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Cardiovascular Research 2007 75(2):229-239; doi:10.1016/j.cardiores.2007.04.013
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Copyright © 2007, European Society of Cardiology

Nitric oxide cell signaling: S-nitrosation of Ras superfamily GTPases

Kimberly W. Rainesa,b, Marcelo G. Boninic and Sharon L. Campbella,b,*

aDepartment of Biochemistry and Biophysics, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, NC 27599-7260, United States
bLineberger Comprehensive Cancer Center, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, NC 27599-7260, United States
cLaboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709, United States

* Corresponding author. Lineberger Comprehensive Cancer Center, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, NC 27599-7260, United States. Tel.: +1 919 966 7139; fax: +1 919 966 2852. campbesl{at}med.unc.edu

Received 29 December 2006; revised 13 April 2007; accepted 18 April 2007


   Abstract
Top
 Abstract
1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
The Ras superfamily of small GTPases cycle between inactive GDP-bound and active GTP-bound states to modulate a diverse array of processes involved in cellular growth control. While the basic mechanisms by which GTPase regulatory proteins regulate GTPase substrates have been revealed through numerous studies, detailed studies into the mechanism(s) of free radical-mediated GTPase regulation have only more recently been tackled. This article reviews the mechanism of free radical-mediated GTPase regulation and shows nitric oxide can serve as important regulator of small GTPase proteins (i.e. Ras and RhoA) through protein modifications such as S-nitrosation.

KEYWORDS Nitric oxide; Redox signaling; Small GTPases


   1. Ras superfamily: Introduction
Top
 Abstract
 1. Ras superfamily: Introduction
2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
The Ras superfamily consists of a number of small monomeric GTPases including Ras, Rho and Rab subfamilies. These GTPases cycle between active GTP- and inactive GDP-bound states to regulate a diverse array of biological processes including cell proliferation, apoptosis, vesicular and nuclear transport [1,2]. Given their critical cellular roles, the GDP- and GTP-bound states of Ras superfamily GTPases are highly regulated by multiple cellular factors. Guanine nucleotide exchange factors (GEFs) [3–6], facilitate exchange of GDP with GTP to produce the active GTP-bound form of the GTPase, whereas GTPase-activating proteins (GAPs) enhance the slow intrinsic rate of GTP hydrolysis to produce the inactive GDP-bound form of the GTPase [5,7,8]. In addition, guanine nucleotide inhibitors (GDIs) down-regulate the activity of a subset of GTPases (e.g., Rho and Rab subfamilies) by preventing membrane association as well as inhibiting guanine nucleotide dissociation [3,5,9,10]. In addition to these regulatory proteins, the free radical nitric oxide (NO), has been shown to react with Ras and other Ras-related GTPases to regulate their activity [11]. While the basic mechanisms by which GEFs, GAPs and GDIs regulate their respective GTPase substrates have been revealed through numerous studies, detailed studies to investigate the mechanism(s) of free radical-mediated GTPase regulation have only recently been tackled since the emergence of protein modifications, such as nitrosation and glutathionation. Nitrosation is often used interchangeably with nitrosylation, but differs slightly because by definition nitrosation is the covalent attachment of a NO group to an amine, thiol or hydroxyl residue, while nitrosylation is the addition of NO without a change in formal charge of the substrate.

A number of GTPases including DexRas [12], Rap1A [11,13,14], Ran [15], Rac1, Cdc42, RhoA [16], Rab3A [11] and H-, K- and N-Ras [17] contain redox-active residue(s) that are sensitive to NO, particularly the NO derivative, nitrogen dioxide (NO2). We have investigated the mechanism by which NO modulates the activity for several of these GTPases, with particular focus on the Ras GTPase, and found that treatment of Ras with NO in the presence of O2 or NO2 produces a Ras-thiyl radical intermediate resulting in GDP dissociation from Ras [11,17]. This, in turn, can lead to activation or inactivation of Ras depending on the concentration of NO and oxidizing potential of the medium.

