(Received for publication, February 14, 1995; and in revised form, May 5, 1995)
From the
Double-stranded RNA-dependent protein kinase (PKR) is suggested
to play an important role in both the antiviral and antiproliferative
arms of the interferon response. To gain insights into the molecular
mechanisms underlying PKR's growth regulatory properties, we
examined the biological and biochemical properties of PKR variants
containing either a mutation in catalytic domain II (PKR-M1) or a
deletion of RNA binding domain I (PKR-M7) in both reticulocyte
translation extracts and in vitro kinase assays with purified
reagents and compared these results with those using the same mutants
stably expressed in vivo. While wild-type PKR (PKR-WT)
efficiently inhibited mRNA translation in a reticulocyte extract, the
inactive PKR-M1 had no effect. The PKR-M7 mutant was modestly
inhibitory in this assay. The PKR-M1 variant was able to reverse the
translational inhibitory effects and increased eukaryotic initiation
factor (eIF)-2
The interferon-induced, ds
PKR was initially identified as
an interferon-inducible enzyme that may become activated during virus
infection and thus play an important role in the regulation of protein
synthesis in infected
cells(12, 13, 14, 15) . More
recently PKR, which is constitutively expressed in mammalian cells, has
been implicated as playing a role in the control of cell growth and
proliferation(16, 17, 18) . The expression of
wild-type human PKR cDNA in yeast caused increased phosphorylation of
eIF-2
The current study was
undertaken to begin to define the molecular mechanisms underlying the
transforming action of two separate PKR variants, the catalytically
dead PKR-M1 (18, 22) and the RNA binding mutant,
PKR-M7, which retains minimal function and is severely deficient in the
binding of dsRNA(21) . Utilizing purified recombinant PKR
variants, we present evidence that PKR-M1 and PKR-M7 have different
effects on protein synthetic activity in reticulocyte lysate, although
both can function in vitro as dominant negative inhibitors of
the wild-type PKR (PKR-WT). However, our in vitro data, taken
together with the eIF-2
Figure 1:
Effects of PKR wild type and
variants on mRNA translation rates and eIF-2 phosphorylation levels in
reticulocyte lysate. A, cell-free extracts of rabbit
reticulocyte were incubated in the presence of PKR-WT, PKR-M1, or
PKR-M7 as indicated. Protein synthetic activity was measured by
[
Figure 2:
Effects of PKR-M1 on translational
inhibition by dsRNA addition and hemin deprivation in the reticulocyte
translation system. A, cell-free extracts of rabbit
reticulocytes were incubated as indicated in the absence or presence of
150 ng/ml dsRNA poly(I): poly(C) and in the presence of 1 µM hemin. Separate incubations were carried out in the absence of
hemin (-h). Indicated amounts of pure PKR-M1 were added
to reactions and protein synthetic activity measured as described
above. B, eIF-2
Figure 3:
Effects of PKR-M7 on translational
inhibition by dsRNA addition and hemin deprivation in the reticulocyte
translation system. A, cell-free extracts of rabbit
reticulocytes were incubated as indicated in the absence or presence of
150 ng/ml dsRNA poly(I): poly(C) and in the presence of 1 µM hemin. Separate incubations were carried out in the absence of
hemin (-h). Indicated amounts of pure PKR-M7 were added
to reactions and protein synthetic activity measured as described
above. B, eIF-2
Figure 4:
Effects of the PKR-M1 variant on purified
functional PKR-WT in in vitro kinase assays. A, 25 ng
of pure PKR-WT was incubated in the presence of 1.0 µg/ml poly(I):
poly(C) and the following increasing amounts of pure PKR-M1: lane
1, 0.0 ng; lane 2, 25 ng; lane 3, 50 ng; lane 4, 100 ng; lane 5, 200 ng; lane 6, 300
ng. The kinase assays were performed as described under
``Materials and Methods.'' B, 10 ng of PKR-WT was
incubated with 200 ng of PKR-M1 with the following increasing amounts
of poly(I): poly(C) in micrograms per ml: lane 1, 0.0; lane 2, 1.0; lane 3, 5.0; lane 4, 10.0.
