From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078
Received for publication, December 20, 2000, and in revised form, January 31, 2001
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ABSTRACT |
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Nitric oxide (NO) has been reported to inhibit
protein synthesis in eukaryotic cells by increasing the phosphorylation
of the Over 2 decades ago, the heme-regulated inhibitor
(HRI)1 of protein chain
initiation in rabbit reticulocyte lysate (RRL) was identified as a
heme-regulated protein kinase that phosphorylated the The amino acid sequence deduced from HRI cDNA indicates that
it is composed of at least five distinct domains (4). The unique
N-terminal domain of HRI contains ~165 amino acids. The second and
fourth domains contain conserved sequence motifs I-V and motifs
VI-XI, which comprise the N-terminal and C-terminal catalytic lobes of
protein kinases, respectively. The third and fifth domains are also
unique, consisting of ~140 amino acids that are inserted between the
two conserved kinase lobes and ~50 amino acids at the C terminus, respectively.
HRI is a hemoprotein that contains two distinct of heme-binding sites
(5-7). Heme binding to the first site is stable, and remains
associated with purified HRI, whereas heme-binding to the second site
is reversible and appears to be responsible for the rapid heme-induced
down-regulation of HRI activity. We have recently
demonstrated that the N-terminal domain of HRI contains the stable
heme-binding site, whereas the unique third domain of HRI appears to
contain the reversible heme-binding site (6, 7), thus raising the
question as to the function of the N-terminal heme-binding domain
(NT-HBD) in the regulation of HRI activation.
Nitric oxide (NO) is now recognized as a major signaling molecule
(8-10). Like NO, carbon monoxide (CO) has also been identified as an
endogenous second messenger in the peripheral and central nervous
system, and has been demonstrated to play an important role in
hemodynamic regulation (10, 11). Both NO and CO have high affinity for
both protein-bound and free heme-iron (12-14). Recently, the
cytostatic activity of NO was found to correlate with NO-induced
increase in eIF2 Protein Synthesis and eIF2 Treatment of Samples with NO and CO--
The NO generator NOC-9
(Calbiochem) was prepared as a 100 mM stock solution in 20 mM Tris-HCl (pH 8.0) and used immediately for experiments
in RRL or in vitro. NOC-9 breaks down to form NO with a
half-life of 3 min at pH 7.4. For CO treatments, samples (RRL or
immunoresins) were gassed in a fume hood with CO (99.0+% CO; Sigma)
for 3 min on ice by directing a stream of CO through a 21-gauge needle
at the surface of the sample with sufficient velocity to cause
continuous mixing of the microcentrifuge tube contents. The tubes were
then sealed with parafilm prior to any further incubations.
De Novo Synthesis, Maturation, and NO or CO Treatment of HRI in
Situ--
Coupled transcription/translation reactions to pulse-label
[35S]HRI and His7-[35S]HRI in
nuclease-treated RRL (TnT RRL, Promega) were carried out as described
previously (21, 22). After radiolabeling, one volume of TnT protein
synthesis mix containing either [35S]HRI and
His7-[35S]HRI was mixed with seven volumes of
normal heme-deficient or hemin-supplemented (10 µM hemin)
protein synthesis mixes containing non-nuclease-treated RRL and the
protein synthesis initiation inhibitor aurintricarboxylic acid (60 µM). The samples were then incubated for 50 min at
30 °C to yield "mature-competent" HRI (50 min in
hemin-supplemented RRL), "transformed" HRI (50 min in
heme-deficient RRL), or "repressed" HRI (40 min in heme-deficient RRL, followed by a 10-min incubation in the presence of 10 µM hemin) (21, 22). The in situ effects of NO,
CO, N-ethylmaleimide (NEM), or dithiothreitol (DTT) on HRI
were then assayed by treating samples with NOC-9, gassing RRL with CO,
or treatment of samples with NEM or DTT followed by an additional
period of incubation at 30 °C as specified in the figure legends.
HRI was then affinity-purified and assayed for kinase activity as
described below.
Assay of the Kinase Activity of Affinity-purified
His7-[35S]HRI--
Immunoadsorption of
His7-[35S]HRI by anti-His5
antibodies (Qiagen) and non-immune control antibodies were done as
previously described (22). Assays for the eIF2
To study the effects of NO or CO on HRI activation in vitro,
HRI was synthesized de novo and matured for 60 min in
hemin-supplemented or heme-deficient RRL, or in heme-deficient RRL (50 min) followed by the addition of 10 µM hemin (for 10 min)
to yield mature-competent, transformed HRI and repressed HRI,
respectively. HRI was then affinity-purified, and autophosphorylation
of HRI was assayed by the incorporation of
[32P]Pi into HRI incubated with
[ Spectral Analysis of NT-HBD of HRI--
Recombinant NT-HBD of
HRI was purified as previously described (7). Spectral analysis was
done using a Shimdazu UV-160 spectrophotometer scanning the NT-HBD
dissolved in 20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl from 200 to 650 nm. The NT-HBD was then reduced by
the addition of several grains of dithionite to the cuvette.
Immediately after the spectrum of reduced NT-HBD was obtained, NOC-9
was added to a concentration of ~1 mM, or the cuvette was
gassed with 99.0+% CO for 3 min to obtain the spectra of NO-bound and
CO-bound NT-HBD, respectively.
Immunoadsorption of HRI from Cultured Cell
Extracts--
Ntera- 2/c1.D1 neuroepithelial (NT-2; Ref. 23) and
C2C12 myoblast cells were cultured in DMEM in the presence of 10%
fetal calf serum, penicillin/streptomycin and 5% CO2 to
~70% confluence. The cells were then grown overnight (16 h) in DMEM
supplemented with 10 µM hemin-HCl. Cells were washed two
times with Hank's buffered saline, lysed directly by scraping cells
from the culture flasks with lysis buffer (~1 ml/2.5 × 107 cells) containing 30 mM HEPES-KOH (pH 7.4),
150 mM NaCl, 1 mM EDTA, 2 mM EGTA,
50 mM NaF, 1 mM DTT, 1% Triton X-100 (v/v),
and protease-inhibitor mixture (Sigma; 100 µl/107 cells
solubilized). The cell lysates were clarified by centrifugation at
15,000 × g for 10 min, and HRI was then immunoadsorbed
with anti-HRI NT-HBD antibody from 10 µl of mouse polyclonal ascites fluid or a control non-immune antibody that had been previously adsorbed to mouse anti-IgG cross-linked to agarose (24). Mouse polyclonal anti-HRI NT-HBD antibody was raised against
affinity-purified recombinant NT-HBD by the HYCABS core facility
(Oklahoma State University, Stillwater, OK). The immune pellets were
washed one time with 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl and 1% (v/v) Tween 20, and two times with 10 mM Tris-HCl (pH 7.4) containing 150 mM NaCl,
and assayed for associated kinase activity as described above.
Levels of eIF2 Effect of NO and CO on Protein Synthesis and eIF2
To determine the effect of CO on protein synthesis, RRL was gassed with
100% CO for 3 min on ice. CO stimulated the rate of protein synthesis
in hemin-supplemented RRL by ~50% (Fig.