Consistent with our proposed mechanism of NO2-mediated Ras guanine nucleotide dissociation [11], reaction of NO2 with glutathione (GSH) and cysteine (CysSH) has been shown to produce a glutathionyl radical (GS) and cysteinyl radical (Cys–S), respectively [18–20]. In addition, NO2-mediated S-nitrosation of GSH and CysSH proceeds through formation of a thiyl radical (i.e., GS and Cys–S) and this process was shown to be dominant over N2O3– or NO-mediated S-nitrosation [18–20]. As we had previously observed that S-nitrosation of Ras does not alter its structure or biochemical activity [21], we speculated that NO2-mediated guanine nucleotide release occurs through a radical propagation mechanism involving Ras thiyl radical conversion to a Ras–GDP guanine radical. The guanine base is particularly sensitive to reaction with free radicals [22–27], and formation of a guanine radical is likely to alter interactions with Ras resulting in release of Ras bound GDP [11]. Our present data is consistent with this mechanism of redox mediated GDP dissociation for Ras as well as other Ras-related proteins [11,14,17,28,29]. Moreover, we have observed that other radical species (i.e., superoxide (O2–)) act on redox active sites within select Ras superfamily GTPases to promote dissociation of GDP via a similar mechanism [11,14,28]. In this context, the mechanism of free radical-mediated GTPase regulation and the target specificity of the free radical toward the redox-active GTPases are reviewed.


   2. Source of NO in the vasculature
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
NO is produced endogenously by a family of nitric oxide synthase [30] enzymes in a 2-step oxidation of L-arginine to NO and L-citrulline [31]. Three distinct isoforms of NOS have been characterized [32]: neuronal NOS (nNOS) and endothelial NOS (eNOS), which are constitutively expressed and activated by influx of intracellular calcium. A third isoform, inducible NOS (iNOS), is regulated by transcriptional activation in response to inflammation and cytokine production. Vascular NO is derived primarily from eNOS in the endothelial cell; however, iNOS can also be induced in many cell types including endothelial and vascular smooth muscle cells, by cytokine stimulation [33,34].


   3. In vivo nitrosative cell signaling
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
One of the best characterized targets of NO is the heme of guanylate cyclase. Nitrosation of guanylate cyclase leads to activation of the second messenger, cyclic guanosine monophosphate (cGMP) which regulates a wide variety of physiological functions, including vascular tone, platelet aggregation, inflammation, neurotransmission, gastric emptying, and hormone release [35,36].

Cysteine residues in proteins are also well known targets of NO. Thiol nitrosation has been detected in cell culture based assays under a myriad of conditions [37–40], and can alter both protein expression and function [41]. For example, cellular analyses have demonstrated that S-nitrosation can inhibit caspases [42,43], c-Jun-N-terminal kinase [44,45], protein tyrosine phosphatases [46,47], and ornithine decarboxylase [48]. In addition, hemoglobin S-nitrosation increases oxygen affinity and modulates vasodilatation [49,50]. Vascular functions regulated by S-nitrosation include ventilation, heart rate, and blood pressure [51].

S-nitrosation can promote post-translational modification of protein thiols in select proteins to modulate their activity. However, NO can also regulate the activity of proteins by mechanisms that are independent of S-nitrosation. Our studies on the Ras GTPase indicate that it is cysteine–thiol radical formation rather that S-nitrosation, that alters Ras activity [14,28]. Although NO can react with protein thiols through both radical and non-radical based mechanisms, we will focus herein on the role of the radical pathway in regulation of Ras superfamily GTPase activity.


   4. NO-mediated thiol nitrosation, the radical pathway
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
NO is a stable free radical gas, which is hydrophobic in nature and extremely small in size. These properties, together with its high reactivity with other radicals and metal centers, render a freely diffusible and selective messenger that can modulate a multitude of physiological processes [30,52–59]. However, NO is a poor nitrosating compound. As physiological levels of molecular oxygen are required for nitrosothiol formation from NO [18,19,60], increasing attention has been devoted to the autoxidative product of NO, NO2, as a cellular nitrosating agent. Both NO and oxygen tend to concentrate in non-polar environments (e.g. plasma membrane) which makes NO2 production (reaction 1) particularly efficient in hydrophobic compartments. NO2 is a moderate oxidant [61] with extremely high reaction rate constants for its reaction with biothiols (k=107 M–1s–1) (Table 1). The product formed in this reaction is invariably the biothiol-derived thiyl radical (i.e., RS) which is nitrosated by recombination with NO in a diffusion controlled reaction (reactions 1–3, Table 1) [62].