Kinase assays were then performed as described under ``Materials
and Methods.'' The position of PKR and eIF-2
Figure 5:
Effects of the PKR-M7 variant on purified
functional PKR-WT in in vitro kinase assays. A, 10 ng
of pure PKR-WT (left side) or 10 ng pure PKR-M7 (right
side) were separately incubated with 0.0 µg/ml poly(I):
poly(C) (lanes 1 and 5), 0.1 µg/ml (lanes 2 and 6), 1.0 µg/ml (lanes 3 and 7),
and 10 µg/ml (lanes 4 and 8). Kinase assays were
carried out in the presence of purified eIF-2 as described under
``Materials and Methods.'' B, 10 ng of pure PKR-WT
was incubated in the presence of 1.0 µg/ml poly(I): poly(C) and the
following concentrations of PKR-M7: lane 1, 0.0 ng; lane
2, 10.0 ng; lane 3, 20.0 ng; lane 4, 50.0
ng.
Figure 6:
Comparison of growth rates and eIF-2
We have presented evidence that two PKR variants possess
distinct biological activities in reticulocyte extract, although both
can act in vitro as dominant negative inhibitors of PKR. These
data lead us to speculate that the inhibition may be occurring through
different mechanisms. First, it is unlikely that either mutant is
working by sequestration of the eIF-2 substrate, since neither
prevented phosphorylation of eIF-2
It was important to
address the biological relevance of these in vitro observations. As earlier mentioned, overexpression of either
PKR-M1 or PKR-M7 in NIH 3T3 cells induced their malignant
transformation and allowed these cells to cause tumors in nude mice (18, 21) . However, important differences do exist
between the PKR-M1 and PKR-M7 cell lines, and this may relate to the
variants' differing modes of action in vitro. Somewhat
unexpectedly we found that the PKR-M7 cell lines grew faster and to
higher densities than the PKR-M1 overexpressing cell lines. More
importantly, levels of eIF-2
The scenario
appears to be different for PKR-M1. Despite the variant's in
vitro properties, PKR-M1 does not cause a dramatic reduction of
eIF-2 phosphorylation in overexpressing cell lines nor does it
appreciably up-regulate growth rates of these cells in culture (Fig. 6). Furthermore, our earlier study showed that the
presence of PKR-M1 failed to prevent excessive phosphorylation of
eIF-2
We are grateful to Olga Savinova for technical
assistance, the Henshaw laboratory for eIF-2
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
phosphorylation levels caused by addition of
double-stranded RNA to reticulocyte extract, whereas PKR-M7 could not.
Both PKR-M1 and PKR-M7 functioned as transdominant inhibitors of PKR-WT
in our in vitro kinase assays. While the inhibition by PKR-M1
required a vast excess of mutant to shut down PKR function, PKR-M7
inhibited PKR-WT at approximately stoichiometric levels. To complement
these experiments, we compared growth rates and
phosphorylation
levels in transformed cell lines overexpressing either PKR-M1 or
PKR-M7. Levels of endogenous eIF-2
phosphorylation were
significantly more diminished in PKR-M7 overexpressing cells compared
with PKR-M1. These paradoxical data will be discussed in terms of the
potential molecular mechanisms underlying malignant transformation
caused by the PKR variants.
RNA-dependent protein
kinase, (
)PKR, is the most studied member of the
eIF-2
-specific kinase subfamily(1, 2) . Other
members of this family include the reticulocyte lysate heme-sensitive
eIF-2
kinase, commonly referred to as HCR or HRI (3) and
the yeast GCN-2 protein kinase which is involved in the translational
regulation of GCN-4(4) . PKR is a cAMP-independent,
serine-threonine kinase, characterized by two distinct kinase
activities: first an autophosphorylation, which represents the
activation reaction; and second, phosphorylation of
eIF-2
(5, 6) . This second phosphorylation event
can lead to limitations in functional eIF-2 and a resultant inhibition
in protein synthesis inhibition(7, 8, 9) . In
addition to its translational regulatory role, recent evidence now
suggests that PKR may play a role in signal transduction and
transcriptional control, possibly through the I-
B/NF-
B
pathway(10, 11) .
