases including Rho, Rac, and Cdc42 are pivotal regulators of the actin cytoskeleton and act as molecular switches in various signal transduction pathways (1-3). Moreover, Rho proteins are the preferred targets for posttranslational modification by various bacterial toxins, comprising C3-like transferases (4, 5), large clostridial cytotoxins (6-8), the cytotoxic necrotizing factors CNF1 from Escherichia coli (9-11), and the dermonecrotic toxin DNT from Bordetella bronchiseptica (12).

Clostridium botulinum exoenzyme C3 was the first bacterial agent identified to modify Rho GTPases (5, 13, 14). C3 and related C3-like transferases ADP-ribosylate RhoA at Asn-41 thereby inhibiting the biological activity of RhoA (15, 16). ADP-ribosylation by C. botulinum C3 (C3bot), by the related transferases from Clostridium limosum (C3lim), and Bacillus cereus (C3cer) appear to be highly specific for RhoA, -B, and -C. In vitro, Rac and Cdc42 are poorly or not at all modified by C3-like transferases (17). Moreover, there is no evidence that GTPases other than RhoA, -B, and -C are ADP-ribosylated by C3bot, C3lim, and C3cer in intact cells. Because C3-catalyzed ADP-ribosylation appears to be highly specific, these transferases are widely used as tools to elucidate the cellular functions of Rho GTPases. Recently, we identified a C3-related transferase (C3stau2) from Staphylococcus aureus that differs in substrate specificity from other C3-like transferases, because it additionally modifies RhoE/Rnd3 (4).

Ral proteins, which belong to the Ras subfamily of low molecular mass GTPases, occur in the two isoforms RalA and RalB, which are about 85% identical. RalA is more than 50% identical with Ras and shares about 33% sequence identity with RhoA. Ral has been implicated in control of cell proliferation and Ras-mediated cell transformation (18, 19). This GTPase is suggested to be involved in vesicle trafficking (20) and is proposed to regulate the cytoskeleton either by targeting filamin (21) or via RalBP1, the Cdc42/Rac-GTPase-activating protein (22, 23). A further proposed effector protein of Ral is phospholipase D1 (PLD1),1 whose regulation by RalA is largely nucleotide-independent (19, 24, 25). Thus, it is tempting to speculate that both RalA and PLD1 may act in concert to modulate receptor endocytosis and vesicle transport. We report here that C3bot and C3lim directly interact with high affinity with RalA. This interaction inhibits glucosylation of RalA by Clostridium sordellii lethal toxin that takes place in the switch I-region of the GTPase. Moreover, C3bot blocked the activation of phospholipase D by RalA, suggesting that the interaction of C3 exoenzymes with RalA has important functional consequences for signal processes mediated by the Ral GTPase.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- The ralA gene was cut from pTacRalA kindly provided by P. Chardin (Valbonne, France) (26) and cloned into pGEX-2T vector. The recombinant GTPases Ral, Rap1, Ras, RhoA, Rac1, and Cdc42 were prepared from their GST fusion proteins as described (27). For studies on the PLD activity, recombinant sRalA was prepared by transforming E. coli with RalA (C-terminally truncated; amino acids 1-177) subcloned into pGEX-4T vector (a kind gift of Dr. R. H. Cool), purified by adsorption of the GST fusion proteins to glutathione-Sepharose, and loaded with Gpp(NH)p as described previously (28). Likewise, human PLD1, subcloned into pAcGHLT (a kind gift of Drs. A. Morris and M. Frohmann), was expressed in Sf9 insect cells and purified as described before (29). C3bot from C. botulinum (17, 30), Clostridium difficile toxin B, and C. sordellii lethal toxin (31) were prepared as reported. Recombinant C3lim, C3stau2, and C3cer were purified as described (4, 32, 33). The W18L mutant of C3lim was constructed using the pMalc2-C3 expression vector (32) by the Seamless Cloning kit (Stratagene) according to the instructions of the manufacturer. The F169A mutant of C3bot was constructed using the Quick-Change kit (Stratagene, Germany) according the manufacturer's instructions. Human platelet membranes were prepared as described (34). 1-Palmitoyl-2-[3H]palmitoyl-glycerophosphocholine ([3H]PtdCho, 37.5 Ci/mmol) was purchased from PerkinElmer Life Sciences. Unlabeled PtdCho and TNM-FH insect medium were obtained from Sigma. PIP2 was purchased from Roche Molecular Biochemicals.

