From the Sealy Center for Oncology and Hematology, Department of Internal Medicine, The University of Texas Medical Branch at Galveston, Galveston Texas 77555-1048
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The double-stranded (ds)
RNA-dependent protein kinase (PKR) regulates protein
synthesis by phosphorylating the Eukaryotic cells rapidly and reversibly halt protein synthesis in
response to a variety of stresses including virus infection and
cytotoxic chemical injury (1, 2). This fundamental homeostatic mechanism is thought to involve the phosphorylation of the We previously reported that in interleukin-3
(IL-3)-dependent cells, IL-3 deprivation induced activation
of PKR in close association with a decreased rate of total protein
synthesis (7). Recently, it has been reported that stresses such as
Ca2+ depletion from the endoplasmic reticulum, sodium
arsenite, or hydrogen peroxide treatments rapidly induce PKR activation
(8-10). It was suggested that although PKR is activated by dsRNA
in vitro, a dsRNA-independent activation mechanism may exist
in cells because it is difficult to envision that such diverse stresses
are likely to rapidly change the intracellular levels of dsRNA species.
Thus a novel cellular regulator of PKR was sought. By screening a mouse cDNA library using the yeast two-hybrid interacting cloning system, we discovered a PKR-associating protein RAX. RAX appears to be a mouse
homologue of PACT, a recently isolated direct activator for PKR (11).
We now find that RAX can activate PKR in the absence of dsRNA in a
cell-free system as reported for PACT. However, results here indicate
that the RAX-PKR association and any resulting cellular activation of
PKR may be regulated by an unique, stress-induced signaling mechanism
featuring RAX phosphorylation.
Cloning of RAX--
Using polymerase chain reaction-based
mutagenesis, the mouse PKR cDNA (12) was first mutated at lysine
271 to arginine to generate the catalytically inactive PKR(K271R), the
mouse equivalent to human PKR(K296R) mutant (13), and then subcloned
into pGBT9 (CLONTECH). A random primed NFS/N1.H7
cell cDNA library was generated in pGAD10
(CLONTECH). The yeast two-hybrid screening of the
cDNA library was performed according to the Matchmaker Two-Hybrid
System protocol (CLONTECH).
Northern Blot Analysis--
A 405-base pair mouse RAX cDNA
fragment (nucleic acid positions 943-1348) was amplified by polymerase
chain reaction and radiolabeled using the Prime-A-Probe kit (Ambion,
Inc., Austin, TX). A mouse multiple tissue blot was obtained from
CLONTECH, and hybridization was performed according
to the manufacturer's instruction.
Bacterial Expression of RAX and Antibody Production--
The RAX
cDNA was cloned into pRSET vector (Invitrogen) to generate a
polyhistidine-tagged RAX and used to transform Escherichia coli BL21 (DE3) pLysS (Novagen, Madison, WI). The protein was induced and partially purified with TALON metal affinity resin according to the manufacturer's instruction
(CLONTECH). The fraction containing RAX was
incubated with poly(I·C)-agarose beads (Amersham Pharmacia Biotech),
and the protein was dissociated by boiling in SDS-PAGE sample buffer
and further purified by SDS-PAGE. The gel slice containing the
recombinant RAX was used to immunize a rabbit to raise anti-RAX
antiserum (COVANCE Research Products Inc., Denver, PA).
Mammalian Expression of RAX--
The murine
IL-3-dependent NFS/N1.H7 cells (14) were maintained in RPMI
1640 medium supplemented with 20% WEHI-3B cell conditioned medium as
described previously (7). The hemagglutinin (HA) epitope-tagged RAX
cDNA was subcloned into the expression vector pcDEF3 (15) and
transfected into NFS/N1.H7 cells by electroporation. The clones stably
expressing HA-RAX were selected as described previously (16).
Immunoprecipitation and Immunoblotting--
Cells (1 × 107) were treated with 1 mM sodium arsenite
(Sigma), 1 mM hydrogen peroxide (Sigma), or 1 µM thapsigargin (Calbiochem) and lysed in 1 ml of buffer
A (10 mM HEPES, pH 7.2, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 20 mM sodium
fluoride, 20 mM sodium pyrophosphate, 20 mM
Nucleic Acid Binding Assays--
5 µg of polyhistidine-tagged
RAX was incubated with 30 µl of poly(I·C)-agarose, native
DNA-cellulose (Amersham Pharmacia Biotech), or poly(C)-agarose (Sigma)
beads in Tris-buffered saline (50 mM Tris-HCl, pH 7.4, 150 mM NaCl) containing 1% Triton X-100 for 1 h at
4 °C. The beads were washed three times before boiling in Laemmli
buffer. The eluted protein was loaded onto a 10% SDS-PAGE gel followed
by Coomassie Blue R-250 staining.
