From the
Calcium depletion from the endoplasmic reticulum inhibits
protein synthesis and correlates with increased phosphorylation of the
Utilization of eukaryotic initiation factor-2 (eIF-2)
In mammalian cells, two related
serine/threonine protein kinases that phosphorylate eIF-2
The endoplasmic reticulum (ER) is the site where initial
folding and processing of newly synthesized secretory proteins occurs
and is also the major site of calcium storage in the
cell(24, 25) . In response to hormonally generated
inositol triphosphate, calcium is released to the cytoplasm as part of
the signal transduction cascade(24) . Sequestered calcium can be
released experimentally by treating cells with calcium ionophores (26) or inhibitors of the microsomal calcium-dependent ATPase
such as thapsigargin(27) . Depletion of calcium from the
endoplasmic reticulum correlates with inhibition of protein synthesis
and increased eIF-2
Exponentially growing
cells were treated with calcium ionophore A23187 (Sigma) in
Dulbecco's modified essential medium containing 10%
heat-inactivated fetal bovine serum for 15 min at 37 °C. Cells were
rinsed with methionine-free and cysteine-free medium and then pulse
labeled in methionine-free and cysteine-free Dulbecco's modified
essential medium containing 50 µCi/ml
[
The
phosphorylation status of eIF-2
NIH3T3 cells were derived that overexpress either eIF-2
The synthesis of
eIF-2
The effect of calcium depletion on
phosphorylation of eIF-2
The data presented here demonstrate that PKR mediates
inhibition of protein synthesis in response to calcium depletion by
phosphorylation of eIF-2
Overexpression of eIF-2
PKR is activated
by binding to dsRNA(14, 46, 47) . The shortest
RNA-RNA duplexes capable of binding PKR are approximately 30 base
pairs; however, the binding efficiency increases with increasing length
up to 85 base pairs(14) . In addition, some single-stranded RNAs
by virtue of their stem-loop structures can bind and either promote or
prevent activation of PKR(19) . Following stable dsRNA-PKR
complex formation, a conformational change likely occurs with
subsequent autophosphorylation and activation(19) . PKR can also
be activated by polyanion molecules (48), although the mechanism is
poorly understood. A transient expression system was used to
characterize the inducer of PKR-mediated eIF-2
Recent results suggest that PKR may phosphorylate I
subunit of eukaryotic initiation factor 2 (eIF-2
) by a
mechanism that does not require ongoing protein synthesis. To elucidate
whether protein synthesis inhibition requires eIF-2
phosphorylation and whether eIF-2
phosphorylation is mediated by
the double-stranded RNA-dependent protein kinase (PKR), we studied
protein synthesis in response to calcium depletion mediated by calcium
ionophore A23187 in cell lines overexpressing wild-type eIF-2
, a
mutant eIF-2
(S51A) that is resistant to phosphorylation, or a
dominant negative mutant PKR (K296P in catalytic subdomain II).
Expression of either mutant eIF-2
or mutant PKR partially
protected NIH3T3 cells from inhibition of protein synthesis upon A23187
treatment. In contrast, overexpression of wild-type PKR increased
sensitivity to protein synthesis inhibition mediated by A23187
treatment. In a COS-1 monkey cell transient transfection system,
increased eIF-2
phosphorylation in response to A23187 treatment
was inhibited by expression of the dominant negative PKR mutant.
Overexpression of the PKR regulatory RNA binding domain, independent of
the PKR catalytic domain, was sufficient to inhibit increased
phosphorylation of eIF-2
upon A23187 treatment. In addition,
overexpression of the HIV TAR RNA binding protein also inhibited
eIF-2
phosphorylation upon A23187 treatment. Taken together, our
data show that calcium depletion activates PKR to phosphorylate
eIF-2
, and this activation is likely mediated through the PKR RNA
binding domain.
(
)is an important control point in the regulation of
protein synthesis initiation(1, 2) . eIF-2 is a
heterotrimer composed of
,
, and
subunits and forms a
ternary complex with initiator methionine tRNA
and GTP.
