(Received for publication, November 19, 1996, and in revised form, February 28, 1997)
From the Department of Biochemistry and Molecular
Biology, Indiana University School of Medicine, Indianapolis, Indiana
46202-5122 and the § Laboratory of Eukaryotic Gene
Expression, NICHD, National Institutes of Health,
Bethesda, Maryland 20892
Protein kinase PKR is activated in mammalian
cells during viral infection, leading to phosphorylation of the subunit of eukaryotic initiation factor-2 (eIF-2
) and inhibition of
protein synthesis. This antiviral response is thought to be mediated by association of double-stranded RNA (ds-RNA), a by-product of viral replication, with two ds-RNA-binding domains (DRBDs) located in the
amino terminus of PKR. Recent studies have observed that expression of
mammalian PKR in yeast leads to a slow growth phenotype due to
hyperphosphorylation of eIF-2
. In this report, we observed that
while DRBD sequences are required for PKR to function in the yeast
model system, these sequences are not required for in vitro
phosphorylation of eIF-2
. To explain this apparent contradiction, we
proposed that these sequences are required to target the kinase to the
translation machinery. Using sucrose gradient sedimentation, we found
that wild-type PKR was associated with ribosomes, specifically with 40 S particles. Deletions or residue substitutions in the DRBD sequences
blocked kinase interaction with ribosomes. These results indicate that
in addition to mediating ds-RNA control of PKR, the DRBD sequences
facilitate PKR association with ribosomes. Targeting to ribosomes may
enhance in vivo phosphorylation of eIF-2
, by providing
PKR access to its substrate.
RNA-binding proteins are important participants in
post-transcriptional regulation of gene expression. Sequence
comparisons between these proteins has led to the discovery of several
RNA-binding motifs, including the double-stranded RNA binding domain
(DRBD)1 (1-5). The DRBD is about 65 residues in length, with a lysine-rich sequence at the
carboxyl-terminal end (1, 2, 4). More than 20 different proteins
containing DRBD sequences have been identified (1). One of the best
characterized DRBD-containing proteins is the double-stranded
RNA-dependent protein kinase, PKR, that functions in a
cellular antiviral response and is transcriptionally induced by
interferon (6-10). Upon viral infection in mammalian cells, PKR
inhibits protein synthesis by phosphorylation of the subunit of
eukaryotic initiation factor-2 (eIF-2) (6-8). The eIF-2 bound to
Met-tRNAiMet and GTP associates with 40 S ribosomal subunits and facilitates recognition of the start codon
during translation initiation (11-14). Upon completion of this
process, the GTP complexed with eIF-2 is hydrolyzed to GDP.
Phosphorylation of eIF-2
by PKR impedes the exchange of eIF-2-GDP to
the GTP-bound form that is required for the next round of translation
initiation. The resulting reduction in eIF-2-GTP levels inhibits
cellular protein synthesis and blocks viral proliferation into
neighboring cells (11-13).
Activation of PKR during viral infection is thought to be regulated
directly by double-stranded RNA (ds-RNA) that is produced during viral
replication. Evidence supporting this model is the observation that the
addition of ds-RNA to a purified in vitro system stimulates
both PKR autophosphorylation and phosphorylation of eIF-2 (7, 8,
15-17). Regulation of PKR by ds-RNA is thought to be mediated by two
DRBD sequences, termed motif 1 and motif 2, that are located in the
amino-terminal portion of the kinase (Fig. 1). Numerous reports have
shown that recombinant proteins containing motifs 1 and 2 can bind
ds-RNA in vitro assays (18-25). Motif 1 is a closer match
to the DRBD consensus and has a higher affinity for ds-RNA than motif 2 (18-22, 24, 26). However, both DRBD sequences are thought to be
required for maximal ds-RNA binding to PKR. Accumulatively, these
studies suggest a mechanism for activation of PKR whereby ds-RNA binds
motifs 1 and 2 and alters the conformation of the kinase leading to
stimulation of PKR function (7, 18-22, 24, 25). Although the molecular details of this regulation are not yet clear, it is thought that ds-RNA-induced autophosphorylation of PKR participates in kinase activation, perhaps by enhancing the affinity of PKR for ATP (6, 7, 15,
27). Autophosphorylation may occur between two PKR molecules that are
linked by protein-protein contacts or a ds-RNA bridge (7, 8,
28-32).