2a). Translation in
heme-deficient RRL was stimulated 40% by CO during the first 5 min of
incubation, but shut-off of translation was not prevented by CO (data
not shown). Similarly, pre-gassing of heme-supplemented RRL with CO
stimulated translation by ~40% during the first 5 min of incubation
in NOC-9-treated RRL, but did not prevent translational shut-off (Fig.
2a). These observations suggested that NO and CO have
opposing effects on protein synthesis in RRL.
Effect of NO and CO on HRI Activity--
To determine whether NO
modulated the activation of HRI, His7-tagged
[35S]HRI was synthesized by coupled
transcription/translation in nuclease-treated RRL and then matured in
normal RRL in the presence or absence of hemin to generate specific
populations of HRI molecules (see Refs. 21 and 22 for detailed
descriptions of the HRI populations). Samples were then treated
in situ with increasing concentrations of NOC-9, and the
His7-tagged HRI was affinity-purified and assayed for
kinase activity (Fig. 3a).
His7-tagged [35S]HRI that was matured in
hemin-supplemented RRL (+, mature-competent HRI) was an inactive
kinase. HRI that was matured in heme-deficient RRL was transformed into
an active auto- and eIF2
To establish whether the effect of NOC-9 on the kinase activity of HRI
was through a direct interaction of NO with HRI, mature-competent, transformed, and repressed His7-[35S]HRI were
affinity-purified and assayed for autokinase activity in the presence
of varying concentrations of NOC-9 in vitro (Fig. 3b). The autokinase activity of all the three forms of HRI
were activated upon treatment with NOC-9 in vitro in a
concentration-dependent fashion, with repressed HRI being
the most sensitive to NO-induced activation (3-fold in the
presence of 0.01 mM NOC-9).
Subsequent studies were carried out with repressed HRI, as
stress-induced activation of repressed HRI in hemin-supplemented RRL
occurs independent of changes in heme concentration (18, 22, 24,
27-29). Thus, repressed HRI is the form of HRI that is likely to be
the most physiologically relevant target for regulation by diffusible
gases in vivo.
Order-of-addition studies were carried out to determine whether CO and
NO have competing effects on HRI activation (Fig. 2b). Gassing repressed His7-[35S]HRI with 100% CO
suppressed its autokinase activity below that of the ungassed control,
and dramatically suppressed the autokinase activity of repressed
His7-[35S]HRI that had been affinity-purified
from NOC-9-treated RRL. Furthermore, in vitro treatment with
NOC-9 markedly enhanced the autokinase activity of repressed
His7-[35S]HRI that had been affinity-purified
from CO-gassed RRL. These results suggest that CO and NO compete for a
common binding site in HRI and have opposing effects on HRI activation.
Mechanism of Regulation HRI Activity by NO and CO--
The
physiological effects of NO are mediated through a number of
mechanisms, including (i) S-nitrosylation of proteins (30), (ii) the generation of free radicals and oxidative stress (31), or
(iii) the coordination of NO by protein-bound heme (12, 32). The only
known biological reactivity of CO is as a ligand for protein-bound
heme, and CO is neither sulfhydryl- nor redox-active. Thus, the
observations that CO both blocks and reverses NO-induced activation of
HRI strongly suggest that NO induces the activation of HRI through its
coordination by heme. However, since sulfhydryl-reactive compounds and
oxidative stress are well known to cause the activation of HRI in
hemin-supplemented RRL (1-3, 33), we carried out experiments designed
to test the first two possible mechanisms further.
To examine whether NO activates HRI by modifying sensitive sulfhydryls
of HRI, the effect of NO on the kinase activity of repressed
His7-[35S]HRI that was affinity-purified from
RRL treated (or not) with NEM was examined (Fig.
4a). Sulfhydryl-reactive
compounds, such as NEM, are thought to activate HRI by covalently
modifying some sensitive sulfhydryls of HRI (1-3, 33-35).
NEM-treatment caused an ~3-fold increase in the autokinase activity
of repressed HRI. Treatment of repressed
His7-[35S]HRI with NO in vitro led
to an even more marked increase in the autokinase activity of HRI. The
autokinase activity of HRI affinity-purified from NEM-treated RRL was
also markedly increased upon treatment with NO, indicating that NO can
further activate HRI containing previously modified sulfhydryls.
However, the possibility remained that NO was modifying a different
site on HRI than that which was reactive with NEM.
To further test the hypothesis that the activation of HRI by NO was not
mediated through its effect on sensitive sulfhydryls of HRI or through
the generation of a generalized oxidative stress, the effect of the
reducing agent DTT on NO-induced activation of repressed HRI was
examined (Fig. 4b). DTT reverses the effect of NO on
proteins that are mediated through S-nitrosylation (36). In
addition, DTT protects HRI from activation induced by
sulfhydryl-reactive compounds and reverses the activation of HRI
induced by generalized oxidative stress and sulfhydryl-reactive heavy
metal ions (3, 33, 37). Addition of DTT prior to NOC-9 treatment had no
effect on NO-induced inhibition of protein synthesis (data not shown) or activation of repressed His7-[35S]HRI
(Fig. 4b).
NO Modulates the Activation of HRI by Binding to HRI's
NT-HBD--
To determine whether NO and CO might modulate the
activation of HRI through their coordination by heme, a spectral
analysis of the recombinant NT-HBD of HRI was carried out. Reduction of HRI's NT-HBD with dithionite caused a shift in the absorption maximum
of the Soret band from 414 nm to 428 nm, with the absorption maximum in
the
To address the question of whether the activation of HRI by NO is due
to the NO binding to NT-HBD, we studied the effect of NO on the
activity of HRI from which the NT-HBD has been deleted (HRI/Met-3; Ref.
6). His7-[35S]HRI/Met-3 was synthesized
de novo, transformed in heme-deficient RRL, and repressed by
the addition of hemin. Compared with wild type
His7-[35S]HRI, the kinase activity of
His7-[35S]HRI/Met-3 was only marginally
activated upon NOC-9 treatment (Fig.
6a). In contrast, treatment
with NEM markedly increased the kinase activity of both
His7-[35S]HRI/Met-3 and wild type
His7-[35S]HRI (Fig. 6a). These
results support the hypothesis that the NT-HBD domain mediates
NO-induced activation of HRI. However, we cannot currently rule out the
possibility that NO may have additional effects mediated through an
interaction with heme bound to the second regulatory heme-binding site
in HRI, because NOC-9 treatment reproducibly caused a slight
stimulation in kinase activity of HRI/Met-3.
To further confirm the involvement of NT-HBD domain of HRI in NO
activation, we studied whether [35S]HRI/Met-3 could form
a complex with His7-[35S]NT-HBD, and what
effect NO had upon the kinase activity of [35S]HRI/Met-3
in such a complex. [35S]HRI/Met-3 and
His7-[35S]NT-HBD were co-expressed in TnT
RRL, followed by transformation of the [35S]HRI/Met-3 in
heme-deficient RRL and treatment (or not) with NOC-9. The ability of
anti-His-tag antibodies to co-adsorb [35S]HRI/Met-3 with
His7-[35S]NT-HBD indicated that the NT-HBD of
HRI interacted with HRI/Met-3 in trans, although
the two proteins did not interact quantitatively (Fig. 6b).