Vertical Slab Isoelectric Focusing--
The phosphorylation
state of eIF2 In Vitro Kinase Assays--
PKR was freshly isolated from the
exponentially growing NFS/N1.H7 cells by immunoaffinity purification
using anti-PKR antibody (Ab-2) (7, 19). PKR was recovered by
competitive elution with the synthetic peptide (7). The HA-RAX was
immunoprecipitated with anti-HA antibody as described above and was
recovered from the immunocomplex by competitive elution with 1 mg/ml HA
peptide (Roche Molecular Biochemicals). After dialysis against
Tris-buffered saline, the purified HA-RAX was quantitated by
immunoblotting using bacterially expressed RAX as a reference. Samples
were incubated for 10 min at 30 °C in 50 µl of reaction buffer (25 mM Tris-HCl, pH 7.6, 2 mM MgCl2, 2 mM MnCl2, 1 mM dithiothreitol, 20 µM ATP, 5 µCi of [ DNA Fragmentation Assay--
Cells (1.5 × 106)
were washed twice with Tris-buffered saline and lysed in 100 µl of
lysis buffer (10 mM Tris-HCl, pH 7.5, 10 mM
EDTA, and 0.5% Triton X-100). The lysate was centrifuged at 13,000 rpm
for 10 min, and the supernatant was treated with 1 µg of RNase A and
40 µg of proteinase K for 2 h at 37 °C, respectively. The DNA
was precipitated with 0.5 M NaCl and 50% isopropanol at To identify novel cellular proteins that could potentially
interact with and modulate PKR, we screened a mouse
IL-3-dependent NFS/N1.H7 cell cDNA library using the
yeast two-hybrid interactive cloning system (20). As bait, the mouse
PKR(K271R), which is deficient in kinase activity (13), was used
because wild type PKR inhibits yeast growth (21). From 4 × 106 yeast transformants screened, eight positive clones
were obtained. The sequence analysis revealed that six clones are
derived from the same novel gene. Clone c35 contained the longest (1080 base pairs) cDNA and encoded a putative 313-amino acid polypeptide terminated by TAG stop codon (Fig.
1A). This gene is referred to
as RAX (PKR-associated protein X).
Further 5' and 3' rapid amplification of cDNA ends studies using
poly(A) RNA from NFS/N1.H7 cells showed that full-length RAX cDNA
is 1.6 kilobases. Computer search (BLAST) analysis indicated that both
cDNA and amino acid sequences of RAX are highly homologous to those
of human PACT (95 and 98% identity, respectively), a recently
identified PKR-associating protein (11) (Fig. 1A). The
striking similarity suggests that RAX is a murine counterpart of PACT.
RAX also has significant homology to two other dsRNA-binding proteins,
including a Xenopus dsRNA-binding protein Xlrbpa (22) (69%
homology) and the mammalian TAR-RNA-binding protein (23) and its murine
counterpart, Prbp (24) (60% homology). As featured in these proteins,
RAX also contains three dsRNA-binding motifs arranged in tandem whose
function is not yet clear (25) (Fig. 1A). As predicted,
recombinant RAX was able to specifically bind dsRNA because virtually
no binding to DNA or single-stranded RNA was observed (Fig.
1B).