Formula 1



Formula 2



Formula 3


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  		Table 1 Rate constants for NO, NO2 and RS with select physiologically relevant compounds

 

   5. NO-mediated thiol nitrosation, the ionic pathway
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
Once produced, NO2 may also recombine with available NO to produce N2O3, in an extremely fast diffusion controlled radical–radical recombination reaction (Table 1). N2O3 is considered to be a potent electrophilic compound capable of nitrosating thiol moieties in peptides and proteins [63]. However N2O3 is also subject to hydrolysis via –OH attack on the electrophilic nitrogen atoms, resulting in NO2– generation [63]. Similar to NO2, O2 and NO, N2O3 is likely to be more soluble in hydrophobic environments where it is stable and in equilibrium with its precursors NO and NO2 [64]. Importantly, the rate constant for N2O3 reaction with glutathione has recently been re-determined and was shown to be more than one order of magnitude lower than that for N2O3 hydrolysis [64]. This fact combined with the excess water available in aqueous solution makes thiol nitrosation via N2O3 less likely in hydrophilic environments.

Thus the pKa, location and three dimensional environment of the cysteine within the protein, are fundamental in determining the nitrosothiol yield as well as the prevailing mechanism [65]. Thiol nitrosation may be summarized by the following set of reactions:


Formula 4


   6. NO and regulation of Ras and Ras-like GTPases in vivo
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
In the instance of the GTPase protein family, several GTPases contain a redox-sensitive NKCD motif, and we have shown that three distinct GTPases in the sub-class of Ras superfamily GTPases (i.e., H-Ras, Rap1A and Rab3A) containing this motif, can be regulated by NO and other redox active species [14]. To date, NO-mediated redox regulation is best characterized for Ras proteins.

Several cell based assays have shown that NO can react with Ras, promote guanine nucleotide exchange (GNE) and Ras activation [66–70]. Although limited studies have been conducted on the role of endogenously generated NO in regulation of Ras function, support for such regulation in cells exists. For example, Yun et al. have demonstrated that NO derived from nNOS can lead to Ras activation in primary rat cortical cells [71]. Their results suggested that Ras is a physiologic target of endogenously produced NO through a signaling pathway involving the N-methyl-D-aspartate (NMDA) glutamate receptor, with NO-mediated Ras activation playing an important role in long-lasting neuronal responses in these cells [71,72]. Intriguingly, it has recently been shown that NO-induced activation of Ras causes inhibition of NOS [73], suggesting NO feedback regulation.

Lander and coworkers showed that human T cells treated with exogenously generated NO, contain an increased percentage of Ras in the GTP-bound form [69]. Furthermore, NO-induced activation of Ras led to modulation of signal transduction pathways downstream of Ras. For example, NO-induced activation of Ras led to stimulation of the mitogen-activated protein kinase (MAPK) pathway in rabbit aortic endothelial cells, resulting in tyrosine phosphorylation of various cellular proteins (i.e., focal adhesion kinase, Src kinase, and MAPK) [74]. Moreover, Deora et al. have shown that NO-producing agents trigger recruitment, Ras activation of phosphatidylinositol 3'-kinase (PI3K) and Raf in human T cells, leading to Ras-dependent activation of extracellular signal regulated kinases (ERKs) [75]. They further identified a NO-mediated Ras signaling pathway, whereby activation of Ras by NO, recruits and activates Raf-1, leading to Elk-1 phosphorylation and tumor necrosis factor-{alpha} messenger RNA induction, suggesting that modulation of Ras activity by NO in T cells may play a critical role in Elk-1-induced activation of T lymphocytes, host defense and inflammation [76]. Subsequent work demonstrated that NO released endogenously from NOS and exogenously from the NO-producing agent NOC-18, modulates the expression of Hypoxia-Inducible Factor-1{alpha} (HIF-1{alpha}) via the Ras-mediated MAPK pathway [77]. Consistent with these results, expression of a dominant-negative form of Ras significantly suppressed NO-mediated HIF-1{alpha} activation [78]. Thus, the increased levels of HIF-1{alpha} under hypoxic conditions in prostate cancer cells [77] may result from NO-mediated Ras activation of the MAPK pathway [79].