[35S]HRI/Met-3 and non-His-tagged
[35S]NT-HBD were co-expressed and treated with 1 mM NOC-9, as a control for nonspecific binding. Although
the amount of [35S]HRI/Met-3 protein that coadsorbed as a
complex with His7-[35S]NT-HBD was much less
compared with the amount of
His7-[35S]HRI/Met-3 that was directly
adsorbed by the anti-His-tag antibodies, assays of eIF2 Effect of NO on HRI Activity in Non-erythroid Cells--
Possible
regulatory roles for HRI in nonerythroid cells have yet to be explored
in detail. Although HRI expression was previously believed to be
specific to erythroid cells (4, 38, 39), mRNA encoding HRI has been
reported to be expressed in several nonerythroid tissues (40, 41). A
search of the data base of expressed sequence tags, dbEST (Table
I) indicated that HRI mRNA is
expressed in a wide variety of nonerythroid cells and tissues, as well
as a variety of tumors. A 1862-base pair cDNA (GenBankTM accession
no. AA196697) putatively coding for HRI from human NT-2
neuroepithelial cells was purchased from Genome Systems, Inc., and
sequenced. The predicted open-reading frame, encoding a protein with
80% sequence similarity to rabbit HRI but lacking a start codon, was
subcloned into the pS64T plasmid. The properties of His-tagged NT-2 HRI
protein that was expressed in and affinity-purified from TnT RRL were
indistinguishable from RRL HRI (data not shown).
Subsequent to this work, the predicted amino acid sequences of four
full length human HRI cDNAs isolated from libraries prepared from
bone marrow (AF147094), dermal hair papilla (AF255050), brain
(AB037790) and dermal microvascular endothelial (AF181071) cells have
been entered into GenBankTM. The entries indicate that the predicted
amino acid sequence of human HRI is 80.9%, 81.8%, and 81.6%
identical to rat, rabbit, and mouse HRI, respectively, and that the NT2
cDNA lacked the first 31 nucleotides of the open reading frame.
To address the question of whether HRI protein was expressed in
nonerythroid cells, anti-HRI/NT-HBD antibodies were used to immunoadsorb protein from extracts prepared from NT-2 and C2C12 cells.
Assays of the immunoadsorbed protein indicated that an eIF2
To determine whether NO stimulated eIF2 Mechanisms that control initiation of protein synthesis play
significant roles in regulating cell growth (reviewed in Ref. 43).
eIF2 Our results suggest that NO may be a physiological regulator of HRI
activation. NO was as potent of an activator of HRI as heme deficiency
in RRL. Although the 0.25-0.5 mM concentration of NOC-9
required to inhibit protein synthesis in hemin-supplemented RRL likely
falls outside of the physiological range of concentrations to which NO
accumulates in vivo, the concentration range is reasonable considering the NO-binding capacity of the estimated millimolar levels
of hemoglobin present in RRL. However, under physiological conditions,
much lower levels of NO might be capable of activating HRI in
reticulocytes, as recent work indicates that the reaction of
oxyhemoglobin with NO functions to maintain NO bioactivity (52,
53).
A more likely physiological target of NO is HRI present in red cell
precursors early during hematopoiesis, when significant levels of
hemoglobin have yet to accumulate. NO suppresses hemoglobin synthesis
in K562 cells (54, 55). Although it was speculated that the NO-induced
suppression of hemoglobin synthesis was a result of NO-induced
inhibition of heme biosynthesis (54), our current results suggest it
could be the result of NO directly activating HRI present in K562
cells. In addition, activation of nitric-oxide synthase has been
implicated in tamoxifen-induced apoptosis of K562 cells (56), and NO
has been reported to (i) influence the growth and differentiation of
normal bone marrow cells (57), (ii) induce apoptosis in bone marrow
progenitor cells (58), and (iii) be a mediator of cytokine-induced
hematopoietic suppression (59). Our results suggest that these effects
of NO could, in part, be mediated via NO-induced activation of HRI, considering the well established role that eIF2 The consequences of NO synthesis on the function and fate of cells
varies depending on the rate of NO synthesis, cell type, presence of
free radicals, and the anti-oxidant status of the cell (reviewed in
Refs. 60-62). NO modulates signaling molecules in apoptotic pathways,
contributing to the physiological balance between pro-apoptotic and
anti-apoptotic stimuli (63). NO at high levels generally induces cell
death, whereas low doses of NO rescues many cells from apoptotic
death. NO is cytotoxic to many cells types (Ref. 64, and references
therein), and the cytostatic activity of NO has been found to correlate
with NO-induced increase in eIF2 Opposing effects of NO on protein synthesis and HRI autokinase activity
were also observed in RRL. In situ, low levels of NO were
observed to stimulate translation and suppress, to a degree, the
autophosphorylation of HRI. These observations may reflect the ability
of NO to displace CO from hemoglobin (see below), or a requirement for
both HBDs to be liganded to NO within the HRI dimer for the NO-induced
allosteric activation of HRI to occur.
A search of dbEST indicates that mRNA encoding HRI is present in
many non-erythroid cell types. Our results indicate that the HRI
mRNA present in NT-2 and C2C12 cells is translated into protein,
albeit at levels much lower that that present in erythroid cells. Thus,
NO-induced activation of HRI may contribute to NO-induced inhibition of
protein synthesis in non-erythroid cells that express HRI. NO-induced
activation of HRI, like the effects of PKR activation (44, 45), may
also play a role in mediating NO-induced apoptosis in these cells.
Furthermore, NO may be a principle activator of HRI in non-erythroid
cells, as these cells are not likely to experience large fluctuations
in their heme content.
Our data also suggest that CO is an important physiological regulator
of HRI activation. CO is the product of the first step in heme
catabolism catalyzed by heme oxygenase (HO), and has been proposed to
be a physiological regulator akin to NO (10, 11, 65). HO-1 expression
is activated in virtually all cell types, not only by the presence of
free heme, but also in response to inflammatory cytokines, hypoxia, and
many forms of oxidative stress (65, 66). Induction of HO-1 plays an
important role in protecting cells from the adverse effects of
oxidative stress (11, 36, 65-68). Furthermore, low concentrations of
CO prevent tumor necrosis factor- The spectral analyses of the binding of NO and CO indicate the
potential mechanism through which NO and CO have opposing effects on
HRI activation. Whereas CO binds and forms a 6-coordinate heme complex,
the spectral changes induced upon the binding of NO to the NT-HBD are
similar to those induced upon the binding of NO to the regulatory
heme-binding domain of guanylate cyclase (70). This observation
suggests that NO activates HRI through the same mechanism by which its
activates guanylate cyclase; cleavage of the iron-histidine bond in the
HBD to yielded a 5-coordinate ferrous-nitrosyl complex (70).