, a reduction in protein synthetic rates, and a decreased
proliferation rate(19, 20) . Conversely, the
transfection of cDNAs encoding nonfunctional PKR variants into murine
cells resulted in malignant transformation, as measured by their
ability to produce tumors in nude mice, suggesting a possible tumor
suppressor role for PKR(16, 18) . The nonfunctional
variants of PKR have included a mutant in catalytic domain II, in which
the essential lysine at position 296 has been changed to an arginine (18) , the DII or PKR-M1 variant, as well as a mutant in which
a crucial 6-amino acid segment (
6) has been deleted(16) .
More recently we have determined that introduction of PKR regulatory
domain variants, which completely lack the first RNA binding domain
(referred to as PKR-M7), into NIH 3T3 cells also induced their
malignant transformation (21) .
phosphorylation results acquired from the
PKR-M1 and PKR-M7 overexpressing cell lines, suggests that the
transdominant transforming action of PKR-M7 is through a translational
regulatory pathway, whereas PKR-M1 may trigger transformation through
an eIF-2
-independent pathway.
Expression and Purification of Recombinant PKR
Variants
Expression of recombinant wild-type PKR was carried out
in Escherichia coli as described by Barber et
al.(23) . The PKR-M1 catalytically inactive domain II
mutant (Lys Arg
) was constructed and expressed in
insect cells using the baculovirus expression system as described
previously(24) . The PKR-M7 regulatory domain variant (24) was expressed as a histidine-tagged fusion protein. This
vector was constructed by introducing a NdeI site at the
second ATG methionine at amino acid position 98 with the resultant
fragment cloned into the pET15b vector (Novagen)(25) . Extracts
containing the PKR variants were prepared as earlier
described(23, 24) . Both the PKR-WT and PKR-M1
proteins were purified utilizing the PKR monoclonal antibody (26) as described earlier(5, 22) . The
histidine fusion PKR-M7 variant was purified by Ni(II) column (Novagen)
according to manufacturer's protocol(25) . Protein
concentrations were determined by comparing the purified PKR proteins
with bovine serum albumin standards after SDS-polyacrylamide gel
electrophoresis and Coomassie Blue staining.
eIF-2
PKR
autophosphorylation and substrate phosphorylation assays were conducted
as described by Katze et al.(22) . Briefly purified
recombinant PKR was incubated in low salt buffer containing 100 mM KCl, 25 mM Tris-HCl, pH 7.2, 10% glycerol, 2 mM MgCl-specific Protein Kinase Assays
, 2 mM MnCl
, 1 mM
dithiothreitol, 3 mg/ml bovine serum albumin, and 5 µM [
-
P]ATP, 1000 Ci/mmol). Activator
poly(I): poly(C) was added to the mix together with purified rabbit
eIF-2 as described(22, 27) .
Rabbit Reticulocyte Translation System
Rabbit
reticulocyte lysates were prepared and used as described(28) .
Analysis of eIF-2
For
analysis in PKR overexpressing cell lines, cells at similar densities
were rinsed twice in ice-cold phosphate-buffered saline and lysed in 20
mM HEPES, pH 7.2, 2 mM EDTA, 100 mM KCl,
.05% SDS, 0.5% Elugent, 10% glycerol, 20 µg/ml chymostatin, 50
nM microcystin, 1 mM dithiothreitol. The 10,000
Phosphorylation
g supernatant was clarified with BPA-1000 (Toso-Haas,
Philadelphia). Supernatant (100 µg of protein) was first subjected
to immunoprecipitation using eIF-2
-specific monoclonal antibody.
Immunoprecipitates were then resuspended in the VSIEF sample buffer and
fractionated by vertical slab gel electrophoresis (27) to
separate phosphorylated from nonphosphorylated forms of eIF-2
. For
reticulocyte analysis, aliquots of the lysate were taken and similarly
treated. Proteins were transferred to Immobilon P and subjected to
immunoblotting using monoclonal antibody to
eIF-2
(27, 29) .