Nucleotide Loading-- RalA (72 µM) was incubated for 10 min at 37 °C in 150 µl of a solution containing 5 mM EDTA, 10 mM GTPgamma S, or GDP and 50 mM HEPES (pH 7.5). After adding 10 mM MgCl2, preloaded GTPases were added to the ADP-ribosylation reaction.

ADP-ribosylation Assay-- ADP-ribosylation of Rho in human platelet lysate by C. botulinum C3 exoenzyme was performed as described (30). For ADP-ribosylation of Rho in rat brain lysate, 60 µg of total protein were incubated with 10 nM C3bot or as indicated in a solution containing 0.3 µM [adenylate-32P]NAD, 2 mM MgCl2, and 50 mM HEPES (pH 7.5) for 30 min at 37 °C in the presence or absence of the indicated recombinant GTPases. ADP-ribosylation assays for C3lim, C3cer, and C3stau2 were performed essentially as described (35). Proteins were subjected to SDS-PAGE and further analyzed by PhosphorImaging.

NAD Glycohydrolase Activity-- For detection of NAD glycohydrolase activity of C3bot or C3stau2, toxins were incubated with 200 µM [adenylate-32P]NAD, 50 mM HEPES (pH 7.3), 2 mM MgCl2 and different amounts of the indicated GTPases for up to 2 h at 37 °C. Aliquots (5 µl) of the reaction mixture were analyzed by TLC (Silica Gel 60F254, Merck) with 66% 2-propanol and 0.33% ammonium sulfate. The amount of formed [32P]ADP-ribose was calculated from PhosphorImager data.

Glucosylation Assay-- For glucosylation by C. difficile toxin B rat brain lysate (60 µg of total protein) was incubated with increasing concentrations of toxin B in 25 µl of a solution containing 20 µM UDP-[14C]glucose, 100 mM KCl, and 20 mM HEPES (pH 7.4) and 5 µg of RalA (8.3 µM) or 5 µg of bovine serum albumin for 20 min at 37 °C. For glucosylation by C. sordellii lethal toxin, rat brain lysates (60 µg of total protein) were incubated with lethal toxin (250 ng) in 25 µl of a solution containing 10 µM UDP-[14C]glucose, 100 mM KCl, 200 µM MnCl2, 0.1 mg/ml bovine serum albumin, 10 mM HEPES (pH 7.4) in the presence or absence of C3lim for 20 min at 37 °C (31). PhosphorImager data of the SDS-PAGE are shown.

Precipitation of C3bot with Immobilized RalA-- GST-RalA or GST-Rho immobilized to Sepharose beads (1 µg each GTPase) or GST-Sepharose beads as control were incubated with 300 ng of wild-type C3bot or C3bot F169A in a buffer containing 2 mM MgCl2, 50 mM HEPES (pH 7.5), 1 mM dithiothreitol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 0.5% Nonidet P-40 (total volume 100 µl) for 45 min at 4 °C. Subsequently, beads were washed three times with the same buffer and subjected to SDS-PAGE followed by Western blot analysis with an anti-C3bot antibody. Detection was performed by using ECL.

Affinity Determination by Fluorescence Titration-- The affinity of the binding of RalA to C3 from C. limosum was measured by fluorescence titration according to Ref. 36, using the tryptophan fluorescence of RalA as readout. The fluorescence of RalA, excited at 290 nm and monitored at 348 nm, decreased significantly after addition of C3. The tryptophan-deficient mutant C3lim W18L was used to minimize the background fluorescence. After the binding is saturated, the fluorescence increases linearly with further addition of C3lim W18L due to the fluorescence of the remaining aromatic residues like tyrosine residues. This linear increase was also measured in the control titration of C3lim W18L to a buffer solution without RalA. The resulting straight line was subtracted from the upper data set in Fig. 6. The subtracted data (lower data set) were fitted according to Equation 1. The upper data set can also be fitted using Equation 1 plus a linear slope.


f=<UP>I<SUB>max</SUB></UP>−(<UP>I<SUB>max</SUB></UP>−<UP>I<SUB>min</SUB></UP>)·(0.5 · (a+x+K<SUB>d</SUB>)− (Eq. 1)

<UP>sqrt</UP>(0.25 · (a+x+K<SUB>d</SUB>)<SUP>2</SUP>−a · x))
where Imax is the maximal fluorescence value; Imin is the minimal fluorescence value; a is the concentration of RalA; x is the concentration of C3lim W18L; and Kd is the dissociation constant.