HO-1 expression appears to play a central role in an important
regulatory network between NO and CO (11). The ability of CO to
antagonize NO-induced activation of HRI leads us to propose a novel
feedback mechanism through which protein synthesis may be regulated by
these diffusible gases. Increases in cellular NO concentrations cause
the release of protein-bound heme (71-73), and the induction of HO-1
(11). The HO-1-catalyzed breakdown of heme would not only be critical
in protecting cells from oxidative damage that would result from the
presence of elevated concentrations of free heme, but would also
produce elevated concentrations of CO, which could act as a feedback
inhibitor of HRI activation induced by NO. The overall effect of these
diffusible gases on HRI activity and protein synthesis would ultimately
be determined by the relative concentrations to which they accumulate
within a cell. High levels of NO might stimulate pro-apoptotic pathways to a degree that could not be reversed by a subsequent elevation in CO.
Indeed, NO readily nitrosylates intracellular free heme and prevents
its degradation by HO (74). In contrast, low levels of NO might lead to
the generation of sustained, elevated levels of CO, which might then
promote cell growth. Furthermore, we speculate that the ability of
elevated CO levels to inhibit the activity of HRI and suppress
eIF2 In summary, we acknowledge that the question of whether HRI is present
in cells from tissues of nonerythroid origin is contentious, as it
difficult to assure that the source of the HRI mRNA or protein is
not from occluded blood. Furthermore, the expression of HRI in cultured
nonerythroid cells could be a result of the aberrant phenotype of
immortalized cells. In addition, our results indicate that HRI
expression in NT2 and C2C12 is an order of magnitude or more lower than
its level of expression in reticulocytes. However, with the
acknowledgment of these caveats, the results presented here suggest
that the following hypotheses are worthy of further investigation: (i)
that activation of HRI may contribute to pathophysiologies that result
from chronic exposure of normal cells to elevated NO levels and (ii)
that HRI may be a potential target for design of tumoricidal agents
directed at transformed cell populations expressing HRI.
-subunit of eukaryotic initiation factor (eIF) 2. However, the mechanism through which this increase occurs has not been characterized. In this report, we examined the effect of the diffusible gases nitric oxide (NO) and carbon monoxide (CO) on the activation of
the heme-regulated eIF2
kinase (HRI) in rabbit reticulocyte lysate.
Spectral analysis indicated that both NO and CO bind to the N-terminal
heme-binding domain of HRI. Although NO was a very potent activator of
HRI, CO markedly suppressed NO-induced HRI activation. The NO-induced
activation of HRI was transduced through the interaction of NO with the
N-terminal heme-binding domain of HRI and not through
S-nitrosylation of HRI. We postulate that the regulation of
HRI activity by diffusible gases may be of wider physiological
significance, as we further demonstrate that NO generators increase
eIF2
phosphorylation levels in NT2 neuroepithelial and C2C12
myoblast cells and activate HRI immunoadsorbed from extracts of these
non-erythroid cell lines.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit of
eukaryotic initiation factor eIF2 (reviewed in Refs. 1-3). eIF2
delivers Met-tRNAi in a complex with GTP to 43 S ribosomal
initiation complexes and is released as a complex with GDP at the
completion of the initiation cycle. Recycling of eIF2·GDP and the
formation of eIF2·GTP·Met-tRNAi complexes requires the
action of the guanine nucleotide exchange factor, eIF2B. Under
heme-deficient conditions, HRI is activated and phosphorylates eIF2.
Phosphorylated eIF2 avidly binds eIF2B, sequestering eIF2B in a poorly
dissociable complex, which subsequently leads to the inhibition of the
initiation of translation, as eIF2·GDP complexes that are present in
excess of eIF2B fail to recycle. Thus, HRI functions to coordinate
globin synthesis with heme availability in RRL.
phosphorylation and inhibition of protein synthesis
in a number of cell lines (15). However, the mechanism of this
NO-induced increase in eIF2
phosphorylation was not characterized.
In this report, we examined the possible function of the NT-HBD in the
regulation of HRI activation, and present evidence that HRI activity is
regulated by the binding of the diffusible gases NO and CO to its
NT-HBD.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phosphorylation in Reticulocyte
Lysates--
Protein synthesis was determined by measuring the
incorporation of [14C]leucine into the acid-precipitable
protein in 5-µl aliquots taken from standard RRL mixtures containing
20 µM hemin at 30 °C as described (16, 17). The
phosphorylation of eIF2
in 2 µl of protein synthesis mixes was
analyzed as previously described by Western blotting of one-dimensional
vertical isoelectric focusing (VIEF) slab gels using 1:1000 dilution of
anti-eIF2
monoclonal ascites fluid (18-20).
kinase activity of
His7-[35S]HRI bound to immunoresin were
performed for 4 min at 30 °C as described (21, 22). Samples were
analyzed by 10% SDS-polyacrylamide gel electrophoresis, followed by
transfer to polyvinylidene difluoride membrane and autoradiography as
described previously (21, 22). Autophosphorylation of HRI
was assayed by the incorporation of [32P]Pi
into HRI during eIF2
kinase assays incubated with
[
-32P]ATP. 32P-Labeled HRI and eIF2
were detected by quantitatively quenching 35S emissions
with three intervening layers of previously developed x-ray film (21,
22).
-32P]ATP and treated or untreated with NO or CO as
described above. A non-His-tagged [35S]HRI was similarly
studied for nonspecific interactions to the resin.
phosphorylation present in C2C12 cells were
determined by lysis of cells directly in VIEF sample buffer and Western
blotting of samples separated on VIEF slab gels as described previously
(25). Levels of eIF2
phosphorylation present in NT-2 cells were
determined by Western blotting of cell extracts separated by
SDS-polyacrylamide gel electrophoresis with antibody specific to
phosphorylated eIF2
(Ref. 26); provided by Dr. D. DeGracia, Wayne
State University, Detroit, MI.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Phosphorylation in RRL--
To determine the effects of NO on protein
synthesis varying concentrations of the NO generator NOC-9 was added to
hemin-supplemented RRL (Fig.
1a). Low concentrations of
NOC-9 stimulated protein synthesis slightly, while high concentrations
of NOC-9 caused a rapid and complete shut-off of protein synthesis. To
examine the mechanism by which NO inhibits translation, the
phosphorylation status of eIF2
in NOC-9-treated RRL was analyzed
(Fig. 1b). NO generation stimulated eIF2
phosphorylation
in a concentration-dependent manner with 1 mM
NOC-9 treatment of hemin-supplemented RRL causing a near quantitative
phosphorylation of eIF2
.
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Fig. 1.
Effect of NO on protein synthesis and
eIF2 phosphorylation. RRL was incubated
under conditions for protein synthesis in the presence of 10 µM hemin (0.00) and in the presence of increasing
concentrations of NOC-9 (0.01, 0.10, 0.25, 0.5, and 1.0 mM)
as indicated at 30 °C. A, protein synthesis was measured
by the incorporation of [14C]leucine into as
acid-perceptible protein at times indicated in the figure.
B, after 20 min of incubation, eIF2
phosphorylation
levels were determined as described under "Experimental
Procedures."
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Fig. 2.