Mallis et al. demonstrated activation of the MAPK pathway in NIH-3T3 fibroblasts via S-glutathiolation and S-nitrosation of multiple thiols in H-Ras by exogenous thiol oxidants such as: hydrogen peroxide, S-nitrosoglutathione, diamide, glutathione disulphide and cystamine [80]. Although direct modification of Ras may mediate MAPK activation, it is also probable that indirect mechanisms of MAPK pathway activation occur, for example, by inhibition of associated phosphatases [46].

In contrast to these findings, endogenous production of NO has been shown to block calcium-induced activation of ERK through a mechanism involving S-nitrosation of Ras in human embryonic kidney cells over-expressing nNOS [81]. Moreover, addition of exogenous NO agents led to inhibition of H-Ras mediated ERK activation in mouse fibroblasts [89]. The investigators speculated that H-Ras was inactivated under these conditions by nitrosation of C-terminal cysteines in Ras, which caused enhanced palmitoyl turnover and reduced association of Ras with membranes [89]. Moreover, it has been suggested that vascular smooth-muscle cells treated with NO donors inhibit epidermal growth factor-mediated Ras activation of Raf-1 [82]. Thus, it appears that Ras can be either activated or inactivated by NO depending on the cellular conditions.


   7. NO-mediated regulation of Ras activity through cysteine 118 radical formation
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 
It is widely accepted that thiol modifications, such as nitrosation, oxidation and glutathiolation are recurrent events leading to key redox-based signals which modulate cellular growth. All products mentioned above can be converted back to the reduced thiol (RSH) form at the expense of reduced cellular thiols such as glutathione (dithiols, sulfenic acid) or ATP/thiols (sulfinic acid) [83,84].

Although the cysteinyl radical commonly participates in reactions leading to several chemical modifications, it is rarely proposed as the actual modulator of enzyme activation. This is likely to happen, because contrary to the other species which are relatively long-lived in vivo, thiyl radicals tend to disappear quickly (milliseconds) and are not detectable in a variety of systems. Thus, cysteinyl radicals are likely to be formed as intermediaries of the nitrosation process.

We have recently shown that exposure of Ras to one-electron oxidizing agents (oxidants that will generate protein radical intermediates) induce Ras guanine nucleotide dissociation, a crucial step in the regulation of Ras. Moreover, we have recently conducted EPR experiments that provide unequivocal detection of a Ras thiyl radical, produced under exposure of the protein to NO in aerated buffers. The thiyl radical production paralleled a marked increase in the guanine nucleotide dissociation rate. Importantly, Ras mutated at Cys118 or containing blocked thiols were not subject to free radical induced GNE nor produced an EPR detectable thiyl radical signal. Of note, Ras nitrosated at Cys118 has been shown to exhibit similar structure and GNE rates as the non-nitrosated form of Ras [21]. Contrary to the belief that S-nitrosation leads to an activated state of Ras, recent findings demonstrate that nitrosation is more likely to be a reversible end product of exposure to NO. As previously anticipated [85,86] these data taken together, constitute compelling evidence for the involvement of a thiyl radical intermediate in modulation of Ras activity.

Like Ras, other Ras-related proteins (i.e., Ran, DexRas) have been shown to undergo S-nitrosation in the presence of both endogenous and exogenous sources of NO [17,66,68,69,87,88]. Our results suggest that Ras thiol nitrosation is likely to be mediated by the NO autoxidation product, nitrogen dioxide radical (NO2), to produce a Ras radical intermediate (Ras–Cys118). Further, our data indicate that formation of the Ras radical intermediate is critical for O2– or NO2–mediated Ras guanine nucleotide dissociation [11,14,17]. The mechanism is fundamentally similar for both NO/O2 and O2– in that either NO2 or O2– can react with the Ras–Cys118 thiol to produce a Ras–Cys118-thiyl radical (Ras–Cys118) intermediate [11,14,17]. The Ras–Cys118 then electronically interacts with Ras-bound GDP, possibly via the Phe28 side-chain, to initiate radical-based conversion of Ras-bound GDP into a Ras-bound GDP neutral radical (G–GDP) (Fig. 1) [11]. Ras-bound G-DP can further react with an additional NO2 or O2– to produce GDP–NO2 (5-nitro-GDP) or GDP–O2 (5-oxo-GDP) adducts, respectively [11,14]. As formation of GDP+ and sequential transformation to a G–GDP (neutral radical) will disrupt key hydrogen bond interactions between Ras residues in the NKCD/SAK motif and the guanine nucleotide base, the NO2 or O2– mediated thiyl–guanine radical propagation process promotes GDP release from Ras (Fig. 1).