Analysis of Cell Line Growth Properties
A detailed
description of the PKR-M1 and PKR-M7 cell lines, including an analysis
of functional activity and physical levels of PKR variants, can be
found elsewhere(18, 21, 30) . To determine
growth rates, cells were plated at 5 10
/60-mm dish
and cell density determined at absorbance at 600 nm and recorded every
24 h. Prior to trypsinization, cell monolayers were extensively washed
to remove dead cells. Cell medium (Dulbecco's modified
Eagle's medium, 200 µg/ml G418, plus 10% fetal calf serum)
was replaced daily.
Effects of Wild-type and Variant PKR Proteins on mRNA
Translation in Reticulocyte Extracts
We previously reported that
cell lines overexpressing the PKR-M1 and PKR-M7 variants were
malignantly transformed and tumorigenic in nude
mice(18, 21) . The current study was initiated to
delineate molecular mechanisms of transformation by these PKR variants.
To accomplish this goal, highly purified PKR proteins were prepared and
their activities analyzed both in reticulocyte lysate and in vitro kinase assays. The recombinant PKR-WT and PKR-M1 kinases were
prepared from E. coli(23) and baculovirus-infected
insect cells(24) , respectively, and immunopurified using
PKR-specific monoclonal antibody bound to CnBr-activated
Sepharose(26) . Since this monoclonal antibody fails to react
with PKR variants lacking RNA binding domain I(22) , we
expressed PKR-M7 as a histidine fusion protein which can be readily
purified by passage over a nickel column. The recombinant wild-type and
mutant proteins were first analyzed for their effects on protein
synthesis regulation in reticulocyte extracts (Fig. 1A). While PKR-WT dramatically reduced protein
synthetic rates, the catalytically inactive PKR-M1 had no effect on
mRNA translation in the reticulocyte extracts. The decrease caused by
PKR-WT occurred in the absence of added dsRNA, thus suggesting that
activation is caused by either endogenous RNAs or other polyanions
present in the lysate. Moreover the protein kinase is already partially
active when purified from E. coli(23) .
C]valine incorporation into trichloroacetic
acid precipitable radioactivity after a 30-min incubation at 30 °C. B, cell-free extracts were incubated as above. Samples were
fractionated by VSIEF, transferred to Immobilon-P, and subjected to
immunoautography using eIF-2
-specific monoclonal antibody. Lanes 1 and 2 are standards of eIF-2
to
demonstrate the position of phosphorylated and nonphosphorylated forms
of eIF-2
. Phosphorylation in the absence of added PKR is shown in lane 3. The following amounts of variants were utilized: lanes 4 and 9, 0.5 µg; lanes 5, 10, and 14, 1.0 µg; lanes 6, 11, and 15, 2.0
µg; lanes 7, 12, and 16: 5.0 µg; lanes 8,
13, and 17, 10.0 µg.
In contrast
to PKR-M1, the RNA binding domain variant, PKR-M7, did reduce
translation rates in these extracts at high concentrations, although
minimally compared with PKR-WT. We then correlated the variants'
effects on translation with an analysis of endogenous eIF-2
phosphorylation in these extracts (Fig. 1B). Several
conclusions can be made concerning these experiments. (i) In the
absence of added PKR, levels of
phosphorylation in the lysates
are minimal (Fig. 1B, lane 3). (ii) Addition of 10
µg/ml PKR-WT caused essentially a 100% conversion to phosphorylated
eIF-2
in the reticulocyte extracts (lane 8). (iii)
Addition of PKR-M1 had little effect on endogenous
phosphorylation levels and even appeared to reduce levels at the lower
concentrations tested. (iv) Due to its minimal functional activity,
PKR-M7 did increase
phosphorylation, although modestly compared
with PKR-WT. It should be emphasized that we measure the steady state
levels of
phosphorylation and not the rate of phosphorylation by
PKR in these in vitro assays. We proceeded to utilize the
reticulocyte system to further examine the differing biological
properties of PKR-M1 and PKR-M7.