This ternary complex binds to the 40 S ribosomal subunit and to mRNA to
form a 48 S species. Upon 60 S ribosomal subunit joining to the 48 S
species, eIF-2-bound GTP is hydrolyzed to GDP. For eIF-2 to promote
another round of initiation, GTP must exchange for GDP, a reaction
catalyzed by the guanine nucleotide exchange factor or
eIF-2B(3, 4) . The phosphorylation of serine residue 51
in eIF-2
increases the affinity of eIF-2 for GDP and thereby
prevents GTP exchange and inhibits further initiation
events(5) . In mammalian cells, protein synthesis is immediately
controlled through environmental changes. For example, protein
synthesis is rapidly inhibited in response to heat
shock(6, 7) , amino acid deprivation, glucose
deprivation, serum starvation(8) , or viral
infection(9, 10) . Under these conditions,
phosphorylation of eIF-2
correlates with inhibition of protein
synthesis, although the specific kinases and/or phosphatases that
regulate eIF-2
phosphorylation are poorly
characterized(11) .
are the
hemin-regulated inhibitor and the double-stranded RNA-activated
inhibitor (PKR)(12) . Hemin-regulated protein kinase is
primarily expressed in erythroid cells and is activated by reduced
levels of hemin. PKR is found in most cells, its synthesis is induced
by interferon, and its activity is dependent upon dsRNA. PKR has a
conserved serine/threonine protein kinase catalytic domain in its
carboxyl terminus and two RNA binding motifs in its amino
terminus(12, 13, 14) . After activation, PKR
displays two known distinct substrate specificities:
autophosphorylation (15, 16) and phosphorylation of
eIF-2
(2) . Recent studies also suggest that PKR activates
the nuclear transcription factor NF
B through phosphorylation of
its inhibitor I
B(17) . PKR-mediated phosphorylation of
eIF-2
and subsequent inhibition of protein synthesis is implicated
in the antiviral and antiproliferative effect of
interferons(18, 19) . Interferon-resistant viruses
encode specific gene products that circumvent the translation
inhibition imposed by PKR activation(9, 10) . In
addition to viral gene products, cellular proteins such as the TAR RNA
binding protein (TRBP) (20) and p58 (21) have been
implicated in regulating PKR activation and may regulate eIF-2
phosphorylation to affect global protein synthesis and cell growth in
response to a variety of different stimuli. Expression of a dominant
negative mutant of PKR transforms NIH3T3 cells (22, 23) and suggests that regulation of eIF-2
phosphorylation may represent a significant mechanism for growth
control.
phosphorylation (28, 29), although the
serine/threonine kinase(s) involved is unknown. We have studied the
effect of overexpression of wild-type and mutants of both eIF-2
and PKR on eIF-2
phosphorylation and translation upon calcium
depletion. Our data suggest that PKR mediates eIF-2
phosphorylation and inhibits protein synthesis.
Vector Construction
The eIF-2
wild-type and S51A mutant (30) cDNAs encoded on 1.6-kilobase EcoRI fragments were subcloned into the unique EcoRI
site of the pEDmtx
VA
expression vector, a
derivative of pED-mtx
(31) lacking the adenovirus
VAI RNA gene. An XbaI-SalI fragment from pBS-8.4
(encoding wild-type PKR cDNA (13) and kindly provided by Dr. B.
Williams (Cleveland Clinic, OH)) was subcloned into the expression
vector pMT2 (32). Subsequently, a 2-kilobase PstI fragment
(5`-PstI from the pMT2 polylinker and 3`-PstI in the
3`-untranslated region of PKR) was subcloned into the PstI
cloning site of pED-mtx
VA
. Site-directed
mutagenesis (32) introduced a K296P (codon AAA to CCA) mutation
into the wild-type PKR expression vector. The EcoRI-PstI fragment encoding PKR mutant K296P was
subcloned into the expression vector pETF VA
to yield
pK296P ETF(33) . T7 epitope-tagged versions of wild-type PKR,
K296P, and a truncated PKR comprising amino acid residues 1-243
encoding the RNA binding domain were constructed by polymerase chain
reaction amplification using specific primers to introduce a T7 epitope
tag at the carboxyl-terminal end of PKR or after residue 243. The
5`-primer was 5`-AACTGCAGCCACCATGGCTGGTGATCTTTCA-3`. The 3`-primer for
full-length PKR was
5`-ACGCGTCGACCTAACCCATTTGCTGTCCACC-AGTCATGCTAGCCATACATGTGTGTCGTTCATT-3`.
