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Cardiovascular Research 75 (2007) 229 – 239
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Review
Nitric oxide cell signaling: S-nitrosation of Ras superfamily GTPases
Kimberly W. Raines a,b , Marcelo G. Bonini c , Sharon L. Campbell a,b,⁎
a
Department of Biochemistry and Biophysics, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, NC 27599-7260, United States
Lineberger Comprehensive Cancer Center, University of North Carolina, 530 Mary Ellen Jones Building, Chapel Hill, NC 27599-7260, United States
c
Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences,
National Institutes of Health, Research Triangle Park, NC 27709, United States
b
Received 29 December 2006; received in revised form 13 April 2007; accepted 18 April 2007
Available online 24 April 2007
Time for primary review 33 days
Abstract
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.
© 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
Keywords: Nitric oxide; Redox signaling; Small GTPases
1. Ras superfamily: Introduction
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 GTPbound 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
⁎ 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.
E-mail address: [email protected] (S.L. Campbell).
(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 radicalmediated 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
0008-6363/$ - see front matter © 2007 European Society of Cardiology. Published by Elsevier B.V. All rights reserved.
doi:10.1016/j.cardiores.2007.04.013
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K.W. Raines et al. / Cardiovascular Research 75 (2007) 229–239
U
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
U
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.
U
Consistent with our proposed mechanism of NO2-mediated
U
Ras guanine nucleotide dissociation [11], reaction of NO2 with
glutathione (GSH) and cysteine (CysSH) has been shown to
U
produce a glutathionyl radical (GS ) and cysteinyl radical
U
U
(Cys–S ), respectively [18–20]. In addition, NO2-mediated
S-nitrosation of GSH and CysSH proceeds through formaU
U
tion of a thiyl radical (i.e., GS and Cys–S ) and this process
was shown to be dominant over N2O3− or NO-mediated Snitrosation [18–20]. As we had previously observed that
S-nitrosation of Ras does not alter its structure or biochemical
U
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
U
(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 radicalmediated GTPase regulation and the target specificity of the
free radical toward the redox-active GTPases are reviewed.
2. Source of NO in the vasculature
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
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].
Table 1
Rate constants for UNO, UNO2 and RSU with select physiologically relevant compounds
Reaction
Rate constant
U
2 NO þ O
U NO
2
U
→2 NO
2
U
kf = 1.1 × 10 M s
kr = 8.0 × 104 M− 1s− 1
k ∼ 2.0 × 105
k = 6.6 – 9.4 × 104 M− 1s− 1
N2 O3 þ H2 O→2NO−2 þ 2Hþ
N2 O3 þ GSH→GSNO þ NO−2 þ Hþ
U
[114]
−1 −1
9
þ NO←→N2 O3
2
Reference
k = 2 × 106 M− 1s− 1
U
[120]
[121,122]
3 −1
k = 1.6 × 10 s
[64]
−1 −1
7
GSH þ NO2 →GS þ NO−2 þ Hþ
k = 2 × 10 M s
[123]
GS þ NO→GSNO
k = 1 × 109 M− 1s− 1
[64]
U U
U
U
GS þ GS →GSSG
9
k = 1.5 × 10 M s
U
U
GS þ GS− →GSSG −
U− þ O
GSSG
2 →GSSG
−1 −1
þ O2
U−
U
U
NO þ O − →ONOO−
2
ONOOH=ONOO− þ GSH→GSOH þ NO−2
[124]
−1 −1
kf = 6.6 × 10 – 6.0 × 10 M s
kr = 1.6 × 105 M− 1s− 1
k = 5 × 109 M− 1s− 1
7
8
9
−1 −1
k = 6.7–19 × 10 M s
−1 −1
k = 660 M s
[124]
[124]
[125,126]
[114]
K.W. Raines et al. / Cardiovascular Research 75 (2007) 229–239
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
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:
U
U
2 NO þ O2 →2 NO2
UNO þ NO →N O
2
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
U
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
U
NO2 production (reaction 1) particularly efficient in hydroU
phobic 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
U
(i.e., RS) which is nitrosated by recombination with NO in a
diffusion controlled reaction (reactions 1–3, Table 1) [62].
2 NO þ O2 Y2 NO2
d
d
d NO
2
þ R SHYRS þ NO
2
d
k ¼ 106 108 M1 s1
RS þ NO YRSNOðnitrosothiolÞ
d d
k ¼ 109 M1 s1
ðReaction1Þ
5. NO-mediated thiol nitrosation, the ionic pathway
U
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−
U
generation [63]. Similar to NO2, O2 and NO, N2O3 is likely
to be more soluble in hydrophobic environments where it is
U
stable and in equilibrium with its precursors NO and NO2
[64]. Importantly, the rate constant for N2O3 reaction with
231
2
3
N2 O3 ¼þ ON…NO−2 ðIonic pathwayÞ
U
U
RSH þ NO2 →RS þ NO−2 ðRadical pathwayÞ
U U
RS þ NO→RSNOðRadical pathwayÞ
U
U
RS þ RS →RSSRðRadical termination reactionÞ
6. NO and regulation of Ras and Ras-like GTPases in vivo
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
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K.W. Raines et al. / Cardiovascular Research 75 (2007) 229–239
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-α 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 NOC18, modulates the expression of Hypoxia-Inducible Factor-1α
(HIF-1α) via the Ras-mediated MAPK pathway [77].