Figure 1
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  		Fig. 1 Proposed radical based mechanism of redox mediated Ras guanine nucleotide dissociation. Reaction of NO in the presence of O2 or NO2 with the Ras Cys118 thiol, produces a Ras radical intermediate, proposed to be a Ras thiyl radical (Ras–Cys118). Ras–Cys118 then electronically interacts with Ras-bound GDP, possibly via the Phe28 side-chain, to initiate radical-based conversion of Ras-bound GDP into a Ras-bound GDP neutral radical (G–GDP). Ras-bound G–DP can further react with an additional NO2 to produce GDP–NO2 (5-nitro-GDP). As formation of GDP+– and sequential transformation to a G–GDP will disrupt key hydrogen bond interactions between Ras residues in the NKCD/SAK motif and the guanine nucleotide base. The n–{pi} interaction between the neutral Ras Phe28 phenyl side-chain and the GDP-bound guanine base is shown in wavy lines. Other Ras residues involved in Ras GDP-binding interactions are omitted for clarity.

 
Similar to formation of small molecular weight nitrosothiols (i.e., GSNO), Ras–SNO can be produced by either a N2O3 and/or an NO2-mediated process in the presence of NO. However, our data support NO2-mediated production of SNO via a radical mediated pathway in which reaction of Ras with NO2 produces Ras–Cys118, which can then react with NO to generate Ras–SNO [17]. Moreover, S-nitrosation of Ras at Cys118 does not promote Ras GNE [11]. Thus, we proposed and provided evidence that a Ras–Cys118 intermediate, as opposed to Ras–SNO, electronically couples with Ras-bound GDP to facilitate Ras guanine nucleotide dissociation [11,14].

As GNE is required to generate the biologically active GTP-bound form of Ras, redox mediated GDP dissociation must be followed by GTP binding to promote Ras activation in situ. While NO2 and O2– can facilitate nucleotide dissociation from Ras, nucleotide association does not occur in the absence of a radical quenching agent. Addition of a radical quenching agent (i.e., ascorbate) facilitates NO2 or O2–-mediated Ras GNE [11,14], most likely due to removal of Ras radical intermediate(s). If NO serves as a quenching agent, the resultant reaction product is S-nitrosated Ras (Ras–SNO) [11,14]. Although our studies indicate that Ras–SNO does not directly promote Ras GNE, Ras nitrosation may function to prevent further radical reaction processes, since the S-nitrosated Cys118 thiol moiety can not react with NO2, GS and NO to produce a Ras radical species. If denitrosation of Ras–SNO occurs by transfer of NO to other biomolecules, such as glutathione or ascorbate, denitrosated Ras could then potentially react with NO2 and a second round of GNE may ensue.

Prolonged incubation of NO2 or O2– with Ras in the presence of GTP but in the absence of a radical quenching agent primarily induces formation of an inactive apo-Ras state, as only a small fraction of the available Ras (~10%) is present as Ras GTP. These results imply that redox agents can serve to terminate Ras signaling by promoting fast release of GDP or GTP from Ras to produce an apo-Ras state which cannot undergo Ras GNE in the absence of a radical scavenger. A radical scavenger may then be necessary to recover inactive apo-Ras into a GTP-binding form of Ras. Consistent with this premise, we have observed efficient in vitro GTP re-association in the presence of a radical quenching agent to produce an active GTP-bound Ras (~50%), which in turn, promotes binding of Ras to the Ras binding domain of Raf-1 [14,89].