Effect of CO on protein synthesis and
NO-induced activation of HRI. a, hemin-supplemented
protein synthesis mixes were gassed (open
circles, open triangles) or not gassed
(closed circles, open
squares) with 100% CO for 3 min on ice, followed by
incubation at 30 °C in the presence (open
squares, open triangles) or absence
(open circles, closed
circles) of 0.5 mM NOC-9. Protein synthesis was
measured by the incorporation of [14C]leucine into as
acid-perceptible protein at times indicated in the figure.
b, to study the effect of CO on HRI,
His7-[35S]HRI was transformed in
heme-deficient RRL and repressed by treatment with 10 µM
hemin as described under "Experimental Procedures." Samples were
then treated with 1 mM NOC-9 (NO,
lane 4), buffer (control,
lane 2), or CO (gassing with 100% CO for 3 min
on ice prior to further incubation; CO, lane
3) for 20 min. His7-[35S]HRI was
affinity-purified, and samples were then either not treated (control,
CO, and NO), gassed with 100% CO for 3 min on ice
(NOC-9 CO, lane 6), or
treated with 1 mM NOC-9 (CO
NO,
lane 5) followed by assay for kinase activity.
Lane 1, nonspecific binding of activity from RRL
expressing non-His-tagged [35S]HRI that was treated with
NOC-9 (NI+NOC-9). Upper panel,
autoradiogram of [35S]HRI. Lower
panel, autoradiogram of [32P]HRI.
*HRI, transformed HRI.
kinase (
, transformed HRI]. The kinase
activity of transformed HRI was inhibited by addition of hemin
(
/+, repressed HRI) to the heme-deficient incubations. Addition of 1 mM NOC-9 caused a marked enhancement of the autokinase and
eIF2
kinase activity of mature-competent, transformed and repressed
HRI (Fig. 3a, lanes 12-14). Although 0.01 and 0.1 mM NOC-9 had little stimulatory effect on the
eIF2
kinase activity of transformed HRI, these concentrations of
NOC-9 increased the activity of repressed HRI to that of transformed HRI in untreated heme-deficient RRL. Although 0.1 mM NOC-9
caused mature-competent HRI to become as active an eIF2
kinase as
transformed HRI in untreated heme-deficient RRL, it also caused a small
decrease in the autokinase activity of transformed and repressed
HRI. This decreased autokinase activity may explain the slight
enhancement of protein synthesis that was observed in the presence of
low NOC-9 concentrations in Fig. 1a.
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Fig. 3.
Effect of NO on
[35S]His7 HRI activity in situ
and in vitro. Mature-competent (+),
transformed ( ), and repressed (
/+)
His7-[35S]HRI were generated de
novo in RRL as described under "Experimental Procedures."
a, to determine the effects of NO on the activation of HRI
in situ, samples of RRL were treated with NOC-9 at the
indicated concentrations for 20 min. His-tagged HRI was
affinity-purified and assayed for kinase activity.
His7-[35S]HRI was detected by direct
autoradiography (upper panel), whereas
32P-phosphorylated HRI (middle panel)
and 32P-phosphorylated eIF2
(lower
panel) were detected after quenching 35S
emissions. b, to determine the effects of NO on the
activation of forms of HRI in vitro, His-tagged HRI was
affinity-purified from RRL, and samples were then treated with NOC-9 at
the indicated concentrations in vitro during the assay for
autokinase activity. NS, nonspecific binding of activity
from RRL expressing non-His-tagged [35S]HRI that was
treated with NOC-9. His7-[35S]HRI was
detected by direct autoradiography (top panel),
and 32P-phosphorylated HRI (bottom
panel) was detected after quenching 35S
emissions. HRI*, transformed HRI.
View larger version (47K):
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Fig. 4.
The effect of NEM and dithiothreitol on
NO-induced activation of HRI. Repressed
His7-[35S]HRI was synthesized de
novo as described under "Experimental Procedures."
a, RRL was then treated with 1 mM NEM
(lane 4), 1 mM NOC-9 (lane
5), or not treated (control, lane 3)
for 10 min followed by immunoadsorption with anti-His-tagged
antibodies. Samples were then treated with 1 mM NOC-9
(NEM+NOC-9, lane 6) or treated with
buffer (control, NEM, NOC-9) during
the assay for autokinase activity. Lanes 1 and
2, nonspecific binding of activity from RRL expressing
non-His-tagged [35S]HRI that was treated with NEM and
NOC-9 (lane 2) or not (control,
lane 1). b, samples of RRL containing
repressed His7-[35S]HRI were treated with 1 mM NOC-9 (NOC-9, lane 3),
1 mM DTT (lane 4), 1 mM
DTT plus 1 mM NOC-9 (DTT+NOC-9, lane
5) or not treated (control, lane
2) for 10 min. His7-[35S]HRI was
affinity-purified and assayed for kinase activity. Lane
1, nonspecific binding of activity from RRL expressing
non-His-tagged [35S]HRI that was treated with NOC-9.
His7-[35S]HRI was detected by direct
autoradiography (upper panel, A and
B), while 32P-phosphorylated HRI
(middle panel, A and B) and
32P-phosphorylated eIF2 (lower
panel, B) were detected after quenching
35S emissions. HRI*, transformed HRI.
/
region shifting from 534 nm to distinct
- and
-bands
with absorption maximum at 560 and 530 nm, respectively (Fig.
5A). Addition of NO caused the
Soret peak to shift immediately to 421 nm and decrease in intensity by
~50%, with the
- and
-bands shifting to 572 and 542 nm,
respectively (Fig. 5B). After 15 min, the Soret band
broadened to give an absorption maximum at 402 nm. In contrast, gassing
the reduced NT-HBD with CO (Fig. 5C) caused the Soret band
to shift from 428 to 422 nm and increase in intensity by ~30%, with
the absorption maximum of the
- and
-bands shifting to 568 and
536 nm, respectively. These results indicate that NO and CO become
coordinated to the heme moiety of the NT-HBD of HRI.
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Fig. 5.
Spectral analysis of NT-HBD of HRI incubated
with NO and CO. Spectral analysis of the purified recombinant
NT-HBD of HRI was done by scanning from 200-650 nm (A,
spectrum 1). Dithionite was then added to reduce
the bound hemin and the sample was rescanned (A,
spectrum 2). After the spectral analysis of the
dithionite-treated NT-HBD, NO was generated by the addition of 1 mM NOC-9, and the sample was rescanned immediately
(B, spectrum 1) and after 15 min
(B, spectrum 2), or the
dithionite-treated NT-HBD was gassed with 100% CO for 3 min followed
by spectral analysis (C). Arrows indicate the
Soret band, the insets within the panels show an
expansion of the region of the /
absorption spectrum, and the
asterisk (*) indicates an absorption peak due to the
presence of dithionite.
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Fig. 6.