Several lines of research have supported the in vivo role of
the DRBD in mediating ds-RNA activation of PKR. Expression of human PKR
in yeast Saccharomyces cerevisiae deficient for its endogenous eIF-2 kinase encoded by GCN2 led to
hyperphosphorylation of eIF-2
at serine 51, resulting in a slow
growth defect due to a general inhibition of translation initiation
(18, 33-35). When mutant forms of PKR with deletions in the motif 1 or
2 sequences were expressed in yeast, there was no phosphorylation of
eIF-2
and near wild-type levels of growth (34). Experiments
analyzing the activity of mutant forms of PKR in mammalian cells have
relied on the observation that PKR specifically inhibits its own
translation (29, 36, 37). From studies using biochemical fractionation and immunofluorescent staining, the majority of PKR in mammalian cells was found to be associated with ribosomes (7, 38-40). The
remaining portion, estimated at less than 20% of the total PKR, was
found in the nucleus in proximity to the nucleoli (40). It was proposed
that newly synthesized PKR associates with ribosomes in close vicinity
to its own mRNA, resulting in a localized phosphorylation of
eIF-2
that would preferentially diminish the synthesis of PKR
protein. Transfection of mammalian cells with PKR cDNAs showed that
alterations in an invariant kinase residue or in DRBD sequences decreased its ability to regulate its own synthesis (29, 36, 37).
Interestingly, expression of certain kinase-inactive mutant forms of
PKR in NIH 3T3 cells conferred a malignant transformation phenotype,
and subcutaneous injection of these transfected cells in nude mice gave
rise to rapid tumor growth (41-44). When similar experiments were
carried out with wild-type PKR, no transformed phenotype was observed.
These results suggest that the kinase functions as a tumor suppressor,
since expression of a mutant PKR in NIH 3T3 cells appeared to reduce
the activity of the endogenous wild-type PKR (42, 43). Furthermore, it
suggests that levels of eIF-2 phosphorylation in cells are important
for control of cell proliferation. Consistent with this model, Donze
et al. (45) showed that expression of a mutant form of
eIF-2
that contains an alanine at the PKR phosphorylation site,
serine 51, in NIH 3T3 cells resulted in malignant transformation.
However, other mechanisms for cell transformation by PKR have not
been ruled out. Recent studies have shown that PKR can also
phosphorylate the I
B family of inhibitors that regulate the
transcription factor NF-
B (46, 47).
In this report, we observed that while motifs 1 and 2 are essential for
PKR function in the yeast model system, these sequences are not
required for in vitro phosphorylation of eIF-2. To
explain this apparent contradiction, we proposed that the in
vivo requirement for DRBD sequences is due to their role in
targeting PKR to the translation machinery. Using sucrose gradient
sedimentation, we did indeed find that wild-type PKR was associated
with ribosomes, specifically with the 40 S particles. Deletions or
residue substitutions in motif 1 or 2 blocked kinase interaction with
ribosomes. Together, these results indicate that the DRBD sequences
carry out a dual function in the regulation of PKR phosphorylation of
eIF-2. First, motifs 1 and 2 are proposed to mediate ds-RNA stimulation
of kinase activity. Our results are consistent with the idea that the
DRBD sequences function together to repress PKR kinase activity.
Binding of ds-RNA to these motifs would release their inhibitory
affect, allowing for activation of the kinase. The second function of the DRBD sequences is appropriate targeting of PKR to the translational machinery. Localization of PKR to ribosomes may increase in
vivo access to its eIF-2 substrate.
Human PKR was expressed in yeast
using plasmid p1420 (33) that contains the PKR cDNA inserted into a
modified pEMBLyex4 vector. Plasmid pEMBLyex4 is a high copy number
URA3-based vector containing the galactose-inducible
GAL-CYC1 hybrid promoter (48). Plasmid p1420 was
transformed into yeast strains H1817 (MATa ura3-52 leu2-3 leu2-112 gcn2
sui2 GCN4-lacZ p1098
(SUI2-S51A, LEU2)) and H1816 (MATa
ura3-52 leu2-3 leu2-112
gcn2
sui2 GCN4-lacZ p1097
(SUI2 LEU2)) (49). Strains H1817 and H1816 are isogenic and
differ only in their SUI2 alleles that encode eIF-2
. To
induce expression of PKR, the transformed strains were first grown in synthetic dextrose medium (50) supplemented with 2 mM
leucine, 1 mM isoleucine, 1 mM valine at
30 °C for 30 h. The saturated cultures were then diluted 1:50
into synthetic minimal medium containing 10% galactose and 2%
raffinose, and cells were grown for 15 h at 30 °C. Mutant PKR
alleles were similarly expressed in yeast strains H1817 and H1816 using
pEMBLyex4-derived expression plasmids that were previously described
(34). PKR-A68P and PKR-A158P contain proline substituted for alanine at
positions 68 and 158, respectively (19, 34). The kinase-defective
mutant PKR-K296R contains an arginine for the invariant lysine at
residue 296 (51). PKR-
1 initiates at a methionine at position 98, resulting in a deletion of motif 1 from residues 1-97 (19, 34).