These results suggest that for NO2 and O2– to promote Ras GNE and Ras activation, radical scavenging agents may be required. Hence, if Ras is exposed to physiological levels of redox agents and reducing biomolecules are present at high enough levels to function as radical scavenger agents [90,91], Ras activation may occur. In contrast, exposure to higher concentrations of redox agents and lower reducing potential may result in Ras inactivation. This may occur through distinct mechanisms (Fig. 2), as highlighted above, or by modification of additional residues in Ras that are partially protected, relative to Cys118. For example, nitrosation of carboxyl-terminal cysteines in Ras, normally targeted for palmitoylation, may lead to release of Ras from the membrane and inhibition of Ras activity [89]. Moreover, nitrosation of additional residues in Ras (i.e., tyrosine and other cysteines) may result in Ras destabilization.


Figure 2
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  		Fig. 2 Differential activity of redox active Ras superfamily GTPases in response to reactive oxygen and nitrogen species under different cellular conditions. In vivo redox potential mediates the cellular outcome of redox agents on redox active Ras superfamily GTPase activity. In a high cellular reducing environment, redox agents are likely to activate redox active GTPases, whereas in a strong oxidizing environment redox agents more likely to promote inhibition. Small GTPases (i.e., RhoA) containing a (GXXXCGK(S/T)C) motif, are more susceptible disulfide formation in the presence of redox agents and thus more prone to inactivation.

 

   8. Redox regulation of NKCD containing Ras superfamily GTPases
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
9. Redox regulation of...
 10. Future directions
 References
 
Ras superfamily GTPases encompass several gene families that regulate a number of events in the eukaryotic cell [92]. These proteins share amino acid identity with the classical Ras proteins and have numerous structural features in common. Intriguingly, the spatial orientation of the Phe28 and Cys118 side chains (Ras numbering) within the NKCD motif of Rap1A and Rab3A is similar to that observed for Ras [11]. Thus, a similar mechanism of NO2-mediated guanine nucleotide dissociation is expected for these NKCD-motif containing GTPases. Consistent with this observation, we have shown that NO/O2 and O2– facilitate guanine nucleotide dissociation from Rab3A and Rap1 (Fig. 3) [11].


Figure 3
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  		Fig. 3 Comparison of NO-mediated guanine nucleotide dissociation rates of NKCD motif-containing small GTPases in the presence of O2. Rates of apparent mant-GDP dissociation from the small GTPases (0.5 µM), Rab3A, Rap1A, wtRas and Ras C118S, in the presences of NO2 were determined by fitting the data to a simple exponential decay. The decrease in fluorescence emission at 460 nm was recorded as a function of time. For comparison, intrinsic GDP dissociation rates for Rap1A, Rab3A, wtRas, and the Ras C118S variant were also measured [11]. Reproduced with permission of Elsevier B.V. Copyright 2005.

 

   9. Redox regulation of Rho GTPases
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
10. Future directions
 References
 
Similar to Ras GTPases, Rho family GTPases regulate a host of cellular processes involved in regulation of cellular growth control [93–95]. However, Rho proteins differ from Ras in that they also regulate pathways that modulate cell morphology and motility through actin cytoskeletal rearrangements as well as oxidant production [93,94,96,97]. The Rab and Sar1/Arf subclasses are best known for their role in vesicle trafficking, whereas the Ran sub-class of GTPases is involved in regulation of nucleocytoplasmic transport and microtubule organization [9,98–100]. As molecular switches, these GTPases regulate both temporal and spatial cellular functions [101].

Although Rho GTPases, like Ras, can regulate pathways that control cellular growth, Rho GTPases interact with distinct regulatory factors and cellular targets to mediate pathways that control these processes [93–95]. We have identified a distinct class of redox-active GTPases conserved in nearly 50% of all Rho subfamily GTPases (Fig. 4), 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 sequence is designated the GXXXXGK(S/T)C motif. For comparison, members of the Ras subfamily of GTPases (i.e., H–, K–, N–Ras and Rap1A) possess a NKCD motif which forms interactions with the guanine nucleotide base and contains a redox-active cysteine, Cys118 (Ras numbering) [11,14,17,68]. Inspection of NMR and X-ray crystal structures for GXXXXGK(S/T)C motif-containing Rho GTPases (i.e., Rac1, RhoA and Cdc42) [102–104], indicates that the Cys18 thiol (Rac1 numbering) in the GXXXXGK(S/T)C motif is solvent accessible, suggesting that reactive oxygen species and reactive nitrogen species [105] may be able to target the Cys18 thiol.