Addition of PKR Variants to Reticulocyte Lysates
Compromised by dsRNA Addition or Hemin Deprivation
It is well
established that addition of dsRNA to reticulocyte lysates severely
compromises mRNA translation rates presumably due to activation of the
endogenous rabbit PKR(31) . We took advantage of this system to
test whether PKR-M1 or PKR-M7 could interfere with activation of the
endogenous kinase by the addition of dsRNA. In the presence of 150
ng/ml poly(I): poly(C), protein synthesis became inhibited after a lag
period, reducing the incorporation of [C]valine
by 35-40% after a 30-min incubation (Fig. 2A).
The addition of increasing levels of PKR-M1 restored protein synthesis
nearly to control levels. Approximately 7.5 µg/ml of the PKR-M1
variant, equivalent to to more than 10 pmol of PKR-M1/100 µl, was
needed to completely restore protein synthetic activity, a level
considerably higher than the estimated endogenous level of PKR of less
than 0.1 pmol/100 µl of reticulocyte lysate.
(
)Since further addition of dsRNA could reverse this
restoration (data not shown), it is likely that PKR-M1 is functioning
to inhibit the endogenous PKR by sequestering the activator. In
contrast to the recovery of mRNA translation caused by PKR-M1, the
regulatory domain variant PKR-M7 failed to reverse the inhibitory
effects of dsRNA (Fig. 3A). Consistent with its
inability to bind to and therefore sequester dsRNA, the highest
concentrations of PKR-M7 had modest effects on protein synthetic rates.
Examination of endogenous eIF-2
phosphorylation levels confirmed
the protein synthesis data. Whereas the catalytically inactive PKR-M1
variant quantitatively reduced
phosphorylation levels in the
presence of dsRNA (Fig. 2B), PKR-M7 had no effects on
these phosphorylation levels (Fig. 3B).
phosphorylation levels were measured
separately in the presence of dsRNA (+dsRNA) and the
absence of hemin (-h) after incubation with increasing
amounts of recombinant PKR-M1 as described in the legend to Fig. 1.
phosphorylation levels were measured
separately in the presence of dsRNA (+dsRNA) and the
absence of hemin (-h) after incubation with increasing
amounts of PKR-M7 as described in the legend to Fig. 1.
The
reticulocyte translation system also contains a heme-sensitive
eIF-2 kinase that can be activated by incubation in the absence of
added hemin(3) . This can be used to test whether the different
PKR variants can act as dominant negative inhibitors by binding to
eIF-2
and preventing its phosphorylation. If this occurred one
would expect an enhancement of protein synthesis rates and resultant
decrease in
phosphorylation levels in hemin-deprived lysates.
However, neither PKR-M1 (Fig. 2A) nor PKR-M7 (Fig. 3A) were able to prevent the severe decreases in
protein synthetic rates caused by the omission of hemin. Similarly
neither variant prevented the increases in eIF-2
phosphorylation
levels caused by the absence of hemin (Fig. 2B and
3B). These data strongly suggest that any transdominant
effects of the PKR-M1 or PKR-M7 variants observed in vivo or in vitro is unlikely due to sequestration of the PKR
substrate.
Transdominant Inhibition of PKR-WT by PKR-M1 and PKR-M7
in Vitro
Thus far we have examined the biological properties of
the variants in reticulocyte extracts but have not yet directly
demonstrated that PKR-M7 or PKR-M1 can act as transdominant inhibitors in vitro. To accomplish this and examine the stoichiometry of
such a reaction, we analyzed the enzymatic activity of PKR-WT which was
incubated with either the catalytic or regulatory domain variants. In
the absence of any variant, the PKR-WT efficiently phosphorylated
exogenously added eIF-2 in the presence of poly(I): poly(C) (Fig. 4A, lane 1). The addition of increasing amounts
of PKR-M1 variant reduced the phosphorylation of eIF-2
(Fig. 4A, lanes 2-6). However, levels of PKR-M1
up to 10 times higher than the levels of wild type were required to
significantly reduce
phosphorylation. We could not observe a
decrease in PKR-WT autophosphorylation due to the concomitant increased
phosphorylation of the 68-kDa polypeptide which likely results from
trans-phosphorylation of PKR-M1 by PKR-WT (since both PKR proteins
comigrate) as also observed by others(32) . PKR-M1 alone is
catalytically dead and unable to autophosphorylate itself(22) .
