(Received for publication, December 30, 1994; and in revised form, February 1, 1995)
From the
The protooncogene p21, a monomeric G
protein family member, plays a critical role in converting
extracellular signals into intracellular biochemical events. Here, we
report that nitric oxide (NO) activates p21
in
human T cells as evidenced by an increase in GTP-bound
p21
. In vitro studies using pure
recombinant p21
demonstrate that the activation
is direct and reversible. Circular dichroism analysis reveals that NO
induces a profound conformational change in p21
in association with GDP/GTP exchange. The mechanism of
activation is due to S-nitrosylation of a critical cysteine
residue which stimulates guanine nucleotide exchange. Furthermore, we
demonstrate that p21
is essential for NO-induced
downstream signaling, such as NF-
B activation, and that endogenous
NO can activate p21
in the same cell. These
studies identify p21
as a target of NO in T
cells and suggest that NO activates p21
by an
action which mimics that of guanine nucleotide exchange factors.
Reactive oxygen species are known to mediate signal transduction
events in lymphocytes (1, 2, 3) and have
recently been implicated in the signaling process of a wide range of
stimuli(4) . The reactive nitrogen species, nitric oxide (NO), ()plays critical roles in many diverse biological processes
such as vasoregulation, host defense and synaptic
plasticity(5, 6, 7, 8) . Recently,
we identified a role for NO as a positive signaling molecule in the
immune system(9, 10) . The protooncogene
p21
has been identified as a key molecular
switch involved in regulating T cell activation triggered by various
mitogens(11, 12, 13) . Therefore, we
hypothesized that the immune stimulatory properties of NO may be
mediated through p21
, and thus we examined
whether NO could activate p21
in human T cells.
Figure 1:
Effect of NO on
p21 activation in Jurkat T cells. The human
Jurkat T cell line was stimulated with various amounts of NO for 10 min (A) or with NO (0.01 µM) for various times (B). At the appropriate times, cells were washed,
p21
was immunoprecipitated, and the bound
nucleotides analyzed. Data are expressed as the percentage of GTP-bound
p21
of control. Control samples had a base line
value of 12.4 ± 3.6% GTP bound. Data represent the mean and
standard deviation from four to six
experiments.
The activating
effect of NO could be mediated either by direct interaction with
p21 or indirectly through additional factors. Therefore,
we mixed NO with pure recombinant p21
in vitro and measured its intrinsic GTPase activity. Surprisingly, NO
enhanced the GTPase activity of p21
in vitro,
indicating a direct effect (Fig. 2A). Co-incubation
with catalase (1000 unitsml) or superoxide dismutase (30 units/ml) had
no effect on the /ability of NO to enhance p21
GTPase
activity (data not shown), indicating that the activating species is
not hydroxyl radical, superoxide, or peroxynitrite. The amount of NO
which gives a maximal response (75 µM) in vitro is much higher than that required in whole cells (0.01
µM, Fig. 1A). We have observed previously
a similar differential sensitivity in leukocyte membranes treated with
NO before or after isolation using a membrane GTPase
assay(10) . This phenomenon may be due to a cellular component
which makes NO more potent and is lacking in purified preparations.
These are likely to be oxidizing conditions which favor formation of
nitrosonium ion (NO
) and facilitate nitrosothiol
formation(23, 24, 25) . In contrast to NO,
carbon monoxide, another gas claimed to be a signaling molecule in the
brain (26, 27) , had no effect on p21
activity (Fig. 2A).
Figure 2:
Effect of NO and carbon monoxide on
p21 enzymatic activity in vitro. Pure
recombinant p21
was incubated with various
concentrations of NO or CO for 10 min (A) after which
p21
GTPase activity was measured.
p21
preloaded with [
H]GDP,
or GDP-loaded p21
mixed with
-
S-GTP, was treated with various amounts of NO for 10
min at 37 °C followed by assay of bound
[
H]GDP or
-
S-GTP (B).
Data represent the mean and standard deviation from three to seven
experiments.
The apparent increase in
GTPase activity could be due to either: 1) an increase in GDP release
from p21 (the rate-limiting step in the catalytic cycle)
or 2) an increase in the intrinsic GTPase activity of
p21
. In vivo, GDP release is controlled by
guanine nucleotide exchange factors (GEF), and represents activation,
whereas GTP hydrolysis is controlled by GTPase-activating proteins and
represents
inactivation(17, 18, 19, 20) . To
distinguish between these possibilities, we preloaded p21
with [
H]GDP and tested whether NO increased
GDP release. As seen in Fig. 2B, concentrations of NO
which enhance GTPase activity (Fig. 2A) induced GDP
release from p21
. Furthermore, at concentrations of NO
where [
H]GDP was released,
-
S-GTP was bound (Fig. 2B). These
data support our findings of an increase in the GTP/GDP ratio on
p21
in whole cells (Fig. 1A) and verify
the concept that NO is an activator of p21
by promoting
guanine nucleotide exchange.