PLD1 Activity Assay-- Recombinant PLD1 activity was measured in the presence of [3H]PtdCho mixed with PIP2 (molar ratio of 8:1) as described previously (29) with some modifying cations. In brief, recombinant PLD1 activity (20 µg of protein) was determined with [3H]PtdCho/PIP2 (200 µM/25 µM) as substrate vesicles in the presence or absence of Gpp(NH)p-loaded RalA (8 nM) for 60 min at 37 °C. To measure the effect of C3 transferase, RalA and C3 transferase were preincubated for 5 min at 30 °C at a molar ratio of 1:2. Stopping the reaction and isolating the specific PLD product, [3H]phosphatidylethanl, were as described before (28).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

RalA Effects on C3-catalyzed ADP-ribosylation-- Addition of recombinant RalA to human platelet lysates blocked the ADP-ribosylation of Rho by C. botulinum exoenzyme C3 (C3bot). The inhibition by RalA was concentration-dependent and depended on the native structure of the protein, because heat inactivation of RalA (5 min, 95 °C) abolished the effect completely (Fig. 1A). Fig. 1C shows the influence of RalA on the time course of the ADP-ribosylation of RhoA by C3bot. Time to inhibition of the ADP-ribosylation of Rho was identical independently whether RalA was present at the start of the reaction or was added during the ADP-ribosylation reaction, suggesting an immediate effect. Several C3 isoforms produced by different bacteria have been described (37). Therefore, we compared the effects of RalA on the ADP-ribosylation of Rho catalyzed by C3-like transferases from C. botulinum (C3bot), C. limosum (C3lim), B. cereus (C3cer), and S. aureus (C3stau2) in rat brain lysate. Whereas RalA inhibited C3bot-induced ADP-ribosylation by about 90%, ADP-ribosylation by C3lim and C3cer was reduced by 63 and 24%, respectively. By contrast, RalA did not inhibit the ADP-ribosylation of Rho by transferase C3stau2 (Fig. 2). Similar to the observed inhibition of ADP-ribosylation in cell lysate, ADP-ribosylation of recombinant RhoA by C3bot, C3lim, and C3cer was also inhibited by RalA, whereas C3stau-catalyzed ADP-ribosylation was not altered by RalA (data not shown).


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  		Fig. 1.   Influence of RalA on the ADP-ribosylation of Rho in human platelet membranes by C. botulinum C3 exoenzyme (C3bot). Human platelet membranes (about 200 µg of total protein) were ADP-ribosylated in the presence of 60 nM C3bot and increasing concentrations of RalA or with heat-inactivated (95 °C, 5 min) RalA for 30 min at 37 °C. Thereafter, labeled proteins were analyzed by SDS-PAGE (B). The corresponding autoradiography is shown in A. C, time course of the ADP-ribosylation of Rho in human platelet membranes (about 100 µg of total protein) by 60 nM C3bot in the presence or absence of 1.8 µM RalA. Arrows indicate the addition of RalA.


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  		Fig. 2.   Influence of ADP-ribosylation of RhoA in rat brain lysate by C3-like transferases. Rat brain lysates (about 60 µg of total protein) were incubated with the ADP-ribosyltransferases from C. botulinum (C3bot), C. limosum (C3lim), B. cereus (C3cer), or S. aureus (C3stau2) (10 nM each) in the presence (+) or absence (-) of RalA (1.8 µM). Radiolabeled probes were resolved and subjected to SDS-PAGE and PhosphorImaging. ADP-ribosylation is given in % of the maximal incorporation of radiolabel in the absence RalA.