subunit of eukaryotic initiation
factor-2. PKR is activated by viral induced dsRNA and thought to be
involved in the host antiviral defense mechanism. PKR is also activated
by various nonviral stresses such as growth factor deprivation,
although the mechanism is unknown. By screening a mouse cDNA
expression library, we have identified an ubiquitously expressed
PKR-associated protein, RAX. RAX has a high sequence homology to human
PACT, which activates PKR in the absence of dsRNA. Although RAX also
can directly activate PKR in vitro, overexpression of RAX
does not induce PKR activation or inhibit growth of interleukin-3
(IL-3)-dependent cells in the presence of IL-3. However,
IL-3 deprivation as well as diverse cell stress treatments including
arsenite, thapsigargin, and H2O2, which are
known to inhibit protein synthesis, induce the rapid phosphorylation of
RAX followed by RAX-PKR association and activation of PKR. Therefore,
cellular RAX may be a stress-activated, physiologic activator of PKR
that couples transmembrane stress signals and protein synthesis.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
subunit of eukaryotic initiation factor-2
(eIF2
),1 which regulates
protein synthesis rate at translational initiation. Phosphorylation of
eIF2
increases the stability of complexes formed between eIF2 and
eIF2B, a guanine-nucleotide exchange factor. eIF2B converts eIF2-GDP to
eIF2-GTP binary complex, which further forms a ternary complex with a
Met-tRNA and becomes associated with the 40 S ribosomal subunit to
initiate translation of mRNA. Because eIF2B exists in cells in
relatively low molar quantities with respect to eIF2, phosphorylation
of only a limited amount (i.e. 20-25%) of eIF2
is
apparently sufficient to sequester virtually all of the eIF2B,
resulting in inhibition of protein synthesis (3-5). The
interferon-inducible double-stranded (ds) RNA-dependent kinase (PKR) is an ubiquitously expressed eIF2
kinase, which was
first identified as a component of the host defense mechanism induced
by interferon (5). In vitro, PKR can be activated by synthetic dsRNA, such as poly(I)·poly(C), and natural dsRNA forms, such as reovirus genomic RNA (5). In interferon-treated cells, virus
infection leads to activation of PKR by autophosphorylation, followed
by eIF2
phosphorylation and inhibition of protein synthesis (5, 6).
Thus, protein synthesis inhibition occurring after virus infection is
thought to be due to the direct activation of PKR by dsRNA species.
However, the mechanism of eIF2
phosphorylation in the absence of
infection is not clear.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-glycerophosphate, 20 mM sodium molybdate, 20 µg/ml
chymostatin, and 1 µM microcystin-LR).
Immunoprecipitation and immunoblotting were performed using anti-PKR
antibody (Ab-1) or anti-HA antibody (12CA5, Roche Molecular
Biochemicals) using protein A-agarose beads, as described previously
(7). For metabolic labeling, cells were incubated with 0.1 mCi/ml
[32P]orthophosphoric acid (ICN) at 1 × 107 cells/ml in phosphate-free RPMI 1640 medium for 2 h. Phosphoamino acid analysis was performed as described (17).
in NFS/N1.H7 cells was analyzed by vertical slab
isoelectric focusing and immunoblotting essentially as described
previously (7, 18). After treatment, cells (3 × 106)
were lysed in 100 µl of buffer A. The acetone-precipitated extract (100 µg) was subjected to the isoelectric focusing and immunoblotting using ECL kit (Amersham Pharmacia Biotech) and a rabbit eIF2
polyclonal antibody raised against a KLH-coupled synthetic peptide corresponding to amino acid residues 298-315 of human eIF2
. For analysis of RAX phosphorylation, Bio-lyte 6/8 (Bio-Rad) and 10 mM ethylenediamine (Sigma) were used for the ampholytes and
the cathode buffer, respectively. Before transfer, the gel was treated with 0.1 M Tris-HCl, pH 6.8, 2% SDS, and 10 mM
dithiothreitol for 30 min at room temperature to enhance the elution efficiency.
-32P]ATP, 0.1 µg
of rabbit reticulocyte eIF2
). Proteins were separated by SDS-PAGE
(10%), and 32P incorporation was quantitated by
auroradiography or directly measured by InstantImagerTM (Packard,
Meriden, CT).
70 °C and analyzed by electrophoresis in a 2% agarose gel
containing 10 µg/ml ethidium bromide.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (49K):
[in a new window]
Fig. 1.
RAX is a ubiquitously expressed protein with
dsRNA-binding motifs. A, amino acid sequence of RAX
aligned with that of human PACT. Only those amino acids that differ in
the two proteins are indicated for PACT. The three dsRNA-binding motifs
(bold letters) are aligned. The sequences have been
deposited in GenBankTM under the accession number AF083032.
B, purified RAX protein (Input) was incubated
with DNA, dsRNA (Poly I·C), or single-stranded RNA
(Poly C), and bound proteins were analyzed by SDS-PAGE as
described under "Materials and Methods." C, Northern
blot analysis of poly(A) RNA harvested from various mouse tissues.