PKR-
2 has a deletion of residues 103-158 that includes motif 2 (19, 34), and PKR-
14-257 is deleted for both DRBD sequences between residues 14 and 257. PKR-
K is deleted for the kinase catalytic domain between residues 271 and 551 (34).
Transformants of H1817
(SUI2-S51A) expressing different alleles of PKR were grown
in synthetic medium containing 10% galactose and 2% raffinose for
15 h at 30 °C. Cells were chilled, collected by centrifugation,
and resuspended in kinase buffer (20 mM Tris-HCl (pH 7.9),
50 mM KCl, 10 mM MgCl2, 2 mM MnCl2, and 5 mM
-mercaptoethanol) and protease inhibitors (1 µM
pepstatin, 1 µM leupeptin, 0.15 µM
aprotinin, and 100 µM phenylmethylsulfonyl fluoride).
Cells were lysed using glass beads and a Vortex mixer, and the samples were clarified by centrifugation at 12,000 × g.
Protein concentrations were determined using the Bradford dye-binding
procedure (52).
Kinase reaction samples containing 5 µg of protein were first
prephosphorylated with 20 µM nonradiolabeled ATP in 90 µl of kinase buffer supplemented with protease inhibitors. This step reduced the incorporation of 32P into endogenous yeast
proteins and can be eliminated without affecting the measured specific
activity relative to wild-type PKR. After incubating the samples at
30 °C for 20 min, 2 µg of recombinant eIF-2 substrate and 20 µCi of [
-32P]ATP in a final concentration of 40 µM ATP were added to the kinase reactions. In experiments
measuring PKR activation by ds-RNA, 0.1 or 1 µg/ml poly(I)·poly(C)
(Sigma) was also added to the assay. As described previously (53), the
eIF-2
substrate is a modified form of yeast eIF-2
that is deleted
for residues 200-304 and contains a polyhistidine "tag" for rapid
purification. The deletion of the carboxyl-terminal residues of
eIF-2
removes three phosphorylation sites for casein kinase II (54,
55). The final volume of the kinase reaction was 100 µl, and 15-µl
aliquots were removed from the sample after 1, 2, 4, 10, and 20 min of
incubation at 30 °C. The aliquots were mixed with an equal volume of
2 × SDS sample buffer, heated at 95 °C for 5 min, and analyzed
by electrophoresis in a 10% SDS-polyacrylamide gel. All in
vitro kinase assays were carried out in at least three independent
experiments. The eIF-2
in the kinase assays often showed some modest
proteolytic degradation resulting in two bands of phosphorylated
substrate. PKR immunoprecipitation kinase assays were carried out as
described previously (56).
Levels of eIF-2 phosphorylation were quantitated by measuring
32P incorporation using a Bio-Rad model GS-250 molecular
imager. The 32P levels/mg of total protein for each sample
were plotted versus time, and the initial velocity,
presented in arbitrary units and normalized to wild-type PKR, was
determined by measuring the slope of the linear portion of the curve
(Table I). The specific activity for each PKR protein was determined by
dividing the initial velocity by the level of kinase protein relative
to wild-type PKR.
|
Another often used measure of PKR activity is autophosphorylation. In
our in vitro assay we used 5 µg of cell lysate in a 100-µl reaction volume. This lysate concentration was in the linear range of the assay. Under these conditions, we detected very little autophosphorylation of PKR. When 100 µg of lysate was used in similar
reaction conditions, we observed incorporation of 32P in
wild-type PKR that was absent in the kinase-deficient mutant PKR-K296R.
Under similar assay conditions, PKR-1 showed a modest reduction in
autophosphorylation compared with wild-type PKR when normalized for
steady-state protein levels. Mutant PKR-
2 and PKR-
14-257 showed
larger reductions in the levels of kinase autophosphorylation. Correlating autophosphorylation and catalytic activity is difficult because these deleted sequences in the PKR mutants may remove potential
autophosphorylation sites. Taylor et al. (57) identified three in vitro autophosphorylation sites in PKR, Ser-242,
Thr-255, and Thr-258. Two of these sites are absent in PKR-
14-257.
Remaining sites of autophosphorylation are not currently characterized, and their role in activation of PKR is not yet fully understood. For
these reasons we relied on the exogenous eIF-2 substrate as a more
reliable measure of PKR catalytic activity.