Ral belongs to the Ras subfamily of GTPases (38). Therefore, we tested whether other members of this protein family (e.g. Ras and Rap) were capable of inhibiting the C3bot-catalyzed ADP-ribosylation of RhoA. As shown in Fig. 3A, neither Ras (~1.8 µM) nor Rap (~1.8 µM) affected C3bot (10 nM)-induced ADP-ribosylation, whereas RalA (~1.8 µM) blocked labeling of Rho. Also Rac1 and Cdc42 (each ~1.8 µM) did not inhibit ADP-ribosylation of Rho. Nucleotide binding causes major conformational changes of low molecular mass GTPases. Therefore, we studied whether the RalA-induced inhibition of the C3-catalyzed ADP-ribosylation depended on the nucleotide-bound state of RalA. To this end, RalA was loaded with GDP or GTPgamma S before starting the ADP-ribosylation reaction. Fig. 3B shows that inhibition of the C3bot-catalyzed ADP-ribosyltransferase activity was detected with GDP- and GTPgamma S-loaded RalA, indicating no major effects of the nucleotide-binding state. To test whether increasing concentration of C3 could overcome the effect of RalA, we studied the ADP-ribosylation of Rho in rat brain lysate without and with 1 µM RalA in the presence of increasing concentrations (10-500 nM) of C3. As shown in Fig. 3C, the inhibiting effect of RalA depended on the concentrations of C3bot and was overcome by high concentrations of the transferase.


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  		Fig. 3.   A, influences of various low molecular mass GTPases on C3bot-catalyzed ADP-ribosylation of Rho. Rat brain lysate (60 µg of total protein) was incubated with 10 nM C3bot for 30 min at 37 °C with of Rac1, Cdc42, RalA, Rap, or Ras (each ~1.8 µM). The total volume was 25 µl. B, influence of the nucleotide bound on Ral-induced inhibition of C3bot-catalyzed ADP-ribosylation. Shown is the time course of C3bot (10 nM)-catalyzed ADP-ribosylation of Rho in rat brain lysate (60 µg of total protein) in the presence of GDP- or GTPgamma S-preloaded RalA, untreated RalA (1.8 µM RalA), and with lysate without the addition of RalA (Con), respectively. C, rat brain lysate (60 µg of total protein) was incubated with increasing concentrations of C3bot in the presence of RalA (1 µM). PhosphorImager data of the SDS-PAGE are shown.

Like other bacterial ADP-ribosyltransferases C3-like exoenzymes possess NAD glycohydrolase activity (37). We studied whether RalA inhibited not only the transferase but also the NAD glycohydrolase activity of C3bot. Surprisingly, addition of RalA did not inhibit but rather increased NAD glycohydrolase activity of C3bot (Fig. 4A). By contrast, the NAD glycohydrolase activity of C3stau2 was not affect by RalA. Also the stimulating effect of RalA on NAD glycohydrolase activity of C3bot was specific, because Ras protein did not affect the activity of C3bot (Fig. 4B).


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  		Fig. 4.   Influences of RalA on C3-catalyzed hydrolysis of NAD. A, time-dependent NAD glycohydrolase activity of C3bot (upper panel) or C3stau2 (lower panel) in the presence or absence of RalA. At the indicated times, probes were taken and separated by TLC. The amount of formed [32P]ADP-ribose was calculated from PhosphorImager data. B, specificity of the effects of RalA on C3-catalyzed NAD glycohydrolase activity. C3bot or C3stau2 were incubated without (con) or with 32P-labeled NAD and various amounts of RalA or Ras for 2 h at 37 °C. Probes were analyzed as described above.

Direct Interaction of Ral and C3 Transferases-- Next, we wanted to know whether C3bot directly interacts with RalA to sequester the transferase. For this purpose a precipitation assay with GST-RalA-Sepharose beads was performed. Fig. 5A shows that GST-RalA-Sepharose beads effectively precipitated C3bot, indicating a direct interaction of RalA with C3bot. Furthermore, we tested whether the presence of NAD did affect the precipitation of C3bot by GST-RalA. Apparently, NAD reduced the affinity of C3bot to RhoA, whereas the precipitation of C3bot by RalA immobilized to GST beads was not altered (Fig. 5A). Recently, Han and co-workers (39) described a novel ADP-ribosylating toxin-turn-turn motif (ARTT motif) in the crystal structure of C3bot, and they proposed that this motif is involved in the recognition of RhoA. Therefore, we addressed the question whether the ARTT motif is also involved in the recognition of RalA. We changed Phe-169 (note: Phe-169 of C3bot is termed Phe-209 in the report by Han et al. (39)) in C3bot to alanine. Phe-169 is located in the ARTT motif and is suggested to play a major role in RhoA recognition. As shown in Fig. 5B, GST-RhoA-Sepharose beads precipitated wild-type C3bot but were unable to precipitate the F169AC3bot mutant. By contrast, GST-RalA-Sepharose beads efficiently precipitated wild-type and mutant C3bot, suggesting that in contrast to enzyme-substrate binding, the ARTT motif is not involved in binding of RalA to C3bot.


