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Originally published In Press as doi:10.1074/jbc.M504768200 on June 30, 2005

J. Biol. Chem., Vol. 280, Issue 35, 31003-31010, September 2, 2005
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Mechanism of Redox-mediated Guanine Nucleotide Exchange on Redox-active Rho GTPases*

Jongyun Heo{ddagger} and Sharon L. Campbell{ddagger}§

From the {ddagger}Department of Biochemistry and Biophysics, §Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599

Received for publication, May 2, 2005 , and in revised form, June 6, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Rho GTPases regulate multiple cellular processes including actin cytoskeletal rearrangements, transcriptional regulation, and oxidant production. The studies described herein demonstrate that small molecule redox agents, in addition to protein regulatory factors, can regulate the activity of redox-active Rho GTPases. A novel (GXXXXGK(S/T)C) motif, conserved in a number of Rho GTPases, appears critical for redox-mediated guanine nucleotide dissociation in vitro. A detailed molecular mechanism for redox regulation of GXXXXGK(S/T)C motif-containing Rho GTPases is proposed.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Rho GTPases comprise a large branch of the Ras superfamily of small guanine nucleotide-binding proteins and include the well characterized family members Rac1, RhoA, and Cdc42 (1). They are involved in regulation of a plethora of cellular processes, including cell morphology, movement, and proliferation (2).

The guanine nucleotide bound state of most Rho GTPase family members is regulated by three distinct types of protein modulatory agents, which alter GTPase activity by regulating cycling of the GTPase between inactive GDP and active GTP-bound forms (2-4). In particular, guanine nucleotide exchange factors (GEFs)1 facilitate exchange of GDP with GTP to promote GTPase activation, whereas GTPase-activating proteins deactivate the GTPase protein by stimulating hydrolysis of bound GTP to GDP. Deactivation can also be achieved by association with guanine nucleotide dissociation inhibitors, which prevent membrane association and GDP dissociation. Similar to Ras GTPases, exchange of GDP for GTP leads to a conformational change in the GTPase that greatly enhances affinity to downstream effectors. The interaction between the GTPase and effector leads to activation of GTPase effector-mediated signal transduction pathways.

A fourth distinct type of modulatory agent has been shown to regulate Ras GTPase activity (5-9). Similar to the action of GEFs, various redox agents, including both reactive oxygen species (ROS) and reactive nitrogen species (RNS), have been shown to stimulate Ras guanine nucleotide dissociation in vitro and up-regulate Ras function in vivo. We have recently elucidated the molecular mechanism by which certain redox agents modulate Ras activity (10-12). Intriguingly, in addition to Ras, Rho GTPase signaling is sensitive to the presence of ROS and RNS and to the redox state of the cell (13-23). However, it is unclear whether redox agents can act directly on Rho GTPases to modulate their activity or whether modulation of Rho GTPase function occurs indirectly.

ROS include the superoxide radical anion () and hydroxyl radical (OH·). In addition, ROS can be generated or interconverted into other ROS by enzyme- and transition metal-mediated catalytic processes. For example, in the presence of transition metals (i.e. Cu2+ and Fe3+), H2O2 is converted into the redox-active free radical, OH·, by the Fenton reaction (24). Moreover, can be converted to OH· and H2O2 by superoxide dismutase (25) or the transition metal-catalyzed Harber-Weiss reaction (26). The activity of NADPH oxidase enzyme complexes can be regulated by Rap and Rac GTPases, and upregulation of NADPH oxidase activity leads to the production of (27). In addition, xanthine oxidase (XO), a terminal enzyme of purine catabolism in the cell, catalyzes the hydroxylation of hypoxanthine to xanthine and xanthine to urate, resulting in reduction of O2 to (28). XO is also known to produce H2O2 (29). RNS include nitric oxide (NO), nitrogen dioxide (·NO2), and dinitrogen trioxide (N2O3) (30). Reaction of NO with O2 produces ·NO2 and N2O3 (30). Nitric-oxide synthases (NOS) catalyze the oxidation of L-arginine to NO and L-citrulline (31). However, when the substrate L-arginine is not present, NOS produces (32). Despite the overwhelming current interest in NOS as a NO-producing enzyme, several studies have implicated XO in the production of NO (33). Peroxynitrite is a precursor of both ROS and RNS, as it is formed by the reaction of NO with , which can then be decomposed into ·NO2 and OH· (30).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Preparation of Chemicals and Proteins—The chemicals used for all experiments were of the highest grade unless otherwise noted. Radio-labeled guanine nucleotides ([3H]GDP and [3H]GTP) were diluted with unlabeled guanine nucleotides prior to use, giving ~1000 dpm/µM nucleotide. Rac1-(1-177), Rac1 F28L, Rac1 C18S, RhoA-(1-181), and Cdc42-(1-188) proteins were expressed and purified as described previously (34). The final proteins were >95% pure as determined by SDS-PAGE. The protein concentration was determined by the Bradford method (35). XO was purchased from Sigma.