Importantly, the ability of PKR-M1 to prevent phosphorylation of
eIF-2
by PKR-WT could be reversed (
phosphorylation
increasing 3-5-fold) by increasing the concentration of dsRNA
again, suggesting that the variant functions by sequestering activator (Fig. 4B, lanes 2-4).
are
indicated.
We next examined the
activity of recombinant PKR-M7 in a similar assay. When assayed alone,
the PKR-M7 variant possessed minimal activity compared with PKR-WT (Fig. 5A). Although autophosphorylation of the
histidine fusion PKR protein is detectable at the highest dsRNA
concentrations, this mutant cannot appreciably phosphorylate the
eIF-2 substrate (Fig. 5A, lanes 7 and 8).
PKR-M7 is, however, an effective inhibitor of wild-type kinase function (Fig. 5B). Moreover, roughly equal amounts of PKR-M7
compared with PKR-WT can significantly reduce both the
autophosphorylation and eIF-2
phosphorylating ability of the
wild-type kinase. Furthermore, excess dsRNA was not able to reverse
this inhibition (data not shown). This is in marked contrast to the
previous experiments in which vast excesses of PKR-M1 were required for
a similar effect that was reversed by increasing activator
concentration. It is relevant to note that PKR-WT and PKR-M7
coincidentally comigrate due to the presence of the histidine tag
despite the latter's smaller size. In contrast to the PKR-M1
variant, however, little trans-phosphorylation of PKR-M7 by PKR-WT was
observed for unknown reasons.
Comparison of NIH 3T3 Cell Lines Overexpressing PKR-M1
and PKR-M7 Variants
The data presented thus far demonstrated
that both PKR-M1 and PKR-M7 can function as transdominant inhibitors of
the wild-type kinase in vitro, resulting in reduced
phosphorylation. An important question that needed to be answered is
whether these variants functioned in a similar way in vivo,
inside a mammalian cell. We therefore analyzed both the growth rates
and endogenous eIF-2
phosphorylation levels in PKR-M1 expressing
NIH 3T3 cells and compared these cells with those expressing the PKR-M7
regulatory domain variant. A detailed description of the PKR-M1 and
PKR-M7 cell lines, including analyses of PKR variant levels, can be
found elsewhere(18, 21, 30) . Although we
previously tested the tumorigenicity of these PKR-M1 cell
lines(18) , we never compared PKR-M1 and PKR-M7 growth rates
nor did we examine
phosphorylation levels in the absence of virus
infection or interferon treatment(30) . Our present analysis
revealed that the growth rate of the PKR-M1 overexpressing cells did
not differ from the control cells expressing the neomycin-resistant
gene alone as revealed by the slope of the curve (Fig. 6A). The PKR-M1 cells did, however, grow to
somewhat higher densities which quickly leveled off after approximately
a week in culture. In contrast, the PKR-M7 cell lines grew both at a
faster rate and to higher densities with growth rates not levelling
off. We then compared endogenous eIF-2
phosphorylation levels in
the three different cell lines at both day 5 and day 7 after plating (Fig. 6B). At both days, phosphorylation levels were
more drastically reduced in the PKR-M7 cell line compared with the
PKR-M1 cell lines. Indeed phosphorylated eIF-2
was barely
detectable only at day 7 in the PKR-M7 cell extracts. Laser
densitometry quantitation at day 7 revealed that eIF-2
phosphorylation levels in PKR-M1 cells were approximately 2.5-fold
lower than in control cells expressing the neomycin-resistant gene
alone, whereas levels in PKR-M7 cells were reduced approximately
50-fold.
phosphorylation levels in control cells expressing only the
neomycin-resistant gene and PKR-M1 and PKR-M7 overexpressing NIH 3T3
cells. A, cells were grown in Dulbecco's modified
Eagle's medium supplemented with 10% fetal calf serum and G418.