The 3`-primer for truncated PKR was
5`-ACGCGTCGACCTAACCCATTTGCTGTCCACCAGTCATGCTAGCCATTGCCAAAGATCTTTTTGC-3`.
Amplified fragments were subcloned into the TA cloning vector
(Invitrogen). PstI/SalI fragments were isolated from
the TA cloning vector and subcloned into pETF VA
(33). DNA sequence analysis confirmed the correct sequence at the
5`- and 3`-ends of the polymerase chain reaction amplified and cloned
fragments.
Derivation and Characterization of Cell
Lines
eIF-2 wild-type, mutant S51A, or PKR K296P
contained within the pEDmtx
VA
vector were
cotransfected with pSV
Neo (ratio of 10:1) into NIH3T3 cells
(provided by Dr. S. Aaronson), and transformants were selected for
growth in G418 (1 mg/ml, Life Technologies, Inc.). NCTC clone 929 cells
(ATCC CCL 1) were cotransfected with pED-mtx
VA
PKR and pSV
Neo (ratio of 10:1), selected for growth
in G418 (1.0 mg/ml), and subsequently selected for gene amplification
by growth in sequentially increasing concentrations of methotrexate up
to 0.3 µM. All cell lines used in this study were cloned
from selected pools by limiting dilution.
S]methionine and
[
S]cysteine (DuPont NEN) for 15 min in the
presence of drug. Cells were rinsed in cold phosphate-buffered saline
and harvested in NP4O lysis buffer as described(34) . Protein
concentrations were determined(35) , and equal amounts of
protein were analyzed by reducing SDS-polyacrylamide gel
electrophoresis (PAGE). Autoradiography was performed after treatment
with EN/HANCE (DuPont NEN). Expression of PKR and eIF-2
were
measured by Western blot analysis using polyclonal anti-PKR antibody or
polyclonal anti-eIF-2
antibody (kindly provided by Drs.
Hovanessian and Hershey).
COS-1 Cell DNA Transfection and
Analysis
COS-1 cells were transfected by the DEAE-dextran
procedure(36) . At 60 h post-transfection, cells were treated
with calcium ionophore A23187 for 15 min at 37 °C and then labeled
with [S]methionine and
[
S]cysteine in methionine and cysteine-free
minimal essential media (Life Technologies, Inc.) for an additional 15
min. Cell extracts were harvested in Nonidet P-40 lysis buffer in the
presence of the protease inhibitors soybean trypsin inhibitor (1.0
mg/ml), aprotinin (1.0 mg/ml), and phenylmethylsulfonyl fluoride (1.0
mM). eIF-2
was immunoprecipitated from cell extracts
using monoclonal anti-eIF-2
antibody (kindly provided by the late
Dr. Henshaw) and protein A-Sepharose as the immunoadsorbent. Steady
state levels of eIF-2
were measured by SDS-PAGE and Western
immunoblotting procedures using anti-eIF-2
polyclonal
antibody(37) . T7 epitope-tagged proteins were detected by
immunoprecipitation from cell extracts using anti-T7 antibody (Novagen,
Madison, WI) and protein A-Sepharose as the immunoadsorbent.
expressed in COS-1 cells at 60 h
post-transfection or in NIH3T3 cells was monitored by labeling cells
with 2 ml of [
P]phosphate (400 µCi of
orthophosphoric acid, DuPont NEN) for 4 h. Thapsigargin (300
nM, Sigma) or A23187 were added during the last 15 min of the
labeling. Cycloheximide (10 µg/ml, Sigma) was added 15 min prior to
the drug treatments and was maintained through the course of the
labeling. Cell extracts were prepared and immunoprecipitated with
monoclonal anti-eIF-2
antibody using protein A-Sepharose as the
immunoadsorbent. Samples were analyzed by SDS-PAGE and autoradiography.
All band intensities were quantified using NIH Image 1.55b program.
wt or a mutant eIF-2
S51A in which the site of PKR-mediated
phosphorylation was mutated to alanine. Western blot analysis
demonstrated that the steady state level of eIF-2
was
significantly elevated in both wild-type (6.4-fold, Fig. 1A, lane2) and mutant eIF-2
S51A (8.8-fold, Fig. 1A, lane3)
transfected cell lines compared to a cell line transfected with the
original vector alone (Fig. 1A, lane1). The overexpression of eIF-2
wild-type or S51A
mutant did not significantly alter the cell growth rate or saturation
density of the cells (data not shown). NIH3T3 cells were derived that
overexpress a PKR mutant K296P in catalytic subdomain II. Western blot
analysis using an anti-PKR polyclonal antibody detected expression of
PKR K296P migrating at 72 kDa under conditions where the endogenous PKR
was not detected in nontransfected cells (Fig. 1A, lanes4 and 5). Overexpression of PKR K296P
increased the growth rate and increased saturation density similar to
previous observations(22, 23) .