Experimental Conditions and Preparation of Redox Agents—All assays and sample preparations were performed in sealed cuvettes and vials to avoid diffusion of volatile redox agents into the open atmosphere (10). To prevent transition metal-mediated conversion of the radical into H2O2 and OH· (26) as well as NO into the nitrosonium ion, all buffers used for kinetic and biochemical assays were passed through a metal-chelating Bio-Rad Chelex 100 cation exchange column (10), and diethylenetriaminepentaacetic acid (DTPA, 0.1 mM) was added to the buffers prior to performing the experiments, unless otherwise noted. Detailed methods for generation and quantification of using XO and a methylene blue-coupled assay system under aerobic conditions have been described previously (12). NO gas was purified by passing it through a scrubbing column with 5 M KOH, and quantification of NO in the assay solution was achieved by using a hemoglobin-coupled assay under anaerobic conditions (10). Authentic ·NO2 was used for ·NO2-mediated Rho GTPase guanine nucleotide dissociation assays, and quantification of ·NO2 in the assay solution was achieved by using the NO2/NO3 assay kit C II (Dojindo) under anaerobic conditions. N2O3 was prepared by mixing NO gas and authentic ·NO2 gas (1:1, mol:mol) under anaerobic conditions. Although the content of N2O3 in the assay solution was not directly determined, the quantity of N2O3 in the assay solution was estimated to be similar to the NO content (i.e. 5 µM NO gas, which is mixed with 5 µM ·NO2 gas) in the assay, based on the assumption that NO reacts with ·NO2 stoichiometrically 1:1. H2O2 was used in the presence and absence of CuCl2 (H2O2/Cu2+) under aerobic conditions. Following the Fenton reaction (24), the transition metal, Cu2+, catalyzes conversion of H2O into OH·

Kinetic Measurements of Guanine Nucleotide Exchange on Redox-active Rho GTPases—A transition metal-free standard assay solution for the measurements consisted of 50 mM NaCl, 5 mM MgCl2, and 0.1 mM DTPA in ammonium acetate buffer (10 mM, pH 7.5, in the presence of DTPA to remove transition metals). However, when CuCl2 was added, DTPA was omitted from the assay solution. To mimic cellularly abundant nucleotide concentrations, the standard assay solution contained excess unlabeled GDP (20 mM) for all Rho GTPase GDP dissociation assays. Moreover, the presence of excess unlabeled GDP in the assay solution prevented precipitation of Rho GTPases upon treatment with redox agents (i.e. or ·NO2). To monitor -mediated Rho GTPase GDP dissociation, XO (unit activity: ~0.5 µM s-1) was incubated with its substrate xanthine (10 mM) for 30 min in the presence of O2 to accumulate in solution prior to the addition of 1 µM [3H]GDP-loaded Rho GTPase (e.g. Rac1, Rac1 C18S, Rac1 F28L, RhoA, or Cdc42). To monitor Rho GTPase GDP dissociation by other potential redox agents, NO (~5 µM) in the absence of O2, H2O2 (0.1% v/v) in the presence of O2, a mixture of H2O2 (0.1% v/v) and CuCl2 (1 µM) in the presence of O, authentic ·NO2 (~3 µM) in the absence of O2, or a mixture of NO (5 µM) and ·NO2 (5 µM) in the absence of O2 was incubated for 2 min prior to the addition of [3H]GDP-loaded Rho GTPase (1 µM). As a control, the intrinsic GDP dissociation rates of Rho GTPases were also measured using experimental conditions identical to those described above, except that XO or redox agents were not added. To monitor -mediated Rac1 GTP association, unlabeled GDP-loaded Rac1 (1 µM) and 20 mM [3H]GTP were added simultaneously to the assay solution containing (generated by XO assay system; see above). To determine the effect of a radical-quenching agent on Rac1 GTP association, ascorbate (1 mM) was added to the assay mixture at assay time 500 s. As a control, the intrinsic rate of Rac1 GTP association was measured using experimental conditions identical to those described above, except that XO was not added. For all assays including both Rho GTPase GDP dissociation and GTP association, aliquots were withdrawn at specific time points and spotted onto nitrocellulose filters. The filters were then washed three times with assay buffer, and radioactivity was determined using a Beckman-Coulter scintillation counter. The resultant radioactivity (dpm) values associated with Rho GTPase-bound [3H]nucleotide (either [3H]GDP or [3H]GTP) were converted into the fraction of mol of nucleotide per mol of total Rho GTPase. The data presented in all figures represent mean and standard error values from triplicate measurements. The rates of redox agent-mediated Rho GTPase GDP dissociation and GTP association were obtained by fitting the data to a simple exponential decay and exponential association, respectively. The regression values associated with the fit were r2 > 0.7595.

