(Received for publication, July 26, 1996, and in revised form, October 31, 1996)
From the Departments of We have identified the site of molecular
interaction between nitric oxide (NO) and
p21ras responsible for initiation of signal
transduction. We found that p21ras was singly
S-nitrosylated and localized this modification to a
fragment of p21ras containing
Cys118. A mutant form of p21ras, in
which Cys118 was changed to a serine residue and termed
p21rasC118S, was not
S-nitrosylated. NO-related species stimulated guanine nucleotide exchange on wild-type p21ras,
resulting in an active form, but not on
p21rasC118S. Furthermore, in contrast to
parental Jurkat T cells, NO-related species did not stimulate
mitogen-activated protein kinase activity in cells transfected with
p21rasC118S. These data indicate that
Cys118 is a critical site of redox regulation of
p21ras, and S-nitrosylation of this
residue triggers guanine nucleotide exchange and downstream
signaling.
It is well known that signal transduction pathways initiated by
extracellular ligands are dependent on protein-protein interactions for
propagation and amplification of their signal. Many of these interactions lead to phosphorylation events. For example,
receptor-tyrosine kinases require a series of protein interactions
utilizing SH2 and SH3 domains of adaptor proteins to generate an
activated form of p21ras, a critical signaling
enzyme (1-3).
Recent studies have identified reactive free radicals as central
participants in certain signaling events (4-7). Enhancing free radical
destruction, either enzymatically or chemically, prevented
ligand-stimulated transcription factor (8) and mitogen-activated protein (MAP)1 kinase (4) activation and
also prevented smooth muscle cell mitogenesis and chemotaxis (4). Thus,
a role is emerging for reactive free radicals in mediating signal
transduction.
Among the many recently discovered functions of NO, a role in signaling
has surfaced (9). Although soluble guanylyl cyclase is an important
target of NO in mediating some of its physiologic functions such as the
regulation of blood pressure (10, 11), other signaling events, some
culminating in transcriptional activation, may be cGMP-independent
(12-15). Our studies have focused on how NO initiates cGMP-independent
signaling within cells (16, 17). We have identified
p21ras as a critical target of NO and other
redox modulators (17-19). Here, we sought an understanding of the
structural basis of the NO-p21ras interaction in
the hope of gaining insight into how redox signaling is achieved.
p21ras(1-166) was expressed and
purified as described previously (20).
p21rasC118S(1-166) was expressed and purified
similarly.
Codon 118 of truncated (codons 1-166)
Ha-ras cDNA was mutated from TGT (cysteine) to TCT
(serine) using the polymerase chain reaction. The generated cDNA
fragment was then sequenced (Sequenase) and cloned into the pATras
bacterial expression vector. To generate full-length
p21rasC118S an NcoI/BamHI
fragment (encoding residues 111-166 of the ras(1-166)
mutant) was exchanged for a 0.8-kilobase fragment encoding residues
111-189 plus 3 One small crystal of CNBr (Fluka) was added to 100 pmol of
p21ras in 20 µl of 0.1 N HCl in a
0.5-ml polypropylene tube. Digestion was carried out at room
temperature for 10 min prior to analysis by ESI-MS. After 10 min,
samples were directly electrosprayed into a Finnigan-MAT TSQ-700 triple
quadrupole instrument for analysis of S-nitrosylation
exactly as we described previously (21).
NO solutions were prepared as
we described previously (17). Briefly, a solution of 20 mM
ammonium bicarbonate solution, pH 8.0, in a rubber-stoppered tube was
sparged for 15 min with N2 and then 15 min with NO gas
(Matheson Gas, East Rutherford, NJ). This resulted in a saturated
solution of NO (1.25 mM). This solution also contained
higher oxides of NO which were not quantified.
GDP-preloaded p21ras or
p21rasC118S was analyzed for guanine nucleotide
exchange activity as we described previously (17). Basal rates of
hydrolysis of [ The human T cell line Jurkat
was grown in RPMI 1640 containing 2% L-glutamine and 10%
fetal calf serum. For transfection cells were washed in serum-free
medium and resuspended to 1-5 × 106 cells/ml in
serum-free medium. Liposomes (50 µl of Lipofectin, Life Technologies,
Inc.) were mixed with a solution of 10 µg of p21rasC118S plasmid in 150 µl of sterile
water. This was then added to 1 ml of culture and placed into culture
flasks. After incubation for 24 h at 37 °C and 5%
CO2, the cells were supplemented with 3 ml of selection
medium (RPMI 1640 containing 10% fetal calf serum, 2%
L-glutamine, and 1 mg/ml G418 (Life Technologies, Inc.)). After another 24 h, cells were resuspended and maintained in the selection medium. Mock transfected cells did not receive any DNA. After
3-4 weeks in selection medium, transfected cells were analyzed for
p21ras levels via Western blotting.