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  		Fig. 5.   Influence of NAD on the interaction of Ral with C3. A, GST-RhoA or GST-RalA (each 0.3 µM) immobilized to glutathione-Sepharose beads were incubated with C3bot (300 ng) in the presence (+) or absence (-) of NAD (200 µM final concentration) for 45 min at 4 °C in a volume of 100 µl. Subsequently, beads were washed three times and subjected to SDS-PAGE followed by Western blot analysis with anti-C3 antibody. Detection was performed by using ECL. B, the same experiment was performed without NAD using C3bot wild-type (WT) or C3bot F169A mutant. GST beads were used as control for unspecific binding (GST), and 100 ng of C3bot wild-type or C3bot F169A were used as a control for the antibody (con). Note that C3bot F169A has a different migration behavior on SDS-PAGE.

To get quantitative data on the affinity of the interaction of C3 with RalA, we applied fluorescence titration and utilized quenching of the protein fluorescence of RalA by interaction with C3. For these studies W18LC3lim, which has wild-type enzyme activity (not shown), was employed. This mutant has no tryptophan residue and, therefore, minimal protein fluorescence. The data are exhibited in Fig. 6. Addition of W18LC3lim at low concentrations caused reduction of RalA protein fluorescence caused by quenching. Addition of W18LC3lim at high concentrations to RalA resulted in an increase in the protein fluorescence again. This increase in total protein fluorescence was caused by residual protein fluorescence of the mutant C3lim. Therefore, we subtracted the residual protein fluorescence of C3lim from the total fluorescence to get the specific quenching effect. The best fit for the fluorescence quenching data was obtained with a Kd value of 12 nM, indicating a high affinity interaction between RalA and C3lim.


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  		Fig. 6.   Fluorescence titration of RalA versus C3limW18L. The fluorescence of RalA (80 nM) was excited at 290 nm and monitored at 348 nm at the indicated concentrations of C3limW18L (upper data set; circles). The fluorescence of C3limW18L alone was determined in the absence of RalA and then subtracted from the upper data set (lower data set; squares). Curves were fitted to the data as described under "Experimental Procedures." The best fit was obtained with a Kd value of 12 nM.

Effects of C3 on Toxin-catalyzed Glucosylation-- Rho GTPases, including Rho, Rac, and Cdc42, are glucosylated by C. difficile toxin B (6). To test the specificity of the effect of RalA on modification of Rho, we studied whether RalA also affected glucosylation of Rho GTPases by toxin B in rat brain lysate. As shown in Fig. 7, addition of RalA neither altered glucosylation of Rho (upper band on SDS-PAGE) nor of Rac/Cdc42 (lower band on SDS-PAGE) by toxin B. These findings indicated that the inhibiting effect of RalA is restricted to C3-catalyzed ADP-ribosylation of Rho.


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  		Fig. 7.   Influence of RalA on glucosylation of Rho GTPases by C. difficile toxin B. Rat brain lysate (60 µg of total protein) was incubated in the presence of the indicated concentration of toxin B with or without RalA (8.3 µM) for 20 min at 37 °C. PhosphorImager data of the SDS-PAGE are shown. The upper band belongs to the 14C-glucosylated RhoA and the lower to the 14C-glucosylated Rac and/or Cdc42.

To address the question whether the interaction of RalA and C3 has any consequences for the biochemical and/or biological properties of Ral, we used C. sordellii lethal toxin. In addition to Rac, lethal toxin also glucosylates Ras proteins, including Rap and Ral (40). To study whether C3 affects the ability of RalA to serve as a substrate for lethal toxin, we glucosylated RalA in rat brain lysate in the presence of C3lim and C3bot, respectively. In rat brain lysate, lethal toxin radiolabels at least two major bands in the presence of UDP-[14C]glucose. Recently, we showed that the upper band belongs the glucosylation of Ral, whereas the lower band mainly represents the modification of Rac (Cdc42) and Ras (41, 42). C3lim (Fig. 8A) blocked lethal toxin-catalyzed glucosylation of RalA (upper band). The same was observed in the presence of C3bot (not shown). By contrast, glucosylation of Rac/Cdc42/Ras (lower bands) by lethal toxin was not affected by addition of C3 isoforms, underlining the specificity of the interaction of C3lim and C3bot with RalA. Moreover, these findings suggested that C3lim and C3bot interact with RalA in a region essential for the interaction of RalA with C. sordellii lethal toxin.