Mass Spectrometry Analysis—Mass spectrometry (MS) sample preparations and analyses of the nucleotide derivatives released from Rho GTPases were identical to those described previously (12), except the Rac1 and Rac1 variants, C18S and F28L (1 µM, 500 µl), were employed instead of Ras and Ras variants.


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Redox-active Rho Subfamily GTPases—We have identified a distinct class of redox-active GTPases conserved in nearly 50% of all Rho subfamily GTPases that contain a cysteine, Cys18 (Rac1 numbering), located at the end of the P-loop (GXXXXGK(S/T), residues 10 to 17, Rac1 numbering). This cysteine-containing P-loop motif will be referred to herein as the GXXXXGK(S/T)C motif. For comparison, select members of the Ras subfamily of GTPases such as H-, K-, and N-Ras and Rap1A possess an NKCD motif that forms interactions with the guanine nucleotide base and contains a redox-active cysteine, Cys118 (Ras numbering) (5, 10-12). Inspection of the available NMR and x-ray crystal structures for the GXXXXGK(S/T)C motif-containing Rho GTPases Rac1, RhoA, and Cdc42 (36-38) indicates that the Cys18 thiol in the GXXXXGK(S/T)C motif is solvent-accessible, suggesting that ROS and RNS (e.g. and ·NO2, respectively) may be able to target the Cys18 thiol. Consistent with this premise, results shown in Fig. 1A demonstrate that facilitates WT Rac1 guanine nucleotide dissociation, whereas the Rac1 C18A variant is insensitive to produced from XO. To confirm that is the redox species that facilitates Rac1 guanine nucleotide dissociation, bovine liver Cu,Zn-superoxide dismutase (~1000 units) was added to the assay system. Superoxide dismutases catalyze the conversion of into O2 and H2O2 (39). When bovine liver Cu,Zn-superoxide dismutase is present, XO-mediated Rac1 guanine nucleotide dissociation is abolished (not shown), indicating that , but not H2O2, generated from XO facilitates Ras guanine nucleotide dissociation. Thus, treatment of Rac1 with enhances the intrinsic rate of GDP dissociation by over 600-fold (Table I). Results shown in Fig. 1B indicate that, in addition to Rac1, other GXXXXGK(S/T)C motif-containing GTPases such as RhoA and Cdc42 are also sensitive to . These results indicate that Cys18 in the GXXXXGK(S/T)C motif of Rac1, RhoA, and Cdc42 is a redox-active target site of .


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  		TABLE I
Rho GTPase GDP dissociation rates in the absence and presence of various redox agents The data presented are mean values of experiments performed in triplicate (Fig. 1). Rates of H2O2-, H2O2/Cu2+-, NO-, ·NO2-, and NO/·NO2-mediated GDP dissociation from RhoA and Cdc42 were also measured as described under "Experimental Procedures" in this table but not shown in Fig. 1. Standard errors for each data point are <29%, and the regression values associated with the fit correspond to r2 > 0.8095.

 
The redox-active Cys118 thiol in the NKCD motif of H-Ras faces the Phe28 side chain and is ~12 Å away from the center of the Phe28 side chain (11, 12). Coupling between Cys118 and Phe28 appears critical for redox-mediated guanine nucleotide dissociation (11, 12). Intriguingly, the Cys18 thiol in the GXXXXGK(S/T)C motif of Rac1 also faces the Phe28 side chain (Rac1 and Ras numbering are the same) and is only ~3.6 Å away from the center of the Phe28 side chain (36-38). We have previously proposed a mechanism whereby reaction of either NO/O2 (via ·NO2) or with Ras generates a Cys118-thiyl radical (Cys118·), which in turn promotes formation of a guanine radical intermediate resulting in perturbation of Ras guanine nucleotide interactions and accelerated release of GDP as GDP derivatives from Ras (11, 12). We further proposed that Phe28 may act as a conduit between the Ras Cys118· and the guanine nucleotide base to facilitate electron transfer (11, 12). If the mechanism of redox-mediated guanine nucleotide dissociation of GXXXXGK(S/T)C motif-containing Rho GTPases (see below) is similar to that of Ras GTPases (11, 12), electronic coupling between the Rac1 Cys18-thiyl radical (Cys18·) and the Phe28 side chain is expected, with the Phe28 side chain serving as an electron conduit between the Rac1 Cys18· and the bound GDP guanine base.