D, protein samples (100 µg) from various cell lines were
subjected to SDS-PAGE and analyzed by immunoblotting using anti-RAX
antiserum. RAX/COS7, COS7 transiently transfected with RAX
cDNA; Pre, probed with pre-immune serum.
Northern blot analysis of poly(A) RNA prepared from mouse tissues showed that RAX mRNA is expressed in all tissues tested (Fig. 1C). Furthermore, an polyclonal antibody raised against recombinant RAX recognized a 35-kDa protein in the lysates from cell lines derived from different species including mouse NIH3T3, IL-3-dependent NFS/N1.H7, rat pheochromocytoma PC-12, and monkey COS7 cells (Fig. 1D). The migration at the 35-kDa position on SDS-PAGE gel corresponds well with a calculated molecular mass of RAX (34 kDa). Importantly, an increased signal was observed in COS7 cells after transfection of RAX cDNA, indicating that the 35-kDa immunoreactive protein is RAX. The 35-kDa band was detected in human HeLa and MCF-7 cell lysates, indicating that the antibody can react with human PACT (Fig. 1D). These results indicate that RAX/PACT is an ubiquitously expressed and well conserved gene between the species.
To investigate the biological function of RAX, we first transfected HA
epitope-tagged RAX into IL-3-dependent NFS/N1.H7 cells (14)
and obtained more than 10 clones that stably express different levels
of HA-RAX (Fig. 2A). There was
no significant correlation between the expression levels of HA-RAX and
the cell growth rate, suggesting that overexpression of RAX is not
growth suppressive in these cells in the presence of IL-3 (Fig.
2B). Interestingly, however, we found that HA-RAX
overexpression accelerates cell apoptosis following withdrawal of IL-3
(Fig. 2, C and D). This apoptosis promoting
effect appears to be dependent on the expression levels of the
exogenous HA-RAX protein because high expressing clones form fragmented
internucleosomal DNA faster than the low expressing clones or vector
controls. These results suggest that RAX has a pro-apoptotic function
that may be suppressed in the presence of growth factor.
|
Results from the yeast two-hybrid assay suggest that RAX can bind to
PKR. To examine the effects of RAX on PKR activity, an in
vitro reconstitution kinase assay was developed using proteins isolated from mammalian cells. HA-RAX was immunopurified from exponentially growing cells (clone 13; Fig. 2). The purified HA-RAX contained no associated kinase activity, indicating that any
potentially associated PKR was removed during purification (Fig.
3A, first lane).
PKR was purified separately from exponentially growing parental
NFS/N1.H7 cells. We found that HA-RAX could potently activate PKR as
indicated by eIF2 phosphorylation and PKR autophosphorylation in the
absence of dsRNA (Fig. 3A). Furthermore, if suboptimum amounts of dsRNA are added to the reaction, RAX synergistically activates PKR (Fig. 3B). Thus, we find HA-RAX purified from
growing cells can activate PKR in vitro. However,
overexpression of HA-RAX apparently does not affect the growth of
NFS/N1.H7 cells in the presence of IL-3, suggesting that the
overexpressed RAX in vivo may not be activating cellular PKR
efficiently under the growing condition. In support of this notion,
co-immunoprecipitation studies using anti-PKR and anti-HA reveal that
the RAX-PKR association is barely detected in exponentially growing
cells even when highly expressed as in the case with clone 13 (i.e. expresses approximately 5-10-fold more than the
endogenous level) (Fig. 4A).
However, an increase in the association occurs when the cells are
deprived of IL-3 for 2.5 h, a time by which protein synthesis is
significantly inhibited by PKR activation (7). Moreover, more dramatic
and rapid RAX-PKR association is observed when the cells are exposed to
various chemical stresses including sodium arsenite (As),
H2O2, or thapsigargin (TG), an inhibitor of
endoplasmic reticular Ca2+-ATPase (Fig. 4A),
agents known to potently inhibit protein synthesis (1, 10).
Importantly, the total amount of HA-RAX in the cell lysate is not
affected by any of these stress treatments, confirming that any
increased association is not due to increased RAX expression (Fig.
4A, lower panel). Furthermore, as previously
reported (2, 7, 26), the stress treatments were also found to induce
phosphorylation of eIF2
, clearly indicating that PKR, its
physiologic kinase, has been activated (Fig. 4B).