Samples containing 20 µg of protein prepared for the in vitro kinase assays were analyzed by electrophoresis in a 10% SDS-polyacrylamide gel and then transferred to a nitrocellulose filter. Immunoblot filters were blocked in a TBS-T solution containing 20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 0.1% Tween 20, and 5% nonfat dry milk and then incubated in TBS-T solution containing either PKR monoclonal antibody 70-10 (58) or rabbit polyclonal antiserum PKR K-17 that was prepared against a carboxyl-terminal polypeptide of PKR (Santa Cruz Biotechnology, Inc.). Western blot results obtained with polyclonal antiserum PKR K-17 were independently confirmed with a monoclonal antibody recognizing an epitope in the carboxyl-terminal kinase domain of PKR. Filters were washed in TBS-T, and PKR-antibody complex was detected using horseradish peroxidase-labeled secondary antibody provided in the ECL Western blotting analysis system (Amersham Corp.). Relative amounts of PKR in the immunoblot experiments were quantitated by measuring band intensities using a Bio-Rad model GS-670 imaging densitometer from autoradiographs generated by different length exposures.
Ribosome AssociationTransformants of H1817 containing different alleles of PKR were grown in synthetic medium containing 10% galactose and 2% raffinose for 15 h, and 50 µg/ml cycloheximide was added to the culture 5 min before harvesting. Cells were chilled on ice, collected by centrifugation, and washed once with breaking solution (20 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 50 µg/ml cycloheximide, and 200 µg/ml heparin). Each of the subsequent steps was performed at 4 °C, and protease inhibitors (1 µM pepstatin, 1 µM leupeptin, 0.15 µM aprotinin, and 100 µM phenylmethylsulfonyl fluoride) were added to all solutions. Cells were resuspended in breaking solution, lysed by glass beads using a Vortex mixer, and clarified by centrifugation at 12,000 × g for 25 min. Supernatant samples containing 20 A260 units were loaded onto a 5-47% sucrose gradient in breaking solution without heparin, and ultracentrifugation was performed using a Beckman rotor SW41 at 39,000 rpm for 3 h (59). Gradients were fractionated using an ISCO UA-6 absorbance monitor set at 254 nm, and 0.5-ml aliquots were collected. Proteins from each aliquot were precipitated by adding 5% trichloroacetic acid and separated by electrophoresis in a 10% SDS-polyacrylamide gel. PKR was detected by immunoblot analysis. To characterize PKR association with ribosomes in the absence of MgCl2, no cycloheximide was added before harvesting the cells, and MgCl2, cycloheximide, and heparin were omitted from the breaking solution. Sucrose gradient sedimentation and SDS-polyacrylamide gel electrophoresis were carried out as described for the fractionation studies performed in the presence of MgCl2.
Yeast is a
useful model system to study the in vivo role of PKR
sequences in the control of its kinase activity (18, 33-35). Dever
et al. (33) showed that PKR expressed from a
galactose-inducible promoter in yeast leads to high levels of
phosphorylation of eIF-2 and a severely reduced rate of cellular
growth. Consistent with these previous reports, we found this growth
defect is substantially alleviated in yeast containing a
kinase-defective PKR-K296R or mutants deleted in motif 1 or 2 (Fig.
1 and Table I). Romano et al.
(34) found that these mutant PKR proteins were unable to phosphorylate
eIF-2
in vivo. These observations were interpreted as
supporting the model that ds-RNA endogenous in yeast interacted with
the two DRBD sequences and stimulated PKR phosphorylation of
eIF-2
.
We next wished to measure the in vitro activity of the
different mutant versions of PKR to determine whether these proteins were impaired for phosphorylation of eIF-2. As noted earlier, translational expression of PKR is autoregulated in mammalian cells.
This appears to be also true in the yeast system, as illustrated by the
observation by Romano et al. (34) that the protein levels of
wild-type PKR were greatly reduced compared with the kinase-defective PKR-K296R mutant. We have confirmed this observation, with wild-type PKR expressed in strain H1816 (
gcn2) showing less than
5% of the protein levels of mutant PKR-
1, PKR-
14-257, or
PKR-K296R as judged by immunoblot analysis (data not shown). To avoid
these large differences in PKR protein levels, we used strain H1817 (
gcn2) that contains an alanine substituted for the
eIF-2
phosphorylation site serine 51. Expression of this
nonphosphorylatable form of eIF-2
blocks both the slow growth
phenotype associated with hyperphosphorylation of eIF-2
and
autoregulation of PKR expression (34). Cell lysates were prepared from
H1817 expressing the different PKR alleles, and the kinase protein
levels were measured by immunoblotting (Fig. 2).
Different PKR mutant protein levels ranged from 0.5 to 1.5 times that
measured for wild-type PKR (Table I).
Cell lysates were prepared from H1817 expressing different PKR mutant
proteins and analyzed for phosphorylation of recombinant eIF-2
substrate. As illustrated in the autoradiogram shown in Fig.