To evaluate whether Phe28 is involved in redox-mediated regulation of Rho GTPase activity, we determined the rate of -mediated guanine nucleotide dissociation associated with a Rac1 F28L variant. The Rac1 F28L variant shows a faster intrinsic rate of guanine nucleotide dissociation (Fig. 1A) relative to that of WT Rac1, similar to that observed previously for the Ras F28L mutant (40). However, the rate of guanine nucleotide dissociation was not further enhanced in the presence of . The Cdc42 F28L variant has been characterized previously by NMR, and results from this study indicate that the structure of Cdc42 is not significantly altered by substitution of leucine for phenylalanine (41). Rather, loss of the packing interaction between Phe28 and the guanine nucleotide base alters dynamics of the Cdc42 protein guanine nucleotide-binding site, resulting in reduced guanine nucleotide-binding affinity and enhanced guanine nucleotide dissociation. Given the conservation of this residue and the structural homology between Cdc42 and Rac1 (36, 38), it is anticipated that the Rac1 F28L mutant will share similar nucleotide-binding properties to that of Cdc42.



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  		FIG. 1.
Effect of redox agents on the rate of guanine nucleotide dissociation from GXXXXGK(S/T)C motif-containing Rho GTPases. A, -mediated guanine nucleotide dissociation rates of wt Rac1 and Rac1 variants. B, -mediated guanine nucleotide dissociation rates of wt Rac1, RhoA, and Cdc42. C, rates of Rac1 guanine nucleotide dissociation by various redox agents. Experimental conditions, assays, and data analysis methods are described under "Experimental Procedures." The rates of Rho GTPase GDP dissociation in the presence and absence of various redox agents are denoted in Table I.

 
These results support the involvement of Phe28 in -mediated Rac1 guanine nucleotide dissociation. Moreover, we would also expect that the shorter distance between the Phe28 side chain and the Cys18 thiol contained within the GXXXXGK(S/T)C motif will result in more efficient redox coupling and consequently enhanced redox-mediated guanine nucleotide dissociation from GXXXXGK(S/T)C motif-containing GTPases relative to NKCD motif-containing Ras GTPases (10-12). In support of this premise, the rates of -enhanced GDP dissociation from both Rac1 and Cdc42 (~6 x 10-3 s-1) (Table I) are at least 2-fold faster compared with that observed for H-Ras (~3 x 10-3 s-1) (12), yet it is not clear whether the faster rate of redox-mediated guanine nucleotide dissociation of Rac1 and Cdc42 compared with H-Ras observed in vitro has implications for their differential regulation by redox agents in vivo. Unlike Rac1 and Cdc42 (36, 38), RhoA possesses another cysteine (Cys18 RhoA numbering; Lys16 Rac1 numbering) in addition to the redox-active cysteine in the GXXXXGK(S/T)C motif (37). Although this additional cysteine faces the redox-active Cys18 with a distance of ~10 Å, it does not face either the Phe28 side chain or the bound GDP guanine base (37). Hence, it is likely that this additional cysteine interacts electronically with the redox-active Cys18 and thus reduces the efficiency of redox coupling between Cys18, Phe28, and the Rho GTPase-bound guanine nucleotide base, resulting in a slower rate of -mediated RhoA guanine nucleotide dissociation compared with that of Rac1 and Cdc42 (Table I).

In addition to , authentic ·NO2, which can be formed by reaction of NO with O2 (10), can stimulate dissociation of GDP from Rac1 (Fig. 1C), RhoA, and Cdc42 (Table I). However, Rac1 guanine nucleotide dissociation is not enhanced in the presence of NO alone (in the absence of O2) or a stoichiometric mixture of NO and authentic ·NO2 (to produce N2O3) (Fig. 1C). These results indicate that the activity of GXXXXGK(S/T)C motif-containing Rho GTPases is sensitive to ·NO2 and but not to NO and N2O3. Furthermore, we show that H2O2 alone (in the absence of a transition metal) does not facilitate Rho GTPase guanine nucleotide dissociation, whereas H2O2 in the presence of a transition metal (H2O2/Cu2+) causes production of OH· (24), which may facilitate guanine nucleotide dissociation from Rac1 (Fig. 1C), RhoA, and Cdc42 (Table I). The results indicate that the activity of GXXXXGK(S/T)C motif-containing Rho GTPases is sensitive to H2O2/Cu2+ (OH·) but not H2O2 alone, providing a reasonable explanation for the previously observed activation of Rho GTPases by H2O2 (13-17).