Significantly, any eIF2
phosphorylation cannot be detected in
growing transfected cells, indicating that the simple overexpression of
exogenous RAX is not sufficient to activate PKR and inhibit growth.
|
|
These results suggest that RAX can directly bind to and activate PKR
following application of stress conditions. The rapid RAX-PKR
association observed after the stress treatments raised the possibility
that RAX may be post-translationally modified as a result of any stress
signal evoked by the treatments. Isoelectricfocusing analysis revealed
that exponentially growing NFS/N1.H7 cells contain only a single
species of RAX with an approximate pI of 8.6 (Fig. 5A). Interestingly, IL-3
deprivation or treatment of cells with As or TG induced the prominent
acidification of RAX to pI 8.3 (Fig. 5A). A similar RAX
mobility shift was also observed when HA-RAX expressing cells were
either treated with As, TG, or H2O2 or deprived
of IL-3 (Fig. 5B). HA-RAX was more acidic (i.e.
pI 7.8) than RAX due to the acidic nature of the HA peptide sequence and was shifted to pI 7.5 following stress treatments. When HA-RAX was
immunoprecipitated from the cell lysate and treated with alkaline phosphatase before loading onto the gel, the TG-induced, shifted band
was no longer observed whereas migration of RAX purified from growing
cells did not change after alkaline phosphatase treatment (Fig.
5C). These findings indicate that growing, unstimulated cells contain only the nonphosphorylated RAX, whereas the stress treatments induced phosphorylation of RAX. Phosphoamino acid analysis revealed that RAX is exclusively phosphorylated on serine residue(s) following As treatment or IL-3 deprivation (Fig. 5D).
|
Both RAX and eIF2 phosphorylation were detected within 3 min after
treatment with TG and As. Significantly, however, phosphorylation of
RAX begins and is maximal before that of eIF2
, indicating that RAX
phosphorylation precedes PKR activation (Fig.
6A). Because PKR was unable to
directly phosphorylate HA-RAX in vitro (Fig. 3), RAX does
not appear to be a substrate for PKR. To test whether cellular RAX
could be phosphorylated by PKR after its association, we examined
whether 2-aminopurine, a selective inhibitor for PKR (27, 28), could
affect RAX phosphorylation. Results revealed that the eIF2
phosphorylation caused by IL-3 deprivation was potently inhibited by
2-aminopurine but had little or no effect on RAX phosphorylation (Fig.
6B). These results therefore suggest that RAX is
phosphorylated by stress-activated, 2-aminopurine-resistant serine
protein kinase(s) before it activates PKR.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The paradigm for PKR activation following viral infection
indicates that dsRNA activates this important regulator of protein synthesis, but the mechanism of activation in the absence of viral infection is not clear. Several studies have shown that various stress
stimuli can induce eIF2 phosphorylation (2, 26), indicating that
activation of PKR may occur as a result of stress signaling, but
whether dsRNA is involved is not known. Our results indicate that RAX
may be a direct, stress-sensitive activator of PKR. The high sequence
similarity between mouse RAX and human PACT suggests that RAX is a
mouse homologue of PACT, which was recently identified using a similar
interactive cloning strategy (11). Furthermore, the in vitro
studies with purified RAX indicate that RAX, like PACT, can directly
activate PKR, confirming that PKR can be activated by cellular proteins
in the absence of dsRNA.
The current model of PKR activation holds that two molecules of PKR bind to a single dsRNA molecule, thereby leading to homodimerization, which results in autophosphorylation via transphosphorylation (5). RAX-mediated autophosphorylation and activation of PKR indicates that RAX may also bind two PKR molecules to cause homodimerization of PKR. We also found that RAX can synergistically activate PKR in the presence of dsRNA (Fig. 3), indicating an unique mechanism. Although further studies are required, the potential for a functionally synergistic interaction between RAX and dsRNA with respect to PKR activation in vivo is considered high because PKR is associated with ribosomes where dsRNA species may be abundant (5, 29).