Consistent with these results, expression of a dominant-negative form of Ras significantly suppressed NO-mediated HIF1α activation [78]. Thus, the increased levels of HIF-1α 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 HRas 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
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 oneelectron 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 (UNO2), to produce a Ras radical intermediate (Ras–
Cys118U). Further, our data indicate that formation of the Ras
radical intermediate is critical for O2U− or NO2U−mediated Ras
guanine nucleotide dissociation [11,14,17]. The mechanism is
U
fundamentally similar for both NO/O2 and O2 − in that either
UNO or O U− can react with the Ras–Cys118 thiol to produce a
2
2
Ras–Cys 118 -thiyl radical (Ras–Cys 118 U) intermediate
[11,14,17]. The Ras–Cys118U 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
U
GDP neutral radical (G –GDP) (Fig. 1) [11]. Ras-bound GU-DP
U
U
can further react with an additional NO2 or O2 − to produce
GDP–NO2 (5-nitro-GDP) or GDP–O2 (5-oxo-GDP) adducts,
U
respectively [11,14]. As formation of GDP+ and sequential
U
transformation to a G –GDP (neutral radical) will disrupt key
hydrogen bond interactions between Ras residues in the NKCD/
U
U
SAK motif and the guanine nucleotide base, the NO2 or O2 −
K.W. Raines et al. / Cardiovascular Research 75 (2007) 229–239
Fig. 1. Proposed radical based mechanism of redox mediated Ras guanine
U
nucleotide dissociation. Reaction of NO in the presence of O2 or NO2 with
118
the Ras Cys thiol, produces a Ras radical intermediate, proposed to be a
U
U
Ras thiyl radical (Ras–Cys118 ). Ras–Cys118 then electronically interacts
28
with Ras-bound GDP, possibly via the Phe side-chain, to initiate radicalbased conversion of Ras-bound GDP into a Ras-bound GDP neutral radical
U
U
U
(G –GDP). Ras-bound G –DP can further react with an additional NO2 to
+U
produce GDP–NO2 (5-nitro-GDP). As formation of GDP − and sequential
U
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–π 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.
233
mediated thiyl–guanine radical propagation process promotes
GDP release from Ras (Fig. 1).
Similar to formation of small molecular weight nitrosothiols (i.e., GSNO), Ras–SNO can be produced by either a
U
N2O3 and/or an NO2-mediated process in the presence of NO.
U
However, our data support NO2-mediated production of SNO
via a radical mediated pathway in which reaction of Ras with
U
NO2 produces Ras–Cys118U, 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–Cys118U 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 GTPbound form of Ras, redox mediated GDP dissociation must be
followed by GTP binding to promote Ras activation in situ.
U
U
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
U
U
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
U
U
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
U
NO2 and a second round of GNE may ensue.
U
U
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].
U
U
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,
234
K.W. Raines et al. / Cardiovascular Research 75 (2007) 229–239
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.
8. Redox regulation of NKCD containing Ras
superfamily GTPases
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
U
Ras [11]. Thus, a similar mechanism of NO2-mediated
guanine nucleotide dissociation is expected for these NKCDmotif containing GTPases. Consistent with this observation,
U
we have shown that NO/O2 and O2 − facilitate guanine
nucleotide dissociation from Rab3A and Rap1 (Fig. 3) [11].
9. Redox regulation of Rho GTPases
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 motifcontaining 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.
U
U
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
U
oxidase activity [96,106–108], that O2 − may stimulate
guanine nucleotide dissociation from Rac in vivo (23). The
U
fundamental mechanistic process for O2 −-mediated GNE
associated with GXXXXGK(S/T)C motif-containing
U
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.
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.
K.W. Raines et al. / Cardiovascular Research 75 (2007) 229–239
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,
U
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.
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
U
U
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
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].
235
Fig. 5. Effects of redox agents on the rate of guanine nucleotide dissociation
from GXXXXGK(ST)C motif-containing Rho GTPases. To monitor O2mediated Rho GTPase GDP dissociation, xanthine oxidase generated O−2 ,
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 NO2U [16]. Reproduced with permission of the
American Society for Biochemistry and Molecular Biology Copyright 2005.
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
236
K.W. Raines et al. / Cardiovascular Research 75 (2007) 229–239
accessible redox-active RhoA Cys 20 thiol, and the αphosphate 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,
U
U
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 RhoAbound 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
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.
Acknowledgements
We thank Jongyun Heo for intellectual contributions and
assistance with figures.
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