MAP kinase activity was measured in an
in vitro kinase assay using myelin basic protein as a
substrate, as we described previously (18). Briefly, serum-starved
cells (24 h, 5 × 106) were treated for 30 min at
37 °C, pelleted, and then resuspended in 300 µl of RIPA buffer
containing 20 mM Tris, pH 7.4, 137 mM NaCl, 2 mM EDTA, 1% Triton X-100, 25 mM
Our earlier studies correlated a single
S-nitrosylation event on full-length
p21ras with enhanced guanine nucleotide exchange
(17). In the present in vitro studies,
p21ras lacking the carboxyl-terminal 23 amino
acids is used. This form of p21ras is commonly
used for in vitro studies and possesses biochemical activity
identical to that of the wild-type enzyme (22). To identify the exact
site of S-nitrosylation, we took advantage of the fact that
cleavage of p21ras at Met residues with CNBr
yields three major fragments each containing a single Cys residue (Fig.
1). We monitored each of the Cys residues for
S-nitrosylation by subjecting p21ras
to CNBr digestion followed by analysis using ESI-MS. In this ESI-MS
assay, the molecular mass of samples treated with NO is compared with
that of untreated samples. An increase in mass of 29 ± 1 Da (the
mass of NO, 30 Da, minus the mass of the substituted proton) and its
lability to increased energy input are indicative of
S-nitrosylation (21). As seen in Table I,
CNBr digestion of p21ras yielded a fragment with
a molecular mass of 6,223 ± 2 Da, corresponding to Fragment 3 (Fig. 1). Upon treatment of p21ras with NO and
subsequent cleavage with CNBr, Fragment 3 had a new mass clearly
indicative of S-nitrosylation (Table I); that is, the mass
of Fragment 3 from NO-treated p21ras was equal
to that of the unmodified Fragment 3 (6,223 Da) plus that of NO (30 Da). This fragment contains Cys118, and thus this Cys
residue is the likely target of NO. It is possible that Fragments 1 and
2 (Fig. 1) were not observed because of inter- and intramolecular
hydrophobic interactions that precluded their solubilization.
ESI-MS analysis of various p21ras preparations
Biochemistry,
¶ Pathology, and
Medicine, Cornell University Medical
College, and the ** Laboratory for Mass Spectrometry,
Department of Biochemistry and Biophysics,
University of North Carolina, Chapel Hill, North Carolina 27599, and
the §§ Department of Biochemistry and Molecular
Biology, Indiana University School of Medicine,
Indianapolis, Indiana 46202
Preparation of p21ras
Proteins
-noncoding region and the coding junction sequenced. A
BglII/BamHI fragment encoding full-length
Ras(C118S) was then subcloned into the BamHI site of the
pCDNA3 mammalian expression plasmid and orientation confirmed by
BstXI digestion.
-32P]GTP were 24.6 ± 6 fmol of
PO4
released/min/mg for the wild-type
enzyme and 18.4 ± 5 fmol/min/mg for
p21rasC118S.
-glycerophosphate, 1 mM NaVO3, 2 mM NaPPi, 20% glycerol, 1 mM
phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. Samples were
vortexed, left on ice for 15 min, and then microcentrifuged for 2 min.
Protein A-Sepharose prebound to anti-ERK1 or ERK2 (Santa Cruz
Biotechnology) was added to supernatants (5 µg/sample). After 1 h at 4 °C, samples were washed twice with RIPA buffer and twice with
kinase buffer (25 mM Hepes, pH 7.4, 25 mM
-glcerophosphate, 25 mM MgCl2, 2 mM dithiothreitol, and 0.1 mM
NaVO3). After the final wash, samples were resuspended in
20 µl of kinase buffer, and 1 µg of myelin basic protein was added
along with 22 µl of 10 µCi/nmol [
-32P]ATP. After
20 min at 30 °C, 4 µl of 6 × Laemmli sample buffer containing 100 mM dithiothreitol was added, and samples
were boiled for 2 min. Samples were run on 15% sodium dodecyl
sulfate-polyacrylamide gels and were analyzed via a PhosphorImager
(Molecular Dynamics).
Localization of the Site of S-Nitrosylation on
p21ras
Fig. 1.
Predicted CNBr fragmentation and location of
Cys residues of p21ras. The sequence of
p21ras(1-166) is indicated. Cleavage at Met
residues with CNBr (indicated by arrowheads) yields three
major fragments. Fragment 1 (green underline) contains
Cys51 and has a mass of 7,203 Da. Fragment 2 (purple
underline) contains Cys80 and has a mass of 4,540 Da.