3, eIF-2
substrate was phosphorylated in the sample
containing wild-type PKR, while no eIF-2
phosphorylation was
detected in lysates prepared from cells expressing no PKR (
PKR) or
the kinase-defective PKR-K296R and PKR-
K proteins (Fig. 3, Table I).
To confirm that phosphorylation of eIF-2
was on serine 51, we
carried out a control experiment using recombinant eIF-2
containing
alanine for serine 51 (eIF-2
-S51A). No phosphorylation of
eIF-2
-S51A was detected in the wild-type PKR reaction (Fig. 3). It
is thought that PKR expressed in yeast is at least partially activated
by endogenous ds-RNA (34, 35). Consistent with the view that PKR in the cell lysates is in the activated conformation, we found that the addition of poly(I)·poly(C) to in vitro kinase reactions
did not further increase phosphorylation of eIF-2
by PKR (data not
shown). In a parallel experiment, we immunoprecipitated PKR from yeast extracts and found that the addition of poly(I)·poly(C) increased less than 2-fold the phosphorylation of eIF-2
by PKR compared with
similar reactions without ds-RNA. These results are in general agreement with Romano et al. (34), who found that the
activity of PKR immunoprecipitated from yeast lysates was not further
stimulated by the addition of poly(I)·poly(C).
When PKR-1 or PKR-
2, deleted for motif 1 or motif 2 sequences,
respectively, were assayed for phosphorylation of eIF-2
, we found
kinase activities slightly elevated over wild-type PKR (Fig. 3 and
Table I). An even more dramatic demonstration that the DRBD sequences
are not essential for kinase activity was found when we assayed the
PKR-
14-257 protein. This mutant kinase, which contains a deletion
of both DRBD sequences, had 9 times the eIF-2
kinase activity of
wild-type PKR. These results suggest that the amino-terminal sequences
of the kinase perform a dual function. First, motifs 1 and 2 mediate
ds-RNA activation of PKR kinase catalytic activity. As further
clarified under "Discussion," the dispensability of the DRBD
sequences for eIF-2
kinase activity would argue against motifs 1 and
2 simply functioning as positively acting regulatory sequences in the
yeast system. A second function of motifs 1 and 2 would not involve
enhancing kinase catalytic activity per se but would
facilitate phosphorylation of eIF-2
in vivo. For example,
the DRBD sequences might target PKR to a cellular location required for
its access to the eIF-2
substrate.
Results from our in vitro kinase assays
suggested that the DRBD sequences carry out a localization role in the
cell that is required for phosphorylation of eIF-2. We hypothesized
that the DRBD sequences target PKR to ribosomes. Several lines of
evidence support the proposal that this kinase associates with the
translational machinery. First, biochemical fractionation studies (7,
38) and immunofluorescent experiments (39, 40) indicate that PKR is
associated with ribosomes in mammalian cells. Second, the kinase substrate, eIF-2
, is localized to ribosomes during initiation of
protein synthesis (59). Finally, Bycroft et al. (4) has determined the NMR solution structure of a DRBD sequence from Drosophila staufen and found a
structure
with many features common to the N-terminal domain of prokaryotic
ribosomal protein S5. Many of the consensus residues shared among
members of the S5 protein family are conserved in DRBD sequences,
suggesting that the two domains share a common evolutionary origin. We
determined the localization of PKR in yeast strain H1817 (
gcn2
SUI2-S51A) using sucrose gradient sedimentation. Strain H1817 was
selected for this analysis because, as noted above, the wild-type PKR
levels were not severely decreased due to autoregulation. Cycloheximide was added to the culture medium 5 min before harvesting and to the
lysis buffer to arrest translation elongation and preserve the
polysomes during preparation and analysis of the sample. Fractions from
the gradient were characterized by immunoblot to determine the
distribution of the eIF-2
kinase. PKR was broadly dispersed in the
sucrose gradient with over 70% of the protein co-migrating in the
ribosomal fractions, primarily with 40 and 60 S subunits and 80 S
particles and to a lesser extent with polysomal fractions (Fig.
4). The remaining portion of PKR was found in the top of the gradient, in fractions free of ribosomes.
PKR extracts were next analyzed in gradients lacking Mg2+, and as expected, we detected only free 40 and 60 S particles (59). As previously observed (59, 60), the ribosomal subunits sedimented more slowly than measured in gradients containing Mg2+ because certain ribosome-associated factors are released upon withdrawal of the cation. Under these conditions, almost all of the PKR migrated with the 40 S subunit (Fig. 4B). To be certain that PKR was not simply associated with mRNA that might still remain coupled with 40 S subunits, we treated the lysates with micrococcal nuclease following a regimen used for preparation of RNA-dependent in vitro translation lysates. PKR was associated with the 40 S subunits after sucrose gradient centrifugation of the nuclease-treated lysate (data not shown). We conclude that PKR is associated with ribosomes in the yeast system.