The ability of NO to induced activation of
HRI is modulated through the NT-HBD. a,
His7-[35S]HRI (wt HRI) or
His7-[35S]HRI/Met-3 was synthesized de
novo, and then matured and transformed in heme-deficient RRL, and
repressed by the addition of 10 µM hemin as described
under "Experimental Procedures." RRL was then treated with 1 mM NEM (NEM; lanes 2,
4, and 7), 1 mM NOC-9
(NOC-9; lanes 2, 5, and
8) or not treated (control; lane 3)
for 10 min. His-tagged wtHRI and HRI/Met-3 were affinity-purified and
assayed for kinase activity. His7-[35S]HRI
was detected by direct autoradiography (upper
panel), while 32P-phosphorylated HRI
(middle panel) and 32P-phosphorylated
eIF2 (lower panel) were detected after
quenching 35S emissions. HRI*, transformed HRI.
NS - nonspecific binding of activity from RRL expressing non-His-tagged
[35S]HRI that was treated with NEM plus NOC-9
(lane 2) or not (control,
lane 1). B,
His7-[35S]HRI/Met-3 (lanes
2 and 4) and non-His-tagged
[35S]HRI/Met-3 (lanes 1,
3, and 5) were synthesized for 20 min in TnT RRL,
followed by the addition of aurintricarboxylic acid. Non-His-tagged
[35S]HRI/Met-3 was then mixed with RRL containing
previously synthesized His-tagged [35S]NT-HBD.
His7-[35S]HRI/Met-3 (lanes
2 and 4) and non-His-tagged
[35S]HRI/Met-3 plus His-tagged [35S]NT-HBD
(lanes 3 and 5) were then matured in
heme-deficient RRL for 50 min followed by a 10-min treatment with 1 mM NOC-9 (lanes 4 and 5).
His-tagged proteins were then affinity-purified and assayed for kinase
activity. Upper panel, autoradiogram of
35S-labeled proteins. Lower panel,
autoradiogram of 32P-phosphorylated eIF2
. NS,
nonspecific binding of activity from RRL expressing non-His-tagged
[35S]HRI/Met-3 and NT-HBD that was treated with
NOC-9.
kinase
activity indicated that the specific activity of the
[35S]HRI/Met-3 (32P incorporated into
substrate per amount of [35S]HRI/Met-3) that was
complexed with the NT-HBD was significantly greater than the specific
activity of His7-[35S]HRI/Met-3 alone (Fig.
6b). Furthermore, NOC-9 markedly enhanced the eIF2
kinase activity of [35S]HRI/Met-3 that was complexed with
NT-HBD. These results suggest that the NT-HBD plays an important
positive role in the NO-induced activation of HRI.
Libraries containing cDNAs encoding HRI identified in dbEST
kinase
activity was specifically adsorbed from the NT-2 and C2C12 cells
extracts (Fig. 7A). Treatment
of immunoadsorbed protein with NOC-9 markedly stimulated
phosphorylation of a protein band, which was specifically
immunoadsorbed from the NT-2 and C2C12 extracts by the anti-HRI/NT-HBD
antibody (Fig. 7B), and co-migrated with RRL HRI (data not
shown). A phosphoprotein that migrated near HRI was immunoadsorbed from
C2C12 extracts. However, the amount of HRI immunoadsorbed from the
C2C12 extracts was below the limit of detection by Western blotting, so
we could not determine whether the protein was HRI by Western blotting.
In addition, our anti-HRI/NT-HBD antibody failed to adsorb any eIF2
kinase or NO-activable autokinase activity from extracts prepared from the blood of HRI-knock out
mice,2 indicating that our
antibody was not cross-reacting with other eIF2
kinases,
particularly PKR, which is known to be present in blood cells (42).
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Fig. 7.
Effect of NO on HRI activity
immunoadsorbed from extracts of from NT-2 and C2C12 cells.
A, HRI was immunoadsorbed from RRL, NT-2, or C2C12 cells
with anti-HRI/NT-HBD (I) or non-immune control
(N) antibody and assayed for kinase activity as described
under "Experimental Procedures." Autoradiogram of
32P-phosphorylated eIF2 . Note that the migration of HRI
is indicated, but the exposure time required to observe phosphorylated
eIF2
in the C2C12 sample overexposes a 32P-labeled band
that migrates near HRI (indicated by * in B). B,
NT-2 and C2C12 cells were grown for 16 h in the presence of DMEM
supplemented with 10 µM hemin. The cells were lysed, and
HRI was immunoprecipitated from cell lysates (NT-2 or C2C12) with
anti-HRI/NT-HBD (I) or non-immune control antibodies
(N). Samples were then treated with 1 mM NOC-9
(+) or buffer (
), and autokinase activity was assayed as described
under "Experimental Procedures." Figure shows autoradiogram of
32P-phosphorylated HRI (arrow); *, unidentified
phosphoprotein that migrates near HRI.
phosphorylation in NT-2
(Fig. 8A) and C2C12 (Fig.
8B) cells, cells were cultured in the presence or absence of
the NO donor SNAP. Treatment of both NT-2 (Fig. 8A) and
C2C12 (Fig. 8B) cells with SNAP caused an increase in
eIF2
phosphorylation in both cell lines. These results suggest that
NO may be an important physiological regulator of HRI activation in
non-erythroid cells.
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Fig. 8.
Effect of NO on eIF2
phosphorylation in NT-2 and C2C12 cells. NT-2 (A)
or C2C12 (B) cells were treated with Me2SO (
,
lane 1) or 1 mM (NT-2) or 300 µM (C2C12) SNAP (+, lane 2) for
4 h and levels of eIF2
phosphorylation determined as described
under "Experimental Procedures." eIF2
bands were quantified by
scanning densitometry and expressed above the lanes as optical density
(O.D.) × mm2.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
phosphorylation performs a critical role in control of cell
proliferation (reviewed in Refs. 44 and 45), with increased eIF2
phosphorylation (20, 46, 47) and down-regulation of protein synthesis
correlating with cell growth arrest and entrance into G0
(48). Cell growth arrest is required for the subsequent terminal
differentiation of a number of cell types (reviewed in Ref. 44).
Increased eIF2
phosphorylation also mediates different forms of
stress-related apoptosis (44, 45, 49, 50). Furthermore, suppression of
eIF2
phosphorylation (44) or expression of a nonphosphorylatable
mutant of eIF2
causes malignant transformation of cells
(51). These observations suggest that eIF2
kinases act
as tumor suppressors in the regulation of cell growth (reviewed in Ref.
44). Specifically, these studies have focused on the double-stranded
RNA-activated eIF2
kinase (PKR), as constitutive or inducible
expression of this eIF2
kinase occurs in most cell types.
phosphorylation plays in suppressing cell growth and inducing apoptotic cell death.
phosphorylation in a number of cell
lines (15).