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  		Fig. 8.   Influence of C. limosum C3 exoenzyme (C3lim) on C. sordellii lethal toxin-catalyzed glucosylation of RalA. Rat brain lysate (60 µg of total protein) was glucosylated by 250 ng of lethal toxin in the presence of the indicated concentrations of C3lim for 20 min at 37 °C. Probes were subjected to SDS-PAGE, and the autoradiography is shown.

It has been reported that RalA is able to activate PLD1 (24). Therefore, we studied whether C3 has any effect on stimulation of PLD1 by RalA. For this purpose, RalA preloaded with Gpp(NH)p was incubated in the absence and presence of C3bot, which was added at a molar ratio of C3:RalA = 2:1, for 5 min at 30 °C. Thereafter, PLD activity was determined for 60 min at 37 °C. As shown in Fig. 9, RalA increased PLD1 activity by about 100%. This increase in enzyme activity was completely prevented after pretreatment of RalA with C3bot, indicating that the binding of C3 to RalA blocks its capacity to stimulate PLD1.


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  		Fig. 9.   Inhibition of RalA-stimulated PLD1 activity by C3 transferase. PLD1 activity was measured as described under "Experimental Procedures" with [3H]PtdCho/PIP2 substrate vesicles in the presence of 8 nM Gpp(NH)p-loaded RalA (note: for this study a C-terminally truncated RalA protein was used) preincubated with or without C3bot transferase. PLD activity in the presence of heat-inactivated (95 °C, 5 min) RalA/C3 transferase was 0.98 nmol/mg-1/h-1. Data are means ± S.E. of three experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

C3-like ADP-ribosyltransferases are potential virulence factors of various bacteria, including Clostridia, B. cereus, and S. aureus. With the exception of C3stau2 from S. aureus that was shown to modify RhoE/Rnd3 (4), C3 from C. botulinum (C3bot), C. limosum (C3lim) and B. cereus (C3cer) appear to ADP-ribosylate only RhoA, -B, and -C but not other GTPases (37). This high specificity is important for their use as tools to study Rho functions in eukaryotic signaling. Our data indicate that C3 transferases directly interact with RalA. Interaction was detected with C3bot, C3lim, and C3cer but not with C3stau2. The RalA-C3 interaction caused inhibition of the ADP-ribosylation of RhoA. Inhibition of C3-catalyzed ADP-ribosylation occurred with Rho in cell lysate and with recombinant RhoA. Ral itself was not ADP-ribosylated by the C3 transferases; however, the precipitation and fluorescence quenching studies indicated that the interaction is of high affinity (Kd 12 nM for C3lim). RalA did not inhibit the NAD glycohydrolase activity of C3bot but caused an approximately 5-fold increase in NAD hydrolase activity. We recently observed a similar but modest increase in NAD glycohydrolase activity of C3bot with RhoA mutants, which are not substrates for ADP-ribosylation, because they lack the acceptor amino acid Asn-41 (43). We propose that binding of RalA favors a conformation of C3 that facilitates the cleavage of NAD. Inhibition of C3-catalyzed ADP-ribosylation was not observed with Ras and Rap, indicating a Ral-specific effect that is not shared by other Ras subfamily proteins. The finding that both GDP- and GTPgamma S-bound RalA inhibited C3 ADP-ribosylation suggests that conformational changes caused by nucleotide binding are not essential for the interaction of RalA with C3 transferases. It has been shown that C3 modifies Rho at the acceptor amino acid asparagine 41 (16). RalA contains a serine residue at the equivalent position (serine 50) and is therefore not a substrate for the transferase. Recently, we characterized the structural requirements of the ADP-ribosylation of RhoA in detail, and we identified several amino acid residues pivotal for modification of Rho by C3 (43). We changed several of the equivalent amino acids of RalA to that of RhoA. However, none of these changes resulted in a labeling of S50N-RalA (data not shown). The finding suggests that either C3 binds differently to Ral as compared with Rho or additional amino acid residues not present in Ral are required for ADP-ribosylation.