DRBD Sequences Mediate PKR Association with RibosomesTo
assess whether DRBD sequences mediate ribosomal association of PKR, we
analyzed PKR mutants containing in-frame deletions in either motif 1 or
motif 2 sequences. Both PKR-1 and PKR-
2 were found exclusively at
the top portion of the gradient, indicating that these mutant proteins
were not associated with ribosomes (Fig. 5). By
comparison, PKR-
K, containing a deletion of the entire kinase
catalytic domain from residue 271 to 551, was located in the ribosomal
portion of the gradient, with about 85% of PKR-
K migrating in the
80 S particle and polysome fractions (Fig. 5A). Wild-type
PKR was predominately found in the fractions containing 40, 60, and 80 S particles (Fig. 4), suggesting that kinase activity may participate
in the ribosomal distribution of PKR. Together, these results indicate
that sequences within the motif 1 and 2 regions are required for
ribosomal association of PKR.
Sequences deleted in the PKR-1 and PKR-
2 mutant proteins are
required to mediate ribosomal targeting. To address whether these
sequences are coincident with the DRBD regions, rather than flanking in
or between these sequences, we characterized two mutant versions of PKR
that each contain a single residue substitution. Both PKR-A68P and
PKR-A158P contain a proline substituted for an alanine residue that is
well conserved among DRBD-containing proteins (1, 2, 4, 19, 34). These
alanine residues are located in the carboxyl-terminal portion of the
DRBD sequences in a region presumed to be
-helical based on the
resolved structures of DRBD sequences from Drosophila
staufen and RNase III from Escherichia coli (1,
4, 61). Green and Mathews (19) showed that both mutant versions of PKR
bind ds-RNA in vitro at 5% or less of the levels measured
for the wild-type DRBD sequences. Cells containing PKR-A68P did not
display the growth defect found in isogenic strains expressing
wild-type PKR, and the PKR-A158P strain showed a partial relief of the
slow growth phenotype (Table I).
Cell lysates containing PKR-A68P were analyzed by sucrose gradient
sedimentation, and the mutant kinase was found free of ribosomes (Fig.
6). PKR-A158P was located in two peaks. The majority of
PKR-A158P was found at the top of the sucrose gradient, and a second
portion of the kinase was located between the free peak and the 40 S
particle (Fig. 6). Analysis of in vitro kinase
phosphorylation of eIF-2 by PKR-A68P and PKR-A1589P mutants revealed
activities slightly elevated compared with wild-type PKR (Fig. 3 and
Table I). These results support the hypothesis that the two DRBD
sequences in PKR directly mediate targeting to the ribosome.
Furthermore, the kinase activity results are consistent with those
previously noted for the PKR-
1 and PKR-
2 (i.e. motif 1 and motif 2 sequences are not essential for PKR catalytic
activity).
Association of DRBD sequences with ds-RNA is proposed to alter the
protein configuration of PKR, leading to induced kinase catalytic
activity. Consistent with this model, motifs 1 and 2 have been shown to
be essential for PKR function in the yeast model system (Table I).
However, mutant versions of PKR containing deletions of DRBD sequences
showed in vitro kinase activities greater than that measured
for wild-type PKR (Fig. 3 and Table I). Given the apparent
contradiction between the in vivo and in vitro
assays, we proposed that in addition to mediating ds-RNA activation of
PKR, the DRBD sequences direct the kinase to a cellular location
important for in vivo phosphorylation of eIF-2. Indeed, characterization of PKR by sucrose gradient sedimentation revealed that
PKR is associated with 40 S ribosomal subunits, and in-frame deletions
or residue substitutions in either of the motif 1 or 2 regions blocked
PKR interaction with ribosomes (Figs. 4, 5, and 6). Our results
indicate that the DRBD-defective mutants are catalytically active but
have impaired in vivo function due to a failure to target
the mutant kinase to the translational machinery. The role of the
ribosomal association in the regulation of PKR may be to provide
enhanced access to its substrate, eIF-2, which associates with 40 S
subunits during the process of translation initiation. In the in
vitro assay, recombinant eIF-2
substrate would be readily
available to the kinase, and thus targeting would not be a prerequisite
for phosphorylation (62).