Figure 4
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  		Fig. 4 Spatial architecture of the Rac and Cdc42 GXXXX(S/T)C18 motif and the RhoA GXXXX(S/T)C16+C20 motif. The distance and spatial orientation of the Phe28 side-chain, nucleotide ligand, and Cys18 thiol group in the GXXXX(S/T)C18 motif of Rac and Cdc42 is presented. The distance and spatial orientation of the Cys16 and Cys20 thiol group in the GXXXX(S/T)C16+C20 motif of RhoA is also presented. The scheme was generated using PDB 1CRQ and RASMOL [109,119].

 
We have recently shown that both NO2 and O2– can enhance the rate of guanine nucleotide dissociation for Rac1, RhoA and Cdc42 GTPases containing the GXXXXGK(S/T)C motif in vitro (Fig. 5) [16], and speculated that since the Rho family GTPase co-localize and modulate NADPH oxidase activity [96,106–108], that O2– may stimulate guanine nucleotide dissociation from Rac in vivo (23). The fundamental mechanistic process for O2–-mediated GNE associated with GXXXXGK(S/T)C motif-containing GTPases appears similar to that of O2–-mediated GNE of NKCD-containing GTPases [16]. The only difference lies in the initial thiyl radical formation site of the GTPases.


Figure 5
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  		Fig. 5 Effects of redox agents on the rate of guanine nucleotide dissociation from GXXXXGK(ST)C motif-containing Rho GTPases. To monitor O2-mediated Rho GTPase GDP dissociation, xanthine oxidase generated O2–, was accumulated in solution prior to incubation with 1 µM [3H]GDP-loaded Rho GTPases (Rac1, RhoA, or Cdc42). Aliquots were withdrawn, spotted onto nitrocellulose filters and radioactivity was determined using a Beckman–Coulter scintillation counter. The resultant radioactivity values of Rho GTPase-bound [3H]GDP were converted into the mole fraction of nucleotide per mole of total Rho GTPase. Data presented represent mean values from triplicate measurements. As a control, the intrinsic GDP dissociation rates of Rho GTPases were also measured using identical experimental conditions in the absence of redox agents. Similar results were obtained in the presence of NO2 [16]. Reproduced with permission of the American Society for Biochemistry and Molecular Biology Copyright 2005.

 
This GXXXXGK(S/T)C motif is also found in other Rho family GTPases (i.e., RhoT and TC10), several Rab GTPases as well as the Ran family GTPase, TC4. Hence, we speculate that O2– and NO2 may modulate temporal and spatial cellular functions of the GXXXXGK(S/T)C motif-containing Rab and Ran GTPases via a molecular mechanism similar to that proposed for redox-active Rho GTPases.

Interestingly, a variant form of the GXXXXGK(S/T)C motif, which contains an additional cysteine (Cys16, RhoA numbering) designated as the ‘GXXXCGK(S/T)C motif’, is found in a subset of redox-active GTPases, including RhoA and RhoB. In RhoA, the redox-active Cys20 (equivalent to Rac1 Cys18) is ~3.6 and ~10.3 Å away from Phe30 (equivalent to Phe28 in Ras and Rac1) and Cys16 (not present in Ras and Rac1), respectively [102]. Although the redox architecture of RhoA [102] is nearly identical to that of Rac1 and Cdc42 [103,104], electronic interaction between Cys16 and Cys20 in RhoA may give rise to distinct redox properties compared to Rho GTPases containing a single cysteine within the GXXXXGK(S/T)C motif.

Consistent with this premise, we have shown that reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the redox-active motif [109]. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to down-regulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity (Fig. 2). The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, we have shown that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA, but not Rac1 or Cdc42, to produce a RhoA thiol–cisplatin–thiol adduct, leading to inactivation of RhoA [16]. Thus, in addition to redox agents, platinated-chemotherapeutic agents also modulate the cellular activity of GTPases containing the GXXXCGK(S/T)C motif.