Rac1 has been shown to regulate the assembly and activation of NADPH oxidase in phagocytes and is implicated in regulation of NADPH oxidase-related enzymes in non-phagocytic cells to generate bactericidal from NADPH oxidase as well as modify gene expression, cell proliferation, adhesion, and many cell-specific functions, respectively (2, 27). It has been shown also that the Rac1 GEF kalirin, as well as Rac1 and Rac2, associates with inducible NOS (42). Moreover, Rac1 colocalizes with inducible NOS in activated macrophages (42). Hence, Rac GTPases may be localized and exposed to levels of and ·NO2 that regulate its activity in situ. The ability of Rac to up-regulate NADPH oxidase activity may lead to enhanced levels of peroxynitrite under certain conditions (42). However, it is not clear whether the OH· radical is present at high enough concentrations in cells under physiological conditions to modulate Rho GTPase activity. Although our results support the possibility that redox agents such , ·NO2, and H2O2/Cu2+ (OH·) regulate Rho GTPase activity in cells, additional studies are required to elucidate the role of these redox agents on Rho GTPase activity in vivo.

Redox Activation of GXXXXGK(S/T)C Motif-containing Rho GTPases—Results in Fig. 1 show that various redox agents facilitate guanine nucleotide dissociation of GXXXXGK(S/T)C motif-containing Rho GTPases. Cellular activation of Rho GTPases requires exchange of bound GDP for GTP. However, to produce the active GTP-bound state of Rho GTPases, GTP association should occur following redox-mediated GDP dissociation from the Rho GTPase to complete guanine nucleotide exchange.



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  		FIG. 2.
Determination of -mediated Rac1 GDP dissociation and GTP association rates. Experimental conditions, assays, and data analysis methods are described under "Experimental Procedures." The rates of Rac1 GDP dissociation and GTP association in the presence and absence of are shown in Table II.

 
As shown in Fig. 2 (and summarized in Table II), association of GTP on Rac1 is not observed after -mediated Rac1 GDP dissociation. These results indicate that facilitates guanine nucleotide dissociation but does not facilitate guanine nucleotide reassociation. However, when the radical-quenching agent, ascorbate, was added to -treated Rac1 in the presence of GTP, binding of GTP on Rac1 readily occurred (Fig. 2). Similar kinetic results are observed for RhoA and Cdc42 in the presence of , authentic ·NO2 and H2O2/Cu2+ (OH·) (not shown), suggesting that exchange of GDP for GTP in the presence of redox agents requires a radical-quenching agent. Although the results suggest that redox-mediated Rho GTPase activation requires synergy with a radical scavenger such as ascorbate, our results also imply that redox agents can serve to terminate Rho GTPase signaling (and other redox-active GTPases) by promoting fast release of GDP or GTP from the GTPase to produce the apo-form, consistent with our previous studies (11, 12). The ability to promote guanine nucleotide exchange and subsequent activation of the GTPase as opposed to GTPase deactivation is likely to be dependent on the cellular conditions, i.e. amount of redox species versus radical-scavenging agents. For example, Rac may be inactivated as a protective cellular response under conditions where or other redox-active species (i.e. ·NO2) are present at high levels relative to radical-scavenging agents. This, in turn, may cause down-regulation of NADPH oxidase activity, thereby providing a mechanism for negative regulation of Rac-mediated NADPH oxidase activity. Further studies are required to delineate GTPase signaling mediated by redox agents with respect to the action of radical scavengers in cells.


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  		TABLE II
-mediated Rac1 GDP dissociation and GTP association rates in the absence and presence of ascorbate The data presented are mean values of experiments performed in triplicate (Fig. 2). Standard errors for each data point are < 38%, and the regression values of the best fits correspond to r2 > 0.7995.

 



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  		FIG. 3.
Proposed mechanism of -mediated guanine nucleotide dissociation for the Rac1 GTPase. GDP is represented in blue, and the oxygen moiety of the nucleotide adduct is shown in red. Multiple resonance states of Rac1-bound {image1} are shown using a blue dotted line. The black dotted lines represent putative hydrogen-bond interactions between Rac1 residues and GDP.

 
Potential Mechanism for Redox Activation of GXXXXGK(S/T)C Motif-containing Rho GTPases—As shown in Fig. 3, we postulate that the mechanism of ROS- and RNS-mediated guanine nucleotide dissociation of GXXXXGK(S/T)C motif-containing GTPases is similar to that of NKCD motif-containing Ras GTPases (11, 12), as our kinetic results, the redox-coupling partners (GXXXXGK(S/T)C, Phe28, and bound GDP), and their structural architecture show a similarity to NKCD motif-containing Ras GTPases (43).