Deletion of motif 1 or 2 sequences that bind ds-RNA, the
presumed activator in the yeast system, reduced the growth defect associated with PKR. When cell lysates containing these mutant versions
of PKR were assayed for the ability to phosphorylate eIF-2, we found
that the mutant kinases displayed activities that were actually greater
than those measured for wild-type PKR (Table I). A previous report (34)
used the yeast model system and also found that the kinase activity of
immunoprecipitated PKR mutants containing residue substitutions in the
DRBD sequences was similar to wild-type PKR. It was suggested that the
concentration of ds-RNA in the immunoprecipitated kinase preparations
may have been high enough to compensate for the reduced ds-RNA in the
DRBD sequences. Alternatively, it was speculated that binding of PKR to
antibodies may have induced the kinase activity independently of
activating ligand. In the studies presented in this report, PKR
activity was measured directly in lysates, without the aid of
antibodies; therefore, the elevated kinase activity associated with
PKR-
1 and PKR-
2 could not be the result of antibody-directed activation. The possibility that elevated concentrations of ds-RNA may
activate these mutant kinases also seems less likely given that the
kinase activity was measured using cell lysates. Even more
definitively, we found that PKR-
14-257, deleted for both ds-RNA
binding motifs, displayed 9 times the kinase activity measured for
wild-type PKR (Table I).
What role do the DRBD sequences play in the regulation of PKR in the
yeast model system? The dramatic increase in kinase activity measured
for PKR-14-257 compared with wild-type kinase indicates that the
amino-terminal sequences of PKR have a negatively acting effect on PKR.
Binding of ds-RNA to both DRBD regions may release this inhibitory
effect on kinase catalytic activity, resulting in increased PKR
autophosphorylation and phosphorylation of eIF-2
. This proposed
negatively acting function of motif 1 and motif 2 could also be
relieved by directly deleting either DRBD sequences. Consistent with
this model, PKR-
1 or PKR-
2 exhibited eIF-2
kinase activity
slightly higher than wild-type PKR, which is thought to be in the
induced conformation (Table I). The fact that kinase activity of mutant
PKR-
14-257 was 9 times greater than wild-type PKR suggests that the
two DRBD sequences may function coordinately to repress kinase or that
sequences between motif 2 and the protein kinase domain also contribute
in conjunction with DRBD sequences to inhibit PKR.
Several reports using transfected cultured monkey kidney cells have
also discussed the proposal that the DRBD sequences function as
negatively acting regulators of PKR (29, 63, 64). Most recently, Wu and
Kaufman (64), observed that expression of the kinase domain from
residues 228-551 was found to have elevated in vitro PKR
activity. A model was proposed that the dsRNA binding domains inhibit
PKR activity, and dsRNA binding induces a conformation change
facilitating activation kinase function. A point of caution, as
discussed in these cited reports, was the difficulty delinating the
degree to which endogenous wild-type PKR contributed to the measured
kinase activity. Concerning the role of sequences between motif 2 and
the kinase domain in the regulation of PKR, Lee et al. (63)
reported that these sequences, described as the third basic region, or
motif 3, are critical for in vitro kinase activity as
measured by autophosphorylation. We observed that the sequences extending to residue 257 were dispensable for in vitro
kinase activity (Table I). In fact, PKR-14-257 phosphorylated
eIF-2
to levels greatly exceeding wild-type PKR (Fig. 3, Table I). One explanation for this apparent contradiction could be that the PKR
mutant deleted for the third basic region removed residues important
for autophosphorylation and would erroneously indicate a loss of kinase
activity (57). Another important difference between these in
vitro kinase studies is that deletion of the third region
described in Lee et al. (63) extended to residue 271, which
would appear to remove sequences within subdomain I of the PKR kinase
catalytic region (65). These kinase domain sequences are retained in
PKR-
14-257.
Regardless of the molecular rationale for why the PKR
mutants depleted of DRBD sequences are catalytically active, it is
striking that these mutant proteins do not phosphorylate eIF-2 in
yeast (34) or cause the accompanying slow growth phenotype (Table I)
(34). To explain this in vivo deficiency, we proposed that DRBD sequences are required to target PKR to the translation machinery, and this localization step is a prerequisite for phosphorylation of
eIF-2
. Consistent with this hypothesis, we found that about 70% of
PKR migrated with ribosomal particles and polysomes fractionated by
sucrose gradient sedimentation (Fig. 4). When Mg2+ and
cycloheximide were omitted from the experiment, resulting in
dissociation of ribosomes into 40 and 60 S subunits, PKR was almost
entirely associated with 40 S particles. These results indicate that
PKR is a ribosome-associated protein with specificity toward the 40 S
subunit.
It is interesting to note that in the presence of Mg2+ and
cycloheximide, PKR was heavily enriched in the 40, 60, and 80 S
fractions, suggesting that the kinase may be preferentially associated
with ribosomal subunits involved in the process of initiation (Fig. 4).