According to the RhoA–GDP crystal structure [102], the buried RhoA Cys16 thiol is ~10.3 Å away from the solvent accessible redox-active RhoA Cys20 thiol, and the {alpha}-phosphate of the bound GDP intercalates between these two RhoA cysteine thiols. Therefore, RhoA Cys16 and Cys20 thiols in the RhoA–GDP complex are not well positioned for disulfide formation or platin–thiol adduct formation. Thus, given our observations that redox agents (NO2 and O2–) promote RhoA guanine nucleotide dissociation, we speculated that RhoA Cys16–Cys20 disulfide formation may require release of the RhoA-bound guanine nucleotide to facilitate interaction between the RhoA Cys16 and Cys20 thiols. Moreover, formation of a RhoA thiol–cisplatin–thiol adduct may require a conformational rearrangement in RhoA, as reaction of cisplatin with the Cys20 thiol may destabilize guanine nucleotide binding interactions leading to release of RhoA-bound GDP to produce GDP-deficient RhoA. Consistent with this premise, we have observed that redox-mediated disulfide formation as well as cisplatin thiol adduct formation results in a form of RhoA that does not bind GDP or GTP [109]. These results are perhaps not unexpected, given recent studies that phenylarsine oxide inactivates RhoA by forming a bridge between Cys20 and Cys16, resulting in a guanine nucleotide deficient form of the protein [110].

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 GNE activity in vitro. Moreover, the radical-based molecular mechanism of Rho GTPase GNE 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.


   10. Future directions
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
References
 
Results from our studies on the Ras GTPase indicate that NO can promote formation of a cysteine–thiol radical, which alters Ras activity through a radical propagation mechanism leading to guanine nucleotide oxidation and release of GDP from Ras. However, S-nitrosation of this cysteine does not affect Ras activity. Hence, the mechanism of NO-mediated regulation of Ras activity is distinct from what has previously been observed in other systems, in which protein S-nitrosation alters cellular function. Thus, the studies described herein form the basis for further investigation of cellular regulation of Ras superfamily GTPases by redox agents. For example, it would be helpful to better characterize conditions by which Ras is activated or inactivated by redox agents in various cellular conditions. It will also be of interest to understand how Ras GEFs and GAPs cooperate in the presence of redox agents. Moreover, we have shown that several Ras related GTPases are redox active. In particular, redox active solvent accessible cysteines are highly conserved in Rab GTPases, and we speculate that Rab GTPases may be sensitive to redox control. Future studies will be required to investigate Rab redox activity. Additionally, although we have demonstrated that redox active Rho GTPases respond differently to redox agents and co-localize with endogenous sources of redox agents, it will be of interest to investigate the regulation of Rho GTPases in vivo.

Thiols are major targets for biological oxidants because of the low oxidizing potential of cysteine and their relatively low pKa, which render them excellent nucleophiles. Whereas Ras nitrosation and other oxidized forms of the Ras thiol may not modulate Ras activity directly, we speculate that oxidation of the redox active cysteine in Rho GTPases may alter GTPase activity due to its location in the phosphoryl binding loop. In support of this premise, direct S-nitrosation of RhoA has recently been shown to inhibit RhoA and ERK signaling to inhibit vascular smooth muscle cell proliferation [111].

In the case of Ras, SNO modification does not appear to directly correlate with changes in Ras biochemical activity. Although other oxidation species of Ras have been observed in cells (Ras SSG [112]), it is unclear whether these alternate modifications directly alter Ras activity. Evidence suggesting that thiyl radicals are recurrent reactive intermediates produced during oxidative stress conditions is mounting [11,86,113–115]. Thus, development of methods such as electron paramagnetic resonance coupled freeze-trap or spin-trap technologies for direct detection of thiyl radicals [114,116–118], rather than SNO or other oxidative thiol end products, should prove useful for detection of redox active GTPases whose activity is modulated under a variety of different redox conditions.

Time for primary review 33 days


   Acknowledgements
 
We thank Jongyun Heo for intellectual contributions and assistance with figures.


   References
Top
 Abstract
 1. Ras superfamily: Introduction
 2. Source of NO...
 3. In vivo nitrosative...
 4. NO-mediated thiol...
 5. NO-mediated thiol...
 6. NO and regulation...
 7. NO-mediated regulation of...
 8. Redox regulation of...
 9. Redox regulation of...
 10. Future directions
 References
 

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