Figure 3:
Effect of hemoglobin and the influence of
blocking antibodies on p21 activity and
nitrosothiol formation. Hb (0.4 mM) was added before or 10 min
after NO (100 µM) addition (A). In antibody
blocking experiments, 10 µg of the indicated antibody was incubated
with 1 µM p21
for 30 min at 22
°C followed by NO (50 µM) addition and GTPase assay or
nitrosothiol assay. Data represent the mean and standard deviation of
three experiments. The asterisk denotes no detectable
nitrosothiols (i.e. less than 0.2
mol/mol).
Release of GDP is effected by
GEF's which bind to regions on p21 that can be
blocked by the monoclonal antibody Y13-259 (29, 30) . To determine if NO induces GDP release in a
manner similar to GEF's, we examined whether preincubation of
p21
with Y13-259 prevents NO activation. As seen in Fig. 3B, antibody Y13-259 substantially prevented
the ability of NO to activate p21
. In contrast,
monoclonal antibody Y13-238, which binds to a different region on
the molecule(31) , had no effect (Fig. 3B).
These data suggest that NO induces GDP release by interacting with a
region of p21
which normally interacts with
GEF's(32, 33) .
Because p21 does not contain a heme group, and Hb reversed the activation by
NO, we examined the possibility that the mechanism of activation was
via formation of a nitrosothiol (Fig. 3B). We found
that NO induced the formation of only one nitrosothiol on
p21
, even though p21
contains 5 Cys
residues. Importantly, the antibody Y13-259, which prevented
activation of p21
by NO, also prevented nitrosothiol
formation, whereas the control antibody Y13-238, which did not
prevent activation, also did not prevent nitrosothiol formation (Fig. 3B). Furthermore, Hg
, a known
thiol-reactive reagent, enhanced p21
GTPase activity
(data not shown). These data strongly suggest that formation of a
nitrosothiol on p21
by NO is responsible for activation.
To examine if NO causes a conformational alteration in
p21, we used circular dichroism (CD) spectral analysis to
assess secondary structure. Treatment of p21
with NO led
to a dramatic change in its CD spectral properties, and computer
analysis revealed a profound reduction in
-helical content (from
60 to 36%) and a concomitant increase in
-sheet content (from 18
to 44%). The basal spectra we obtained for untreated p21
were identical to that published previously(34) . These
data suggest that NO induces a conformational change in p21
concomitant upon nitrosothiol formation, leading to enhanced
nucleotide exchange.
Figure 4:
Role of p21 in NO
signaling. Upper panel, parental PC12 wild type (wt)
or ras-negative (mt) cells were serum-starved
overnight and then treated with the indicated concentration of sodium
nitroprusside (SNP), an NO-generating compound, for 4 h
followed by nucleus isolation and assay for NF-
B binding activity.
The arrow denotes migration of the NF-
B/DNA complex. Lower panel, HUVECs were either untreated or treated 16 h with
lipopolysaccharide (LPS, 30 µg/ml) and interferon-
(IFN-
, 50 ng/ml) or
N-methyl-L-arginine (NMA)
(3 mM) in phosphate-free RPMI 1640 containing 100 µCi/ml
PO
. Then, indicated wells
were treated with L-arginine (ARG, 10 mM)
for 15 min and samples assayed for GTP-bound
p21
.
The physiological
relevance of our findings are strengthened by our demonstration that
endogenous NO activates p21 (Fig. 4, lower
panel). We induced nitric oxide synthesis in HUVEC with LPS and
IFN-
(37) . We found that in the presence of the nitric
oxide synthase inhibitor
N-methyl-L-arginine, providing a
pulse of the substrate, L-arginine (to avoid the
desensitization effect found in Fig. 1B), led to
recovery of an activated form of p21
(Fig. 4, lower panel). Thus, an autocrine loop may exist within cells,
whereby endogenous NO activates cellular p21
, leading to
cell activation and nitric oxide synthase transcriptional regulation.
NO is a highly reactive gas which can be generated in high local
concentrations at sites of inflammation. Thus, our findings suggest
that activation of p21
by NO is likely to be a major
mechanism of amplification of leukocyte-induced local tissue damage.
The present findings identify p21 as a target of NO in
T cells. We propose that NO, via formation of a nitrosothiol, leads to
a conformational change which enhances guanine nucleotideexchange, thus
mimicking endogenous GEF's. The ability of NO to activate
p21
, a non-heme enzyme, represents a novel signal
transduction pathway. Furthermore, because nitrosothiol formation on a
critical cysteine is apparently responsible for its activation,
p21
may be ideally suited to transduce other oxidative
stress signals from both endogenous and exogenous sources in many cell
types. In fact, others have found that redox agents modulate binding of
GTP to p21
immunoprecipitates(38) . Furthermore,
the activation of p21
or other monomeric G proteins by NO
may not be restricted to T cells and conceivably could mediate many
signals transmitted by NO, such as Ca
-independent
vesicle exocytosis (39) and synaptic plasticity in the
brain(8, 40) . Heterotrimeric G proteins may also be a
target of NO as the G
subunits are highly homologous to
p21
(18) . For example G proteins are known to
mediate olfaction, and NO has recently been implicated in this
sytem(41) .