PKR activation leads to protein synthesis inhibition and decreased cell growth (5, 21, 30). Because RAX can efficiently activate PKR, RAX-PKR association during cell growth must be tightly regulated to prevent inadvertent activation of PKR. Co-immunoprecipitation studies suggest that the RAX-PKR interaction can be enhanced by stress signals. The mechanism, however, remains unknown. We found a significant correlation between PKR activity and the phosphorylation state of RAX after various stress treatments. Because RAX phosphorylation clearly precedes PKR activation, these data support the idea that the post-translational modification of RAX may be involved in the RAX-PKR association and activation of PKR in vivo. Phosphoamino acid analysis shows that only serine is phosphorylated, indicating that RAX is the substrate for a stress-activated serine/threonine kinase. Arsenite, thapsigargin, hydrogen peroxide, and IL-3 withdrawal all have been reported to activate the mitogen-activated protein kinase subfamily that includes c-Jun N-terminal kinase/stress-activated protein kinase and p38 kinase (31, 32). Whether RAX is a substrate for these kinases will require further studies.
Interestingly, RAX in growing cells is not heavily phosphorylated (Fig. 5), but even unphosphorylated RAX can apparently activate PKR in vitro (Fig. 3). Therefore, these results suggest that RAX phosphorylation per se is not required for PKR activation but may potentially regulate the RAX-PKR association and affect PKR activation, possibly by altering the affinity or accessibility of RAX to PKR. However, the precise regulatory role for RAX phosphorylation and the mechanism by which RAX can activate PKR remains to be elucidated. Because PKR is a ribosomal protein (29, 33), one possibility is that phosphorylation may facilitate ribosomal localization of RAX and thereby association with PKR. Another is that phosphorylated RAX may have a higher affinity for PKR binding than certain PKR inhibitory molecules such as p58 (34) and TAR-RNA-binding protein (35). Identification of the phosphorylation site(s) and subsequent site-directed mutagenesis analysis of RAX are required to test these possibilities.
Patel and Sen (11) have reported that transient transfection of the
PACT cDNA increased the level of eIF2 phosphorylation (about
2-fold) in HT-1080 fibrosarcoma cells, and the establishment of cell
lines stably overexpressing PACT was unsuccessful. On this basis it was
concluded that simple overexpression of PACT activates PKR and
suppresses cell growth. By contrast, RAX can be overexpressed (more
than 10-fold) in IL-3-dependent NFS/N1.H7 cells with no
effect on PKR activity or cell growth, at least in the presence of
IL-3. Although the reason for the discrepancy between PACT and RAX with
respect to cell growth is not yet clear, it could be due to the
different cell types tested. RAX, in the absence of IL-3, accelerates
apoptosis in direct proportion to the expression levels (Fig.
2B). Therefore, it seems likely that growth factors such as
IL-3 may, at least in part, potently suppress stress signals
(e.g. stress kinase activity) by inhibiting the association
between RAX and PKR. It will therefore be important to determine
whether PACT can be overexpressed in NFS/N1.H7 cells and not result in
cell death in the presence of IL-3.
In addition to PKR, another stress-sensitive eIF2 kinase, termed
PERK (36) or PEK (37), has been recently cloned. PERK is associated
with endoplasmic reticulum and can be activated by endoplasmic
reticulum stresses such as thapsigargin. However, PERK is not activated
by cytoplasmic stresses including arsenite, ultraviolet irradiation,
and heat shock (36). In fact, our recent data indicate that neither
arsenite nor IL-3 deprivation activates PERK in NFS/N1.H7
cells.2 Thus, some stresses
may activate only PKR, whereas some may activate both eIF2
kinases
through different mechanisms.
In summary, we propose that RAX is part of a novel stress-activated
signaling pathway that features inhibition of protein synthesis that
occurs as a result of the direct interaction between RAX and PKR.
Because PKR activation has been implicated in apoptosis and the
stresses applied here can all induce cell apoptosis, activated RAX
appears to be a potent negative regulator of cell growth and survival.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank R. Jagus for providing purified
eIF2, J. A. Langer for pcDEF vector, and D. A. Vasquez and
S. P. Warnken for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grants HL54083 and CA44649.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF083032.
To whom correspondence should be addressed. Tel.: 409-747-1935;
Fax: 409-747-1938; E-mail: tito{at}utmb.edu.
2 T. Ito, D. Ron, and W. S. May, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
eIF2,
subunit of eukaryotic initiation factor-2;
As, sodium arsenite;
IL-3, interleukin-3;
PKR, double-stranded RNA-dependent protein
kinase;
TG, thapsigargin;
ds, double-stranded;
PAGE, polyacrylamide gel
electrophoresis;
HA, hemagglutinin.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|