Recently, Han and co-workers (39) analyzed the crystal structure of C3 and described a motif termed ADP-ribosylating toxin turn-turn motif (ARTT motif), which was suggested to be essentially involved in substrate recognition by C3. These authors proposed that Phe-169 (Phe-209 in the nomenclature of Han et al. (39)) is responsible for the affinity of C3 to its substrate RhoA. We changed Phe-169 of C3bot to alanine. The mutant of C3bot was not able to ADP-ribosylate RhoA (not shown) and exhibited a significant reduction in the interaction with RhoA as analyzed by a precipitation assay. However, the interaction of F169AC3bot with Ral was not changed, suggesting that a different or additional region of C3 is responsible for the interaction with RalA. The interaction of RalA with C3 did not only cause sequestration of the transferase to inhibit modification of RhoA but also affected the functional properties of RalA. RalA is a substrate for glucosylation by C. sordellii lethal toxin, which modifies Rac (possibly Cdc42) and Ras proteins including Ral (40, 42). Binding of C3 inhibited the ability of RalA to serve as a substrate for glucosylation. In Ras, C. sordellii lethal toxin glucosylates Thr-35 that is located in the switch-I region of the protein and blocks the interaction with Ras effectors (44). The equivalent amino acid residue in RalA is Thr-46. Thus, interaction of C3 with RalA, which caused inhibition of glucosylation, most likely occurs in the same region of RalA and, therefore, may affect cellular actions of the GTPase. So far, cellular functions of Ral are still largely enigmatic (45). Evidence has been presented that Ral is activated by Ras via Ral-guanine nucleotide exchange factors by a signal pathway parallel to the Raf/mitogen-activated protein kinase cascade and may be involved in transformation to act downstream of Ras (18, 19, 46). RalA is suggested to be involved in regulation of the actin cytoskeleton. For example, dominant negative RalA blocks filopodia formation induced by Cdc42 (21). A cross-talk between Ral and Rho subfamily GTPases is also suggested, because the Ral effector RalBP1 (also termed RLIP1 or Rip1) acts as a GTPase-activating protein for Cdc42 and Rac at least in vitro (19, 22, 23). Thus, some of these functions may be affected by C3 binding. PLD is one of the best established effectors of RalA (24, 28, 47). The activity of PLD1 is increased by direct binding to RalA (48). Indeed, we observed that C3 inhibited the stimulation of PLD1 by RalA supporting our hypothesis that the interaction of Ral and C3 is of functional importance.

Notably, some effects of C3 on eukaryotic target cells are not simply explained by ADP-ribosylation and inactivation of Rho. For example, it was reported that C3 activates the stress signaling pathways of c-Jun N-terminal kinase and p38 (49). Moreover, C3 was shown to inhibit the activation of the serum-responsive element by EGF. However, this EGF signal pathway is suggested to be independent of Rho (49). As it has been shown recently that PLD and RalA cooperate with the EGF receptor to transform 3Y1 rat fibroblasts (50), modulation of EGF receptor signaling might be the functional link between C3, RalA, and PLD1, and this hypothesis will be addressed in future studies. Our findings indicate, however, that C3 exhibits potent effects on regulatory GTPases without ADP-ribosylation. This activity of C3 has to be considered when using C3bot as a tool to study Rho signaling, especially when high concentrations of C3bot are applied in microinjection studies.

    FOOTNOTES

* This work was supported by the Deutsche Forschungsgemein-schaft (Sonderforschungsbereich 388).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Present address: Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany.

|| To whom correspondence should be addressed: Institut für Pharmakologie und Toxikologie der Albert-Ludwigs-Universität Freiburg Otto-Krayer-Haus, Albertstr. 25, D-79104 Freiburg. Tel.: 761-203-5301; Fax: 761-203-5311; E-mail: aktories@ruf.uni-freiburg.de.

Published, JBC Papers in Press, February 14, 2002, DOI 10.1074/jbc.M201072200

    ABBREVIATIONS

The abbreviations used are: PLD1, phospholipase D1; GST, glutathione S-transferase; PIP2, phosphatidylinositol 4,5-bisphosphate; EGF, epidermal growth factor; PtdCho, 1-palmitoyl-2-[3H]palmitoylglycerophosphocholine; Gpp(NH)p, guanosine 5'-(beta ,gamma -imido)triphosphate; GTPgamma S, guanosine 5'-3-O-(thio)triphosphate.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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