When PKR-K was similarly analyzed, about 85% of the mutant kinase
was found in the 80 S and polysome fractions of the sucrose gradient,
indicating that the kinase domain does not directly participate in
ribosomal targeting (Fig. 5). However, the fact that PKR-
K mutant is
enriched with polysomes, whereas wild-type kinase is primarily located
in the 40, 60, and 80 S fractions, suggests that kinase activity may
affect the association of PKR for certain ribosomal populations.
Building on the model that wild-type PKR interacts primarily with
initiating ribosomes, the large portion of PKR-
K complexed with
polysomes may indicate that kinase activity facilitates the release of
PKR from ribosomes that are beginning the elongation phase of
translation. This proposal explains why almost no PKR-
K protein was
found in the free peak and an increased portion of kinase mutant
complexed with elongating ribosomes.
What is the nature of the interaction between PKR and ribosomes? Both DRBD sequences are required for kinase association with the translational machinery. This conclusion was most specifically supported by the observation that PKR-A68P and PKR-A158P were blocked in their association with ribosomes (Fig. 6). Both mutants have been reported to be severely reduced in their ability to bind ds-RNA (19). These results suggest that motif 1 and motif 2 interaction with rRNA is central to PKR association with ribosomes. The fact that PKR was bound almost exclusively with 40 S subunits indicates a specificity for rRNA sequences, although protein-protein interactions may also participate in ribosomal targeting.
DRBD Sequences May Carry Out Multiple Functions in the Regulation of PKR ActivityThe DRBD sequences appear to perform a dual
function important for regulating PKR. To control kinase catalytic
activity, motifs 1 and 2 would bind activating ligand, triggering
autophosphorylation of the kinase. As discussed above, stimulation of
kinase activity may involve ligand-induced relief of the negatively
acting DRBD regions. Phosphorylation of PKR is thought to lock the
kinase into a catalytically induced conformation that no longer
requires ds-RNA binding for eIF-2 kinase activity (6, 7, 15). The
high level of PKR activity would be retained until the kinase is
dephosphorylated by protein phosphatases (66, 67). Motif 1 and motif 2 sequences would now be available to target activated PKR to ribosomes,
specifically to 40 S subunits. Localization of the kinase to ribosomes
may be essential for PKR to come into contact with its eIF-2 substrate.
The eIF-2 couples with 40 S ribosomal subunits during translation
initiation. Ramirez et al. (59) have utilized sucrose
gradient methods similar to those described in this report to show
directly that yeast eIF-2
migrates in the ribosome fractions. PKR
recognition and phosphorylation of eIF-2 would reduce the exchange of
GDP-GTP exchange catalyzed by eukaryotic initiation factor-2B, leading
to a reduction in the rate of protein synthesis.
As discussed in the Introduction, expression of certain kinase-inactive PKR mutants in NIH3T3 cells leads to a malignant transformation phenotype (42-44). Two different molecular models have been proposed to explain this trans-dominant inhibition of PKR (8, 24, 28, 29, 34, 42). One model suggests that overexpressed mutant PKR binds all of the available ds-RNA in cells and, thus, prevents ligand activation of the endogenous wild-type kinase. The second model proposes that the kinase-defective mutant forms a heterodimer with wild-type PKR and impedes intermolecular autophosphorylation required for activation of the kinase. Both mechanisms have drawn experimental support. The basis for the apparent contradictions between these studies may be that both mechanisms can contribute to trans-dominant inhibition depending on the level of activating ligand found in the particular cell system.
The important role of ribosome targeting in in vivo
phosphorylation of eIF-2 supports the idea that overexpressed mutant kinase could also reduce the function of endogenous wild-type PKR by
competing for ribosomal binding sites. As noted earlier, the PKR-
K
mutant binds efficiently to the ribosomes and may alter the release of
the kinase from the translation machinery. By preventing the endogenous
wild-type PKR from targeting to ribosomes, the mutant kinase would
block phosphorylation of eIF-2
. Experiments are ongoing to determine
whether co-expression of mutant forms of PKR in yeast alters ribosome
association of wild-type PKR.
In closing, the DRBD sequences of PKR appear to be important for proper localization of this kinase to the ribosomes. Given the large number of different proteins containing DRBD sequences, it is interesting to speculate that many of these sequences carry out a similar ribosomal targeting function. Another likely candidate is the yeast protein encoded by YML3. The YML3 protein contains a single DRBD sequence in its carboxyl terminus, from amino acid residue 315 to 375, and has been shown to be associated with the large ribosomal subunit in mitochondria (68). While initial reports suggested that YML3 encodes the L3 protein of the mitochondrial ribosome (68), more recent work indicates that this is not the case (69). The physiological role of the YML3 protein and the possible contribution of DRBD-mediated ribosomal targeting to this function remain to be determined.
We thank Anna DePaoli-Roach, Alan Hinnebusch, and Peter Roach for helpful comments on this manuscript.