Figure 1:
Expression of eIF-2 S51A or PKR
K296P protects cells from A23187-mediated inhibition of protein
synthesis. PanelA, cell extracts were prepared for
Western immunoblot analysis using anti-eIF-2
polyclonal antibody (lanes1-3) or anti-PKR polyclonal antibody (lanes4 and 5) as described under
``Experimental Procedures.'' Clones shown were obtained from
transfection with pED-mtx
VA
vector alone (lanes1 and 4), eIF-2
wild-type vector
(wt 5, lane2), eIF-2
S51A (51(2), lane3), and mutant PKR K296P (KP3A, lane5). PanelB, cells were treated with
increasing concentrations of A23187, and
[
S]methionine and
[
S]cysteine incorporation into trichloroacetic
acid-precipitable protein was measured as described under
``Experimental Procedures.'' The cpm/µg protein are
represented as the percent of control obtained from a parallel dish
without A23187 treatment. PanelC, SDS-PAGE analysis
of extracts from cells pulse labeled with (+) or without (-)
A23187 treatment was as described under ``Experimental
Procedures.'' The figure shows an autoradiogram of the dried gel.
Cell lines analyzed are indicated at the top. Molecular weight
markers are indicated at the right. PanelD,
cell extracts were immunoprecipitated with anti-eIF-2
monoclonal
antibody and analyzed by SDS-PAGE.
The effect of
wild-type eIF-2, eIF-2
mutant S51A, or PKR mutant K296P
overexpression on translation inhibition in response to calcium
depletion was monitored by treating exponentially growing NIH3T3 cells
with the calcium ionophore A23187 for 15 min and metabolic pulse
labeling. Compared to untreated cells, A23187 treatment reduced protein
synthesis to 13% in the original NIH3T3 cells and to 14% in cells that
overexpress eIF-2
wt. In contrast, A23187 treatment reduced
protein synthesis to 31% in cells that overexpress mutant eIF-2
S51A and to 29% in cells that overexpress PKR mutant K296P. Cells
overexpressing either mutant eIF-2
S51A or PKR mutant K296P were
more resistant to inhibition of protein synthesis over a range of
A23187 concentrations compared to control cells that overexpress
eIF-2
wt (Fig. 1B). Overexpression of wild-type
eIF-2
, mutant eIF-2
S51A, or mutant PKR K296P did not
detectably alter the specificity of polypeptides synthesized compared
to control cells (Fig. 1C, lanes1, 3, 5, and 7). In addition, A23187 treatment
did not significantly alter the spectrum of polypeptide synthesized in
these cell lines. These results suggest that expression of either
mutant eIF-2
S51A or mutant PKR K296P protected the cells from
inhibition of global protein synthesis upon A23187 treatment and did
not protect translation of selective mRNAs.
in the control and transfected cells was measured by
immunoprecipitation of equal amounts of protein from the cell extracts
with anti-eIF-2
antibody and analysis by SDS-PAGE. The synthesis
of eIF-2
was increased in cells transfected with eIF-2
wt or
eIF-2
S51A (Fig. 1D, lanes3 and 5) compared to control NIH3T3 or cells that overexpress PKR
K296P (Fig. 1D, lanes1 and 7). eIF-2
synthesis was reduced approximately 5-fold upon
A23187 treatment in control NIH3T3 cells (Fig. 1D, lanes1 and 2) and in cells that overexpress
eIF-2
wt (Fig. 1D, lanes3 and 4). In contrast, eIF-2
synthesis was not significantly
reduced upon A23187 treatment in the cells that overexpress mutant
eIF-2
S51A (Fig. 1D, lanes5 and 6) or mutant PKR K296P (Fig. 1D, lanes7 and 8). The effects of A23187 treatment on
eIF-2
synthesis paralleled the effects on global protein synthesis
in the different cell lines.
was measured in NIH3T3 cells that
overexpress eIF-2
wt. Immunoprecipitation of eIF-2
from
[
P]phosphate-labeled NIH3T3 cells that
overexpress eIF-2
wt detected [
P]phosphate
incorporation into eIF-2
(Fig. 2, lane1).