-induced apoptosis in L929
fibroblasts (69), suggesting that the anti-apoptotic effect of HO-1
expression may be mediated in part via CO. Here we demonstrate that CO
suppresses the activity of HRI and stimulates protein synthesis in RRL,
and has the capacity to reverse NO-induced activation of HRI. These
results suggest that HRI may be regulated through the competition
between NO and CO for a common binding site in the NT-HBD of HRI.
phosphorylation may be a critical event in protecting cells
from NO-induced apoptotic cell death.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. Steven D. Hartson (Oklahoma State University, Stillwater, OK) and Jane-Jane Chen (Massachusetts Institute of Tchnology, Cambridge, MA) for critical reading of the manuscript, the staffs of the Sarkey's Biotechnology Research Laboratory and the HYCABS Core Facility at Oklahoma State University for their technical support, and Dr. Jane-Jane Chen for generously providing HRI plasmid constructs used in these studies.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health NIEHS Grant ES-04299 and by Oklahoma Agricultural Experiment Station Project 1975.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: 246 NRC, Oklahoma
State University, Stillwater, OK 74078-3035. Tel.: 405-744-6200; Fax: 405-744-7799; E-mail: rmatts@biochem.okstate.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M011476200
2 B.-G. Yun, A. Han, J.-J. Chen, and R. L. Matts, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
HRI, heme-regulated
inhibitor (heme-regulated eukaryotic initiation factor 2 kinase);
eIF, eukaryotic initiation factor;
eIF2
,
-subunit of eIF2;
HBD, heme-binding domain;
NT-HBD, N-terminal HBD of HRI;
RRL, rabbit
reticulocyte lysate;
TnT RRL, nuclease-treated reticulocyte lysate with
coupled transcription and translation;
DTT, dithiothreitol;
NEM, N-ethylmaleimide;
VIEF, vertical isoelectric focusing;
NOC-9, 6-(2-hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine;
SNAP, S-nitroso-N-acetylpenicillamine;
HO, heme
oxygenase;
DMEM, Dulbecco's modified Eagle's medium;
PKR, double-stranded RNA-activated eIF2
kinase.
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REFERENCES |
---|
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---|
1. | Chen, J.-J. (1993) in Translational Regulation of Gene Expression 2 (Ilan, J., ed) , pp. 349-372, Plenum Press, New York |
2. | Chen, J.-J., and London, I. M. (1995) Trends Biochem. Sci. 20, 105-108[CrossRef][Medline] [Order article via Infotrieve] |
3. | Hunt, T. (1979) Miami Winter Symp. 16, 321-345 |
4. | Chen, J. J., Throop, M. S., Gehrke, L., Kuo, I., Jayanta, K. P., Brodsky, M., and London, I. M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7729-7733[Abstract] |
5. | Chefalo, P. J., Oh, J., Rafie-Kolpin, M., Kan, B., and Chen, J.-J. (1998) Eur. J. Biochem. 258, 820-830[Abstract] |
6. |
Rafie-Kolpin, M.,
Chefalo, P. J.,
Hussain, Z.,
Hahn, J.,
Uma, S.,
Matts, R. L.,
and Chen, J.-J.
(2000)
J. Biol. Chem.
275,
5171-5178 |
7. |
Uma, S.,
Matts, R. L.,
Guo, Y.,
White, S.,
and Chen, J.-J.
(2000)
Eur. J. Biochem.
267,
498-506 |
8. | Ignarro, L. J. (1990) Annu. Rev. Pharmacol. Toxicol. 30, 535-560[CrossRef][Medline] [Order article via Infotrieve] |
9. | Zhang, J., and Snyder, S. H. (1995) Annu. Rev. Pharmocol. Toxicol. 35, 213-233[CrossRef][Medline] [Order article via Infotrieve] |
10. | Snyder, S. H., Jaffrey, S. R., and Zakhary, R. (1998) Brain Res. Rev. 26, 167-175[Medline] [Order article via Infotrieve] |
11. | Foresti, R., and Motterlini, R. (1999) Free Radic. Res. 31, 549-75[Medline] [Order article via Infotrieve] |
12. | Tsai, A. (1994) FEBS Lett. 341, 141-145[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Hentze, M. W.,
and Kuhn, L. C.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
8175-8182 |
14. | Kim, Y.-M., Bergonia, H. A., Muller, C., Pitt, B. R., Watkins, W. D., and Lancaster, J. R. (1995) Adv. Pharmacol. 34, 277-291[Medline] [Order article via Infotrieve] |
15. | Kim, Y.-M., Son, K., Hong, S.-J., Green, A., Chen, J.-J., Tzeng, E., Hierholzer, C., and Billiar, T. R. (1998) Mol. Medicine 4, 179-190 |
16. | Ernst, V., Levin, D. H., and London, I. M. (1978) J. Biol. Chem. 253, 7163-7172[Medline] [Order article via Infotrieve] |
17. | Hunt, T., Vanderhoff, G., and London, I. M. (1972) J. Mol. Biol. 66, 471-481[Medline] [Order article via Infotrieve] |
18. | Thulasiraman, V., Xu, Z., Uma, S., Gu, Y., Chen, J.-J., and Matts, R. L. (1998) Eur. J. Biochem. 255, 552-562[Abstract] |
19. | Maurides, P. A., Akkaraju, G. R., and Jagus, R. (1989) Anal. Biochem. 183, 144-151[Medline] [Order article via Infotrieve] |
20. |
Scorsone, K. A.,
Panniers, R.,
Rowlands, A. G.,
and Henshaw, E. C.
(1987)
J. Biol. Chem.
262,
14538-14543 |
21. |
Uma, S.,
Hartson, S. D.,
Chen, J.-J.,
and Matts, R. L.
(1997)
J. Biol. Chem.
272,
11648-11656 |
22. |
Uma, S.,
Thulasiraman, V.,
and Matts, R. L.
(1999)
Mol. Cell. Biol.
19,
5861-5871 |
23. | Pleasure, S. J., Page, C., and Lee, M. V.-Y. (1992) J. Neurosci. 12, 1802-1815[Abstract] |
24. |
Matts, R. L.,
Xu, Z.,
Pal, J. K.,
and Chen, J.-J.
(1992)
J. Biol. Chem.
267,
18160-18167 |
25. | Benton, P. A., Barret, D. J., Matts, R. L., and Lloyd, R. E. (1996) J. Virol. 70, 5525-5532[Abstract] |
26. | DeGracia, D. J., Sullivan, J. M., Neumar, R. W., Alousi, S. S., Hikade, K. R., Pittman, J. E., White, B. C., Rafols, J. A., and Krause, G. S. (1997) J. Cereb. Blood Flow Metab. 17, 1291-302[Medline] [Order article via Infotrieve] |
27. | Xu, Z., Pal, J. K., Thulasiraman, V., Hahn, H. P., Chen, J.-J., and Matts, R. L. (1997) Eur. J. Biochem. 246, 461-470[Abstract] |
28. | Matts, R. L., Hurst, R., and Xu, Z. (1993) Biochemistry 32, 7323-7328[Medline] [Order article via Infotrieve] |
29. |
Matts, R. L.,
and Hurst, R.
(1992)
J. Biol. Chem.
267,
18168-18174 |
30. | Broillet, M.-C. (1999) Cell. Mol. Life Sci. 55, 1036-1042[CrossRef][Medline] [Order article via Infotrieve] |
31. | Ducrocq, C., Blanchard, B., Pignatelli, B., and Ohshima, H. (1999) Cell. Mol. Life Sci. 55, 1068-1077[CrossRef][Medline] [Order article via Infotrieve] |
32. | Henry, Y., and Guissani, A. (1999) Cell. Mol. Life Sci. 55, 1003-1014[CrossRef][Medline] [Order article via Infotrieve] |
33. |
Matts, R. L.,
Schatz, J. R.,
Hurst, R.,
and Kagen, R.