Given that Rac GTPases co-localize with -producing NADPH oxidases and regulate their activity (27), Rac1 activity may be regulated by , possibly via a feedback mechanism. Although this mechanism is shown for , our data indicate (Fig. 1 and Table I) that other redox agents, such as ·NO2, can modulate Rac1 activity and possibly other GXXXXGK(S/T)C motif-containing Rho GTPases. Consistent with our data, Rac GTPases have been shown to co-localize and interact with inducible NOS and may facilitate peroxynitrite production (42). Given the data presented here and the mechanism proposed for redox regulation of H-Ras, we postulate that a Rac1 Cys18· intermediate, formed by the reaction of the Rac1 Cys18 thiol and (Fig. 3, step i), withdraws an electron from the Rac1-bound GDP guanine base via the Rac1 Phe28 side chain to produce a Rac1-GDP cation radical ({image1}) (step ii). {image1} is then converted to G·-DP by elimination of a H+ from the N-1 of {image1}, resulting in disruption of key Rac1 guanine nucleotide hydrogen-bond interactions, including the guanine N-1 interaction with the Asp118 side chain (Rac1 numbering) resulting in release of GDP and formation of a GDP-deficient form of Rac1 (apoRac1) (Fig. 3, step iii). When additional is present, G·-DP can further react with to produce an oxygenated GDP adduct (GDP-O2H) (Fig. 3, step iv), and formation of GDP-O2H is likely to interfere with additional Rac1-GDP interactions thereby triggering dissociation of the bound GDP-O2H from Rac1. GDP-O2H can be then decomposed to an unstable 5-diamino-4O-imidazolone ribose diphosphate (5-DIm-DP) (Fig. 3, step v). If reaction of G·-DP with occurs prior to release of G·-DP from Rac1, 5-DIm-DP will be the dominant product formed over 8-diamino-4O-imidazolone ribose diphosphate (8-DIm-DP), as the C-5 site of Rac1-bound G·-DP is exposed (hence is accessible to the C-5 site of Rac1-bound G·-DP), whereas the C-8 site of Rac1-bound G·-DP is not solvent-accessible (36). However, if reaction of G·-DP with ·NO2 occurs prior to the release of G·-DP from Rac1, the 5-DIm-DP is likely to predominate, and this adduct is then released from Rac1 (Fig. 3, step v). Depending on the experimental conditions, the Rac1-released 5-oxo-GDP is further degraded into oxygenated nucleotide derivatives. If 5-oxo-GDP is treated with acid (e.g. formic acid), 5-imino-4O-imidazolone ribose diphosphate (IIm-DP) and (Fig. 3, step vi1) 5-amino-4O-imidazolone ribose diphosphate (AIm-DP) (Fig. 3, step vi2) can be formed (12). If 5-oxo-GDP is treated with methanol prior to acid, 5-diamino-4O-2-methyl-imidazolone ribose diphosphate (DMm-DP) (Fig. 3, step vi3) can be produced. The release of GDP-oxygenated adduct(s) from Rac1 produces a GDP-deficient form of Rac1 (apoRac1). ApoRac1 can be converted to an active guanine nucleotide binding state by the addition of a radical scavenger (i.e. ascorbate) in the presence of GDP or GTP. Given the cellular abundance of GTP, by mass action, the active guanine nucleotide binding form of Rac1 is likely to become GTP-bound leading to Rac1 activation in situ.

According to the proposed mechanism (Fig. 3), the Cys18 and the Phe28 side chain play a key role in radical-based perturbation of Rac1 GDP-binding interactions and generation of oxygenated GDP adduct(s) released from Rac1. Thus, mutation of these key residues should hinder -mediated Rac1 guanine nucleotide dissociation. Consistent with the proposed mechanism as shown in Fig. 1A, treatment of Rac1 with does not facilitate guanine nucleotide dissociation rates associated with the Rac1 variants, C18S and F28L. We have also characterized the guanine adduct(s) released from WT and variant Rac1 proteins upon exposure of ROS and RNS by MS to determine whether the proposed oxygenated GDP derivatives are produced. Adducts released from Rac1 upon exposure to were treated with methanol and acid and then analyzed by MS. Three main peaks with molecular masses of 415.26, 417.26, and 465.26 Da were observed, which correspond well with the proposed end products of -treated Rac1 (Fig. 4A). Given that the predicted molecular masses of AIm-DP, IIm-DP, and DMm-DP are 415.26, 417.26, and 465.26 Da, respectively, we have putatively assigned the three Rac1-released GDP-O2H derivatives as AIm-DP, IIm-DP, and DMm-DP, respectively. We were unable to detect MS peaks corresponding to the molecular weight of 8-DIm-GDP derivatives. The inability to detect these derivatives is consistent with our premise that the C8 site of {image1} is not exposed to solvent, so that the reaction product of the G·-DP C-8 with , 8-DIm-GDP, is unlikely to be produced and detected. The Rac1-derived oxygenated GDP derivatives were not detected when the Rac1 variants, C18S and F28L, were treated with (Fig. 4, B and C), providing further support that the Rac1 residues Cys18 and Phe28 play a key role in the production of the 5-DIm-GDP adduct. Additionally, when NO/O2 (·NO2) was used instead of to promote Rac1 guanine nucleotide dissociation, a nitrated GDP derivative (putatively assigned as 5-guanidino-4-nitroimidazole diphosphate (11)) was detected (not shown). The generation of this adduct is consistent with the proposed mechanism in Fig. 3, where reaction of the redox agent ·NO2 with the Rac1 Cys18 thiol can also produce Cys18·, and the resultant GDP radical then reacts with ·NO2 to produce the nitrated GDP adduct (11).