Medium was changed and cell densities determined every 24 h by
measuring the absorbance at 600 nm. B, cell extracts from day
5 and day 7 were analyzed by isoelectric focusing gel electrophoresis
and immunoblot analysis. Blots were incubated with an
eIF-2
-specific monoclonal antibody. Lanes marked eIF-2
and
eIF-2
-P are standards of purified eIF-2 incubated in the absence
or presence of the heme regulated kinase to demonstrate the position of
phosphorylated and nonphosphorylated forms of
eIF-2
.
by the heme regulated kinase.
The catalytically inactive PKR-M1 mutant is likely inhibiting PKR-WT by
sequestering the dsRNA activator, since (i) PKR-M1, at large excesses,
can reverse the damaging effects of translation in reticulocyte extract
caused by dsRNA addition; (ii) large amounts of PKR-M1 are required for
transdominant inhibition of PKR-WT in our in vitro kinase
assays; and (iii) this inhibition of the wild-type kinase can be
reversed by further addition of excess dsRNA. In contrast, the
regulatory domain mutant, PKR-M7, can inhibit PKR-WT at approximately
equal concentrations, cannot reverse dsRNA effects in reticulocyte
lysate, nor can its action in vitro be reversed by addition of
more activator. We conclude, therefore, that PKR-M7 is probably
inhibiting kinase activity through a direct interaction, forming
inactive heterodimers with the wild-type protein kinase, although we
concede we have no concrete data at this time to support this model.
Others have presented evidence that PKR may need to dimerize to become
fully functional(33, 34) .
phosphorylation were more
dramatically reduced in the NIH 3T3 cell lines overexpressing the
regulatory domain variant, PKR-M7, compared with PKR-M1 expressing cell
lines. This raises the possibility that the two variants may be
triggering transformation through different pathways. The in vivo and in vitro data describing PKR-M7 activity are
consistent and strongly suggest that PKR-M7 functions through the
inactivation of PKR activity by mechanisms that do not involve the
sequestration of activator. This then results in reduced eIF-2
phosphorylation levels and accelerated growth rates in cell lines
expressing the variant. This situation is very similar to cell lines
overexpressing the
6 PKR mutant (16) and the PKR cellular
inhibitor P58(35) . In both these cases, decreases in
endogenous PKR function lead to dramatic reductions in eIF-2
phosphorylation levels, accelerated growth rates, and subsequent
ability of these cells to cause tumors in nude mice.
in interferon-treated cells infected by encephalomyocarditis
virus(30) . Taken together, these results suggest one of two
possibilities regarding the variant's ability to induce malignant
transformation: (i) PKR-M1 may still work by down-regulating PKR
function by sequestering dsRNA activator. However, since eIF-2
phosphorylation levels are not dramatically diminished in
vivo, PKR-M1 also may be inhibiting other, nontranslational
activities of PKR, e.g. those functions involved with signal
transduction and transcription(10, 11) . Consistent
with this hypothesis is the recent report demonstrating that a nearly
identical catalytically inactive PKR molecule can alter the activation
state of NF-
B(10) . (ii) An alternative explanation is
that PKR-M1 transforms cells via mechanisms completely independent of
any known PKR regulatory pathway. It may be that PKR-M1 overexpression
is disrupting a PKR-independent pathway by binding to and/or
sequestering unknown proteins which would then result in malignant
transformation. This would then suggest that the variant does not
function as a bona fide transdominant inhibitor of PKR in
overexpressing cells and would be in agreement with a recent report
showing that the domain II mutant cannot function as a dominant
negative inhibitor in yeast(34) . However it would be in
contradiction to our earlier findings suggesting PKR-M1 can function
dominant negatively in vivo using transient transfection
assays(36) . In any case, it seems unlikely that the in
vitro inhibition of PKR and eIF-2
phosphorylation (this
report) and effects on protein synthetic rates in reticulocyte extracts
caused by PKR-M1 (this report and (37) ) accurately reflect the
only biological properties of the PKR-M1 variant.
antibody, Dr. Leonard
Jefferson for purified eIF-2, and Marjorie Domenowske for help with the
figures. We are grateful to Greg Schaefer for excellent technical
assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.