(1991)
J. Biol. Chem.
266,
12695-12702 |
34. |
Chen, J.-J.,
Yang, J. M.,
Petryshyn, R.,
Kosower, N.,
and London, I. M.
(1989)
J. Biol. Chem.
264,
9559-9564 |
35. |
Yang, J. M.,
London, I. M.,
and Chen, J.-J.
(1992)
J. Biol. Chem.
267,
20519-20524 |
36. |
Foresti, R.,
Clark, J. G.,
Green, C. J.,
and Motterlini, R.
(1997)
J. Biol. Chem.
272,
18411-18417 |
37. | Jackson, R. J., Herbert, P., Cambell, E. A., and Hunt, T. (1983) Eur. J. Biochem. 131, 313-324[Medline] [Order article via Infotrieve] |
38. | Crosby, J. S., Lee, K., London, I. M., and Chen, I. M. (1994) Mol. Cell. Biol. 14, 3906-3914[Abstract] |
39. | Pal, J. K., Chen, J.-J., and London, I. M. (1991) Biochemistry 30, 2555-2562[Medline] [Order article via Infotrieve] |
40. |
Mellor, H.,
Flowers, K. M.,
Kimball, S. R.,
and Jefferson, L. S.
(1994)
J. Biol. Chem.
269,
10201-10204 |
41. |
Berlanga, J. J.,
Herrero, S.,
and De Haro, C.
(1998)
J. Biol. Chem.
273,
32340-32346 |
42. | London, I. M., Levin, D. H., Matts, R. L., Thomas, N. S. B., Petryshyn, R., and Chen, J. J. (1987) in The Enzymes (Boyer, P. D. , and Krebs, E. G., eds), 3rd Ed., Vol. XVIII , pp. 359-380, Academic Press, New York |
43. | Clemens, M. J., and Bommer, U.-A. (1999) Int. J. Biochem. Cell Biol. 31, 1-23[CrossRef][Medline] [Order article via Infotrieve] |
44. | Jagus, R., Joshi, B., and Barber, G. N. (1999) Int. J. Biochem. Cell Biol. 31, 123-138[CrossRef][Medline] [Order article via Infotrieve] |
45. |
Kaufman, R. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11693-11695 |
46. | Rowlands, A. G., Montine, K. S., Henshaw, E. C., and Panniers, R. (1988) Eur. J. Biochem. 175, 93-99[Abstract] |
47. | Duncan, R. F., and Hershey, J. W. B. (1985) J. Biol. Chem. 260, 5493-5497[Abstract] |
48. | Zetterberg, A., and Larsson, O. (1995) in Cell Cycle Control (Hutchinson, C. , and Glover, D. M., eds) , pp. 206-227, IRL Press, Oxford |
49. |
Srivastava, S. P.,
Kumar, K. U.,
and Kaufman, R. J.
(1998)
J. Biol. Chem.
273,
2416-2423 |
50. |
Der, S. D.,
Yang, Y. L.,
Weissmann, C.,
and Williams, B. R.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
3279-3283 |
51. | Donze, O., Jagus, R., Koromilas, A. E., Hershey, J. W. B., and Sonenberg, N. (1995) EMBO J. 14, 3828-3834[Abstract] |
52. |
Gross, S. S.,
and Lane, P.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9967-9969 |
53. |
Gow, A. J.,
Luchsinger, B. P.,
Pawloski, J. R.,
Singel, D. J.,
and Stamler, J. S.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
9027-9032 |
54. |
Rafferty, S. P.,
Domachowske, J. B.,
and Malech, H. L.
(1996)
Blood
88,
1070-1078 |
55. |
Domachowske, J. B.,
Rafferty, S. P.,
Singhnania, N.,
Mardiney, M.,
and Malech, H. L.
(1996)
Blood
88,
2980-2988 |
56. | Maccarrone, M., Fantini, C., Ranalli, M., ., Melino, G., and Agro, A. F. (1998) FEBS Lett. 434, 421-424[CrossRef][Medline] [Order article via Infotrieve] |
57. |
Shami, P. J.,
and Weinberg, J. B.
(1996)
Blood
87,
977-982 |
58. | Selleri, C., Sato, T., Raiola, A., Rotoli, B., Young, N. S., and Maciejewski, J. P. (1997) Br. J. Haematol. 99, 481-489[CrossRef][Medline] [Order article via Infotrieve] |
59. | Maciejewski, J. P., Selleri, C., Sato, T., Cho, H. J., Keefer, L. K., Nathan, C. F., and Young, N. S. (1995) J. Clin. Invest. 96, 1085-1092[Medline] [Order article via Infotrieve] |
60. | Nathan, C., and Xie, Q. W. (1994) Cell 8, 915-918 |
61. | Billiar, T. R. (1995) Ann. Surg. 221, 339-349[Medline] [Order article via Infotrieve] |
62. |
Nathan, C.
(1992)
FASEB J.
6,
3051-3064 |
63. | Melino, G., Bernassola, F., Knight, R. A., Corasaniti, M. T., Nistici, G., and Finazzi-Agro, A. (1997) Nature 388, 432-433[CrossRef][Medline] [Order article via Infotrieve] |
64. |
Farlis-Eisner, R.,
Sherman, M. P.,
Aberhard, E.,
and Chaudhuri, G.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
9407-9411 |
65. | Ponka, P. (1999) Am. J. Med. Sci. 318, 241-256[Medline] [Order article via Infotrieve] |
66. |
Maines, M. D.
(1988)
FASEB J.
2,
2557-2568 |
67. |
Durante, W.,
Kroll, M. H.,
Christodoulides, N.,
Peyton, K. J.,
and Schafer, A. I.
(1997)
Circ. Res.
80,
557-564 |
68. |
Hartsfield, C. L.,
Alam, J.,
Cook, J. L.,
and Choi, A. M.
(1997)
Am. J. Physiol.
273,
L980-L988 |
69. | Petrache, I., Otterbein, L. E., Alam, J., Wiegand, G. W., and Choi, A. M. (2000) Am. J. Physiol. 278, L312-319 |
70. |
Zhao, Y.,
Brandish, P. E.,
Ballou, D. P.,
and Marletta, M. A.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
14753-14758 |
71. |
Nakano, R.,
Sato, H.,
Watanabe, A.,
Ito, O.,
and Shimizu, T.
(1996)
J. Biol. Chem.
271,
8570-8574 |
72. |
Kim, Y. M.,
Bergonia, H. A.,
Muller, C.,
Pitt, B. R.,
Watkins, W. D.,
and Lancaster, J. R., Jr.
(1995)
J. Biol. Chem.
270,
5710-5713 |
73. | Khatsenko, O. G., Gross, S. S., Rifkind, A. B., and Vane, J. R. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 11147-11151[Abstract] |
74. | Juckett, M., Zheng, Y., Yuan, H., Pastor, T., Antholine, W., Weber, M., and Vercellotti, G. (1998) J. Biol. Chem. 272, 23388-2339[CrossRef] |