Treatment with either A23187 or thapsigargin increased the
[
P]phosphate incorporation by 3-fold (Fig. 2, lanes3 and 5). Whereas
treatment with the translation elongation inhibitor cycloheximide (10
µg/ml, a concentration that inhibited protein synthesis greater
than 90%) slightly reduced [
P]phosphate
incorporation in NIH3T3 cells that overexpress eIF-2
wt, it did
not significantly inhibit the increase in phosphorylation observed upon
treatment with A23187 or thapsigargin (Fig. 2, lanes4 and 6). These results show that calcium
depletion mediated by either A23187 or thapsigargin increases
eIF-2
phosphorylation by a mechanism that does not require ongoing
protein synthesis.
Figure 2:
Increased eIF-2 phosphorylation by
calcium depletion does not require ongoing protein synthesis. NIH3T3
cells that overexpress wild-type eIF-2
were labeled with
[
P]phosphate for 4 h and then treated with
A23187 or thapsigargin in the presence (+) and absence (-)
of cycloheximide (CHX) as described under ``Experimental
Procedures.'' Cell extracts were prepared and immunoprecipitated
with anti-eIF-2
monoclonal antibody and analyzed by
SDS-PAGE.
To further characterize the role of PKR in
response to calcium depletion, we studied the effect of wild-type PKR
overexpression on protein synthesis upon A23187 treatment. To date, we
have not been able to obtain wild-type PKR overexpression in stably
transfected NIH3T3 cells, possibly due to its negative effect on cell
growth(38) . However, we were able to establish mouse fibroblast
L929 cell lines that overexpress wild-type PKR. Western blot analysis
of two independent cell lines transfected with wild-type PKR detected
PKR protein under conditions where the endogenous PKR was not detected
in nontransfected cells (Fig. 3A, data not shown).
Treatment with 1 µM A23187 reduced protein synthesis to
40% in control L929 cells and to 17% in cells that overexpress
wild-type PKR (Fig. 3B, lanes4 and 7). Treatment with 5 µM A23187 completely
inhibited protein synthesis in cells that overexpress wild-type PKR,
whereas residual protein synthesis was detected in control L929 cells (Fig. 3B, lanes5 and 8).
Similar results were derived with an independently derived clone of
L929 cells that overexpresses wild-type PKR (data not shown). These
results demonstrate that overexpression of wild-type PKR sensitizes the
cell to inhibition of protein synthesis mediated by calcium depletion.
Figure 3:
Overexpression of wild-type PKR sensitizes
L929 cells to A23187. PanelA, cell lysates from the
original L292 cells and from clone 2 were prepared and analyzed by
Western blot analysis using anti-PKR polyclonal antibody as described
under ``Experimental Procedures.'' PanelB,
cells were pulse labeled with [S]methionine and
[
S]cysteine in the presence (1, 5) or absence (-) of A23187 treatment (1 or 5 µM as indicated), and cell extracts were prepared for SDS-PAGE as
described under ``Experimental
Procedures.''
The role of RNA binding in the ability for PKR mutant K296P to
inhibit eIF2 phosphorylation in response to A23187 was studied by
transient overexpression of either the PKR RNA binding domain (amino
acid residues 1-243) expressed independently or of another RNA
binding protein, the HIV TRBP in COS-1 cells. The PKR mutant K296P and
the amino-terminal 1-243 amino acids of PKR were engineered with
a bacteriophage T7 epitope tag in the carboxyl terminus. The ability of
these engineered molecules to inhibit activation of endogenous PKR was
tested by a cotransfection assay to measure the rescue of translation
of a reporter mRNA encoding adenosine deaminase that is not translated
due to PKR activation(33) . Each construct was cotransfected
with the adenosine deaminase expression vector p9A, and total protein
synthesis was measured by SDS-PAGE analysis of cell extracts. Upon
cotransfection with the T7-tagged wild-type PKR expression vector,
adenosine deaminase translation was not detected (Fig. 4A, lane2), whereas
cotransfection with either the T7-tagged RNA binding
domain(1-243) or the T7-tagged PKR mutant K296P increased
adenosine deaminase translation (Fig. 4A, lanes3 and 4). In addition, a 35-kDa polypeptide and
a 72-kDa polypeptide, the expected size for the T7-tagged proteins,
were detected upon analysis of the total cell extracts (Fig. 4A, lanes3 and 4) and
were specifically immunoprecipitated with anti-T7 antibody (Fig. 4A, lanes6 and 7).