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  		FIG. 4.
Molecular weight determination of -mediated Rho GTPase Rac1-bound GDP dissociation products by mass spectrometry. MS analysis results for WT Rac1 (A), Rac1 C18S (B), and Rac1 F28L (C) are shown. Sample D does not contain Rac1 and thus serves as a GTPase control. Fractions containing the nucleotide derivative products from samples A-C as well as the control D were prepared and analyzed by MS as described under "Experimental Procedures." For each assigned chemical adduct, two additional satellite peaks can be observed because of the presence of isomers of the assigned chemical adducts (11, 12). amu, atomic mass units.

 
The key feature of the proposed mechanism (Fig. 3) is that the redox agents react with the Cys18 thiol in the GXXXXGK(S/T)C motif to produce Cys18·. Given the redox potentials of (E7 ~= 0.9 V versus normal hydrogen electrode (NHE)), ·NO2 (E7 ~= 1.0 V versus NHE) and OH· (E7 ~= 2.3 V versus NHE) (30), it is reasonable to postulate that the redox potential of the Rho GTPase Cys18· intermediate is lower than ~0.9 V (versus NHE). Thus, it is possible that any redox agent that can react with Cys18 thiol and possesses a redox potential higher than ~0.9 V (versus NHE), i.e. carbonate radical anion (E7 ~= 1.8 V versus NHE) (30) can facilitate Rho GTPase guanine nucleotide dissociation. Results from this study also indicate that peroxynitrite, if present at physiological concentrations, may serve as a redox agent for the redox-active Rho GTPases because peroxynitrite can be decomposed into two redox-active molecules, ·NO2 and OH· (30), that can facilitate guanine nucleotide dissociation of Rho GTPases.

Redox-mediated Thiol Modification—We show that treatment of Rac1 with the ROS, (or RNS, ·NO2), promotes guanine nucleotide dissociation (Fig. 1) giving rise to apoRac1 and a modified GDP adduct (Fig. 4) that is released from Rac1. Guanine nucleotide exchange can be achieved by addition of a radical scavenger, which promotes binding of the cellularly abundant GTP to produce active Rac1-GTP. Based on these in vitro observations, we would predict that apoRac1 Cys18· may be generated under conditions where an excess of redox agent (i.e. or ·NO2) is present over radical scavenger. Further reaction with , if present in excess, is expected to produce a Rac1 sulfonate adduct (). If OH· (H2O2/Cu2+) or NO is present instead of , S-sulfenated (Rac1-SOH) or S-nitrosylated Rac1 (Rac1-SNO), respectively, can be formed. Consistent with this premise, the addition of NO instead of ascorbate (Fig. 3) produces S-nitrosylated Rac1 (Rac1-SNO, not shown). Interestingly, radical-based sulfenation of a thiol in bovine serum albumin (BSA-Cys34) has been observed previously, in which the BSA-Cys34 thiol is effectively oxidized by H2O2 in the presence of transition metals to produce a BSA-Cys34 sulfenic acid adduct (i.e. BSA-SOH) (44). In addition, if glutathione (GSH) is present in conjunction with RNS or ROS (i.e. ·NO2, , or OH·), apoRac1-Cys18·, once formed, may react with GSH in the presence of nucleotide to generate Rac1 S-glutathiolation (Rac1-SSG), as radical-based formation of glutathione disulfide has been observed in a mixture of GSH and ·NO2 (45). Furthermore, H2O2 has been shown to promote formation of Ras-SSG in the presence of GSH (46). Because modification of the Rac1 Cys18 thiol (i.e. Rac1-SNO) renders Rac1 insensitive to (and ·NO2) (not shown), prolonged incubation of Rac1 with excess may produce a redox-inactive thiol-modified form of Rac1. In fact, redox-mediated thiol modification of other Rho GTPases may occur, as RhoA and Cdc42 contain the same Rac1 redox-active motif.