Fragment 3 (red underline) contains Cys118 and
has a mass of 6,225 Da.
[View Larger Version of this Image (39K GIF file)]
Analyte
Molecular
mass
Differencea
Da
Da
CNBr-cleaved p21ras
6,223
± 2
CNBr-cleaved p21ras + NO
6,253 ± 2
30
p21ras
18,852 ± 2
p21ras + NO
18,882 ± 2
30
p21ras (C118S)
18,836 ± 2
p21ras (C118S) NO
18,836 ± 2
0
a
Difference refers to the mass difference between the
untreated and NO-treated preparations within each group.
To confirm that Cys118 was indeed the site of S-nitrosylation, we generated a form of p21ras identical to the wild-type enzyme, except that Cys118 was modified to a Ser residue (referred to as p21rasC118S). This modification only changes the sulfur atom of Cys118 to oxygen, thus reducing the mass of the enzyme by 16 Da to 18,836 Da. We treated wild-type p21ras with NO under conditions in which we achieved approximately 50% S-nitrosylation. Analysis by ESI-MS revealed the parent enzyme (mass = 18,852 Da) and a singly S-nitrosylated derivative (mass = 18,882 Da, Table I). Treatment of p21rasC118S with NO under identical conditions resulted in no S-nitrosylated product but only the parent enzyme (Table I). These data identify Cys118 as the molecular target of NO on p21ras.
Cys118 as the Molecular Trigger for NO SignalingHaving established that Cys118 is the site
of interaction of NO on p21ras, we examined
whether this S-nitrosylation was responsible for NO-induced
p21ras activation and subsequent downstream
signaling events. We have found previously that NO and other free
radicals induce guanine nucleotide exchange on
p21ras in cells and in vitro
(17-19). Therefore, we examined whether NO could induce nucleotide
exchange on GDP-preloaded p21rasC118S in
vitro. Direct measurement of exchange relies on a filter binding
assay to which our p21ras(1-166) protein does
not bind quantitatively (22, 23). Therefore, to measure exchange,
GDP-preloaded p21ras is treated with NO in the
presence of [-32P]GTP, and hydrolyzed
32Pi is quantified. We have previously shown
this to be a measure of exchange in our system (17). As seen in Fig.
2, NO potently stimulated [
-32P]GTP
hydrolysis of GDP-preloaded wild-type p21ras
(open circles). In contrast, NO had almost no effect on the
exchange rate of p21rasC118S (Fig. 2,
closed circles). The basal rates of hydrolysis for the two
enzymes were similar (24.6 ± 6 fmol of
PO4
released/min/mg for the wild-type
enzyme and 18.4 ± 5 fmol/min/mg for
p21rasC118S). A clear biphasic curve of
activation and subsequent inhibition by higher concentrations of NO was
seen. We and others have previously seen this type of biphasic behavior
of NO in many systems (15, 17, 24). The inhibitory component may be due
to nonspecific noxious effects of high concentrations of NO and its
higher oxides or due to quenching (25). These data indicate that
interaction of NO with Cys118 is required for NO-induced
guanine nucleotide exchange on p21ras.
Since NO and other redox modulators can stimulate several biochemical
events downstream of p21ras, such as nuclear
factor B translocation and MAP kinase activity (15, 18, 26), we
examined whether Cys118 on p21ras is
a target for NO in cells. Using Jurkat T cells mock transfected or
stably transfected with an expression plasmid encoding a full-length version of p21rasC118S (i.e. residues
1-189), we examined the ability of NO to activate MAP kinase activity.
Immunoprecipitation of the MAP kinases ERK1 and ERK2 from wild-type
cells treated with NO-generating compounds
S-nitroso-N-acetylpenicillamine or sodium
nitroprusside resulted in enhanced phosphorylation of the ERK substrate
(Fig. 3A, open bars). In contrast,
Jurkat T cells stably expressing p21rasC118S(1-189) did not respond to NO in
this in vitro kinase assay (Fig. 3A,
hatched bars). These transfected cells expressed 7-10-fold more p21ras than the wild-type cells as
determined by Western blotting with anti-p21ras
antibody, Y13-259 (Fig. 3B). This antibody cannot
distinguish between wild-type and p21rasC118S,
suggesting that although endogenous p21ras was
not specifically inhibited, ectopic expression of high levels of mutant
p21ras apparently prevented its signaling. This
dominant negative activity of p21rasC118S toward
NO action may be due to its high level of expression. The pool of
effectors available for wild-type p21ras to
interact with may be reduced greatly by overexpression of p21rasC118S, perhaps due to their sequestration.