These results show that addition of the T7 tag to the carboxyl terminus
of either PKR K296P or the RNA binding domain did not interfere with
the ability of these proteins to inhibit endogenous PKR and rescue
adenosine deaminase translation. Previous studies demonstrated
inhibition of endogenous PKR activity by expression of TRBP in COS-1
cells(20) .
Figure 4:
PKR mutant K296P, truncated PKR
1-243, and TRBP inhibit A23187-induced eIF-2
phosphorylation. Human cDNAs encoding PKR wild-type, mutant K296P, or
truncated 1-243 were engineered with a T7 epitope tag in the pETF
VA
vector. PanelA, The T7-tagged
constructs were cotransfected into COS-1 cells with the adenosine
deaminase expression vector p9A (33). At 60 h post-transfection, cells
were pulse labeled with [
S]methionine and
[
S]cysteine, and cell extracts were prepared for
analysis by SDS-PAGE before (lanes1-4) and
after (lanes5-7) immunoprecipitation with
anti-T7 monoclonal antibody. PanelsB and C,
COS-1 cells were cotransfected with the wild-type eIF-2
cDNA in
the expression plasmid pED-mtx
VA
and the
indicated T7-tagged expression vectors and analyzed for eIF-2
phosphorylation as described under ``Experimental
Procedures.''
The effect of PKR mutant K296P, truncated
1-243 PKR, and TRBP on phosphorylation of eIF-2 in response
to A23187 treatment was studied by cotransfection of these expression
vectors with an eIF-2
expression vector. Since the subpopulation
of transfected cells express both plasmid DNA molecules, the effect of
protein expression from one plasmid on the phosphorylation status of
eIF-2
overexpressed from a second plasmid (pED-2
wt) can be
monitored. At 60 h post-transfection, the effect of A23187 treatment on
the phosphorylation status of the overexpressed eIF-2
was
determined by measuring the amount of
[
P]phosphate incorporation into eIF-2
and
comparing it to the steady state eIF-2
level measured by Western
blot analysis. Western blot analysis showed that cells cotransfected
with pED-2
wt had increased levels of eIF-2
(Fig. 4, B and C (bottom), lanes10-16 and 19-22) compared to cells
that did not receive DNA (Fig. 4, B and C (bottom), lanes8, 9, 17, and 18). In the presence of wild-type PKR
cotransfection, the level of eIF-2
was slightly reduced (Fig. 4B (bottom), lane16),
consistent with inhibition of protein synthesis mediated by wild-type
PKR. In addition, A23187 treatment did not alter the steady state level
of eIF-2
(Fig. 4, B and C (bottom), lanes11, 13, 15, 20, 22). Analysis of immunoprecipitated
eIF-2
by SDS-PAGE and autoradiography showed significant
[
P]phosphate incorporation into eIF-2
in
cells transfected with pED-2
wt alone (Fig. 4B, lane10). A23187 treatment increased phosphate
incorporation into eIF-2
by 5-fold (Fig. 4B, lane11). In the presence of wild-type PKR
cotransfection, eIF-2
was significantly phosphorylated (Fig. 4B, lane16) to a degree similar
to that observed by treatment with A23187 (Fig. 4B, lane11). In cells cotransfected with PKR mutant
K296P (Fig. 4B, lane12) or the RNA
binding domain alone (Fig. 4B, lane14), phosphorylation of eIF-2
was reduced and, most
significantly, was not increased upon A23187 treatment (Fig. 4B, lanes13 and 15).
Similarly, cotransfection with the TRBP expression vector with
pED-2
wt showed that TRBP expression inhibited eIF-2
phosphorylation in the absence or presence of A23187 treatment (Fig. 4C, lanes21 and 22)
compared to the absence of TRBP (Fig. 4C, lanes19 and 20). Thus, expression of either the PKR
RNA binding domain or TRBP, another dsRNA binding protein, can inhibit
eIF-2
phosphorylation mediated by calcium depletion.