Rho GTPase Redox Regulation and Biological Relevance—Based on the results described here, we postulate that there exist two distinct mechanisms by which Rho GTPase activity is modulated by stimulation of Rho GTPase guanine nucleotide dissociation, small molecule and protein modulatory agents, i.e. redox agents and GEFs. Activation by one or both of these mechanisms is likely to depend on cellular conditions such as co-localization with a GEF or redox agent, GEF activation state, level of redox agents, and redox environment. Although a defined role for redox agents in direct cellular regulation of Rho GTPases has not yet been established, recent studies provide evidence for in vivo redox regulation of Rho GTPase activity. For example, it has been shown that the activation state of the Rho GTPases, Rac1 and RhoA, responds rapidly to changes in oxygen tension in a phosphatidylinositol 3-kinase and NADPH oxidase-dependent manner, and their coordinated actions regulate endothelial barrier function in endothelial cells of conduit pulmonary arteries (22). It has also been shown that Caki-1 cells exposed to hypoxia (1% O2, low oxygen tension) exhibit increased Cdc42, Rac1, and RhoA protein expression and activity (23). Overexpression and activation of Rho proteins is downstream of and dependent on the production of ROS, because specific inhibition of ROS-producing NADPH oxidase down-regulates GTPase activity (23). In contrast, previous studies indicate that blocking NADPH oxidase did not inhibit Rac1 activity under certain hypoxia-induced conditions in endothelial cells of conduit pulmonary arteries (22). Moreover, human neutrophil adherence to extracellular matrix proteins has been shown to induce an initial inhibition of stimulated ROS formation, and this is believed to result from inhibition of GEF-mediated Rac2 activation (47). Understanding the basic mechanism of GEF and redox-mediated regulation of Rho GTPase activity in vitro should help to delineate the complexities associated with in vivo regulation.

Given our results, we would predict that high levels of redox agent(s) and oxidative conditions may result in Rho GTPase thiol modification and redox inactivation of Rho GTPases. Therefore, thiol-modified forms of Rho GTPases may be insensitive to ROS production by NADPH oxidase. However, high levels of redox agents and oxidative conditions may be more representative of oxidative stress and pathophysiological rather than physiological conditions or conditions that give rise to oxidative burst, for instance, in response to microbial invasion (48).

Other GXXXXGK(S/T)C Motif-containing GTPases—In addition to the redox-active GXXXXGK(S/T)C motif-containing Rho GTPases, we have found that a number of Rab GTPases also possess a GXXXXGK(S/T)C motif. Rab GTPases comprise the largest branch of the Ras GTPase superfamily and are best known for their role in vesicular trafficking (49). Hence, we speculate that (i) redox agents, e.g. , ·NO2, and H2O2/Cu2+ (OH·), may modulate temporal and spatial cellular functions of the GXXXXGK(S/T)C motif-containing Rab GTPase subfamily GTPases; and (ii) the fundamental molecular mechanism of the redox-active Rab GTPases may be similar to that proposed for redox-active Rho GTPases.

In summary, we have identified and characterized a unique redox-active motif present in Rho family GTPases that is important for redox-mediated regulation of guanine nucleotide exchange activity in vitro. Moreover, the radical-based molecular mechanism of Rho GTPase guanine nucleotide exchange appears similar in nature to the mechanism characterized for Ras GTPases. The redox-active motif is present in other Ras superfamily GTPases (i.e. Rab GTPases), suggesting that redox regulation of GTPase signaling is more widespread than previously envisioned. The studies described here form the basis for the investigation of cellular regulation of Ras superfamily GTPases by redox agents.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants RO1CA89614-O1A1 and PO1 HL45100 (to S. L. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, University of North Carolina, 530 Mary Ellen Jones Bldg., Chapel Hill, NC 27599-7260. Tel.: 919-966-7139; Fax: 919-966-2852; E-mail: campbesl{at}med.unc.edu.

1 The abbreviations used are: GEF, guanine nucleotide exchange factor; ROS, reactive oxygen species; RNS, reactive nitrogen species; XO, xanthine oxidase; NOS, nitric-oxide synthase; MS, mass spectrometry; BSA, bovine serum albumin; WT, wild type; 8-DIm-DP, 8-diamino-4O-imidazolone ribose diphosphate; 5-DIm-DP, 5-diamino-4O-imidazolone ribose diphosphate; AIm-DP, 5-amino-4O-imidazolone ribose diphosphate; IIm-DP, 5-imino-4O-imidazolone ribose diphosphate; DMm-DP, 5-diamino-4O-2-methyl-imidazolone ribose diphosphate; NHE, normal hydrogen electrode; DTPA, diethylenetriaminepentaacetic acid. Back


   ACKNOWLEDGMENTS
 
We thank Constance A. Rogers and MinQi Lu for the preparation of protein samples used in this study. We are also grateful for the use of the UNC Michael Hooker Proteomics Core facility, which was supported in part by a gift from an anonymous donor for research targeted to proteomics and cystic fibrosis.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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