Another possibility is that MAP kinase activity is suppressed in the
mutant cells. To test this, we treated parental and transfected cells
with phorbol myristate acetate (100 ng/ml) and the calcium ionophore
A23187 (500 ng/ml) for 5 min. We found that these agents, which bypass p21ras in activating MAP kinase, stimulated MAP
kinase activity in both cell types (i.e. 221 ± 8 versus 261 ± 9% of control, parental versus transfected). Thus, NO donors did not stimulate MAP
kinase activity in p21rasC118S-transfected cells
although these cell harbored a functional MAP kinase system. These data
indicate that Cys118 is indeed the target of NO on
p21ras responsible for triggering downstream
signal transduction.
Redox regulation of signaling pathways currently presents several conceptual riddles. These include identifying the source of regulatory redox species, maintaining specificity, and identifying the redox target. Our earlier work demonstrated a functional requirement for p21ras in NO signaling (17). Here, we have focused on identifying the molecular target of NO on p21ras. We have identified Cys118 as the critical site of S-nitrosylation. The crystal structure of p21ras is well defined (27, 28), and modeling studies show that Cys118 is the most surface accessible of the three Cys residues in our p21ras preparation. Using the program X-PLOR (Molecular Simulations, Inc.) the solvent accessible surface of p21ras complexed to GDP (coordinates obtained from the NMR solution structure) was calculated using a 1.4 Å radius probe. Whereas the solvent accessibility of Cys51 and Cys80 side chains were similar and fairly shielded from the solvent, Cys118 was solvent exposed. The buried nature of residues Cys80 and Cys51 provides a structural basis of why a single S-nitrosylation occurs on p21ras upon exposure to NO.
A mechanistic understanding of how S-nitrosylated Cys118 leads to enhanced guanine nucleotide exchange will likely be provided by solving its x-ray crystal structure. However, some insight is gained from considering what is currently known about p21ras structure and function. Cys118 is located within a highly conserved region (NKXD) of the ras superfamily and indeed all GTP-binding protein sequences. This NKXD motif, in which Cys118 is the variable X residue in p21ras, interacts directly with the guanine nucleotide ring of GTP and GDP and with other nucleotide-binding loops of the protein (29). Independent mutation of residue 116, 117, or 119 leads to an increased dissociation rate of bound nucleotide, resulting in an increased rate of nucleotide exchange (30). Although our p21rasC118S mutant had basal rates of guanine nucleotide exchange similar to those of the wild-type protein, it is possible that S-nitrosylation results in an alteration in protein-GDP contact, resulting in nucleotide exchange.
The presence of a redox-active residue in such a critical domain suggests that its conservation may reveal enzymes and transductional systems that may be similarly regulated. In the ras superfamily, Ha-, Ki-, and N-ras, rap1A, rap1B, rab1, and rab3 contain a Cys residue in this conserved region. In contrast, ral, tc21, R-ras, rap2, and rho gene products do not. Within the ras subfamily, this Cys residue is conserved from slime mold to man. Such conservation suggests that this molecular redox trigger is an important mechanism by which cells respond to reactive free radicals.
It is likely that this molecular switch is regulated by cellular antioxidant levels. We have shown previously that reducing cellular glutathione levels renders the p21ras signaling pathway dramatically more sensitive to redox activation (18). According to this model, as glutathione is oxidized, the Cys switch on p21ras becomes available for modification, and a preprogrammed signaling cascade is initiated. Glutathione or other low molecular weight thiols likely play an important role in S-nitrosylation of p21ras. NO, under anaerobic conditions, cannot participate in S-nitrosylation. NO must be converted to a redox active form such as nitrosonium ion (NO+; Ref. 9). This occurs rapidly in the presence of metals or thiols, and thus S-nitrosoglutathione may be a crucial reservoir of redox active NO.
Such a redox-triggered signaling pathway may also provide a mechanistic understanding of some recent observations identifying a requirement for reactive free radicals in mediating platelet-derived growth factor (PDGF) signaling. In those studies (4), PDGF stimulated H2O2 production in vascular smooth muscle cells. When H2O2 production was blocked, PDGF-induced enhancement of MAP kinase activity, chemotaxis, and DNA synthesis was prevented. Many of these PDGF-dependent events require p21ras, and thus the redox-sensitive target, Cys118, is likely to be involved in this reactive free radical-dependent signaling cascade. Modification of Cys by redox modulators other than NO, such as hydroxyl radical or heavy metals, would necessitate a Cys modification that is chemically different from the S-nitrosylation described herein. However, the structural alterations may ultimately be the same.