. The endoplasmic reticulum is the
principal cellular storage site for calcium and is also the site where
initial folding of newly synthesized secretory proteins occurs.
Intralumenal calcium is required for the folding and secretion of
selective proteins (39). Depletion of intralumenal calcium immediately
disrupts normal protein folding, inhibits further translation
initiation events, and increases phosphorylation of
eIF-2
(40) . Agents that disrupt protein folding in the ER
also induce eIF-2
phosphorylation and inhibit protein
synthesis(41) . The prolonged presence of unfolded proteins in
the ER activates transcription of the glucose-regulated genes through a
mechanism that requires ongoing protein
synthesis(42, 43, 44) . In contrast, the
immediate increase in eIF-2
phosphorylation in response to calcium
depletion studied here did not require ongoing protein synthesis. Thus,
it appears these two events are mediated through different signaling
pathways. It is presently unknown whether the proximal signal to induce
eIF-2
phosphorylation is calcium depletion or the presence of
misfolded proteins in the ER.
mutant
S51A, the site of phosphorylation mediated by eIF-2
kinases PKR
and hemin-regulated protein kinase (12), partially protected the cells
from protein synthesis inhibition mediated by calcium ionophore A23187
treatment. These findings suggest that calcium depletion can inhibit
protein synthesis through an eIF-2
kinase that phosphorylates
serine residue 51. Expression of either the PKR dominant negative
mutant K296P or eIF-2
mutant S51A protected cells to the same
degree from inhibition of protein synthesis mediated by calcium
depletion. In addition, overexpression of wild-type PKR sensitized
cells to ionophore A23187 treatment, further implicating PKR in
mediating the response. This latter finding is consistent with
observations that interferon treatment, known to induce PKR expression,
sensitizes cells to the antiviral effects of A23187
treatment(45) . It is unlikely that altered calcium storage
resulting from transformation mediated by PKR mutant K296P expression (22, 23) elicits protection from the A23187 insult
because similar protection was observed by transient expression of the
PKR K296P in COS-1 cells, and increased A23187 sensitivity was observed
upon stable overexpression of wild-type PKR in L929 cells. The sum of
these results strongly implicates PKR in mediating eIF-2
phosphorylation in response to calcium depletion.
phosphorylation in
response to calcium depletion. Our results demonstrated that expression
of a functionally defective PKR mutant K296P, the RNA binding domain
alone, or the dsRNA binding protein TRBP all inhibited the
A23187-induced phosphorylation of eIF-2
and lead us to propose two
mechanisms by which these proteins inhibit the calcium-mediated
activation of PKR. First, the PKR mutant K296P, the PKR RNA binding
domain, and TRBP may form dimers with endogenous wild-type PKR and
subsequently prevent its trans-autophosphorylation and
activation upon calcium depletion. In support of this possibility, we
have recently demonstrated by coimmunoprecipitation that intact PKR
interacts with both the PKR RNA binding domain (residues 1-243)
as well as TRBP when coexpressed in COS-1 cells.
(
)Alternatively, since the K296P mutant PKR, the RNA
binding domain, and TRBP all have in common the ability to bind dsRNA,
it is possible that calcium depletion induces a unique dsRNA PKR
activator. Increased cytosolic calcium may alter the secondary
structure of some RNA molecules to become good PKR activators. Although
there are several known cellular gene products that inhibit PKR
activity(20, 49, 50, 51) , the finding
that overexpression of mutant PKR K296P, the PKR RNA binding domain,
and TRBP all prevent PKR-mediated phosphorylation of eIF-2
in
response to calcium depletion suggests it is unlikely that calcium
depletion directly inhibits the activity of a cellular PKR inhibitor.
B to
activate the transcription factor NF
B and induce expression from
NF
B-dependent promoters(17) . In addition, calcium
mobilization is a primary mechanism in signal transduction and is
implicated in the activation of specific gene transcription upon cell
activation or differentiation(52) . The question arises as to
why calcium depletion from the ER would be associated with both
transcriptional activation as well as translational inhibition? We
propose that change in a cell program involves a coupling between
disassembly of polysomes mediated by PKR activation and recruitment of
free ribosomes to newly transcribed mRNAs. It is possible that this
coordinated response at both the level of mRNA generation and at the
level of mRNA utilization is required to establish specific changes in
gene expression.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.