From the Department of Cancer Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio 44195 and the Department of Genetics, Case Western Reserve University, Cleveland, Ohio 44106
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ABSTRACT |
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The interferon-induced, double-stranded RNA
(dsRNA)-activated protein kinase, PKR, inhibits protein synthesis via
phosphorylation of the alpha subunit of the translation initiation
factor eIF2. A kinase insert region N-terminal of PKR kinase subdomain
V, which is conserved among eIF2 kinases, has been proposed to
determine substrate specificity of these kinases. To investigate the
function of this kinase insert region, selective PKR mutants were
generated, and kinase activities and eIF2
affinities were analyzed
in vitro. The in vivo function was investigated
by growth inhibitory assays in yeast and translational assays in COS
cells. Among the 13 mutations, 5 lost kinase activity and 3 exhibited
less than 30% of wild-type eIF2
binding activity. The deletion of
the conserved sequence (amino acids 362-370) resulted in a protein
that had no kinase activity and only about 25% of wild-type eIF2
binding, suggesting that this sequence is not only required for PKR
kinase activity but also is important for substrate interaction. It was
determined that the hydrophobicity of the conserved sequence of PKR is
required for kinase activity but is not crucial for eIF2
binding.
The amino acid residue Glu-367 in the conserved motif was shown to be
directly involved in substrate binding but was not important for kinase
activity. These results suggest that the activation of PKR is not a
prerequisite for its binding to the substrate and that the conserved
motif in subdomain V contributes to the interaction of PKR and
eIF2
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INTRODUCTION |
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Protein synthesis is divided into three phases: initiation,
elongation, and termination. The reactions in each phase are promoted by soluble protein factors that transiently interact with the ribosome,
mRNA, and aminoacyl-tRNA (1). The phosphorylation of the subunit of translation initiation factor 2 (eIF2
) is a well
characterized translational control mechanism. During the first step of
translation initiation, a ternary complex eIF2-GTP-Met-tRNA is formed
and subsequently binds to the 40 S ribosomal subunit of the ribosomes.
After one round of initiation, eIF2 is released as an eIF2-GDP complex.
To re-form the ternary complex, the GDP bound to eIF2 must be replaced
with GTP, and this reaction is catalyzed by the guanine nucleotide
exchange factor eIF2B. Phosphorylation of eIF2
sequesters the
function of eIF2B, thus blocking eIF2 recycling, and results in the
inhibition of protein synthesis (1).
Three distinct eIF2 kinases have been characterized in eukaryotic
cells, including the hemin-regulated inhibitor
(HRI,1 or HCR), the
double-stranded RNA activated protein kinase (PKR), and the yeast
kinase GCN2 (general control nondepressible). All three kinases
phosphorylate eIF2
at Ser-51 in response to cellular stress
conditions (2, 3). HRI mediates protein inhibition in heme-deficient
reticulocyte lysate and is also activated by heat shock, sulfhydryl
reagents, and heavy metal ions (4). PKR is up-regulated by interferon
and becomes activated upon viral infections that produce dsRNA or
single-stranded RNA with double-stranded secondary structure (5). GCN2
is activated by uncharged tRNA under amino acid starvation conditions.
Phosphorylation of eIF2
by GCN2 mediates gene-specific translational
control of GCN4, a transcriptional activator of amino acid biosynthetic
genes in yeast. GCN2 is required for increased translation of GCN4
mRNA in amino acid-starved cells (3). Sequence analysis shows that PKR, HRI, and GCN2 share sequence and structural features distinct from
other eukaryotic protein kinases. Most notable is the presence of an
insert sequence located between kinase subdomains IV and V, consisting
of 24 amino acid residues for human PKR, 11 amino acid residues for
murine PKR, 128 amino acid residues for HRI, and 112 amino acid
residues for GCN2 (Fig. 1A) (6). Although subdomain V is
less well conserved among eukaryotic protein kinases (6), the eIF2
kinases share significant homology in this domain, suggesting that this
motif might contain a putative substrate-specific recognition domain
(7, 8).
PKR is a serine-threonine kinase that mediates the antiviral and
antiproliferative effects of interferon through phosphorylation of
eIF2 (5). Discovered as an enzyme responsible for the inhibition of
translation in response to dsRNA in reticulocytes (9), PKR has two
distinct kinase activities: autophosphorylation, requiring the presence
of dsRNA, divalent cations (Mn2+ and Mg2+), and
ATP (10, 11), and phosphorylation of exogenous substrates. This second
activity is independent of dsRNA but dependent on ATP and divalent
cations (4). As phosphorylation of eIF2
by PKR provides a mechanism
for cell defense against virus infection, many viruses have evolved
specific mechanisms to inactivate PKR, including degradation of PKR
(12), sequestering of dsRNA activator (13), binding to PKR to inhibit
its activation (14), or activating a cellular PKR inhibitor (15). DNA
transfection of certain plasmids can also activate PKR and result in
poor translation of plasmid-derived mRNAs. Translation can be
restored by coexpression of PKR inhibitors of a mutant form of eIF2
where serine residue 51 has been replaced by alanine (16). Because of
the homology between human and yeast eIF2
, PKR is also able to
phosphorylate yeast eIF2
at Ser-51 and inhibit yeast growth (7).
Human PKR is a 551-amino acid protein consisting of an N-terminal
regulatory domain and a C-terminal catalytic domain (17). The
regulatory domain contains two dsRNA binding motifs that are homologous
to those in other dsRNA-binding proteins (18). Motif I (amino acids
55-75) is essential for dsRNA binding, whereas motif II (amino acids
145-166) plays a less important role (19-21). The catalytic domain
contains 11 conserved kinase subdomains characterizing PKR as a
serine/threonine kinase (17). The invariant lysine (Lys-296) in
subdomain II is essential for the phosphate transfer reaction. Mutation
of this lysine to arginine or proline results in a catalytically
inactive protein (7, 22). As mentioned above, PKR has a 24-amino acid
kinase insert between subdomains IV and V and the highly conserved
eIF2 kinase motif LFIQMEFCD in subdomain V. A full-length PKR
containing a deletion of six amino acids (LFIQME) at this motif was
unable to undergo autophosphorylation or to phosphorylate eIF2 (23),
underlining the importance of this region to PKR activity.
To delineate the individual contribution of conserved individual amino
acids in and around the kinase insert region of PKR to eIF2 binding
and kinase activity, we generated 13 PKR mutants in this region,
analyzed their in vitro auto- and substrate phosphorylation activities and eIF2
affinity, and examined the function of selected mutants in yeast and in mammalian cells. We found that the
hydrophobicity of the conserved motif in subdomain V is essential for
PKR kinase activity but is not necessarily required for the interaction
with eIF2
. We also identified a specific amino acid (Glu-367) that is involved in eIF2
binding but not in PKR auto- and substrate phosphorylation. We conclude that the conserved motif in subdomain V is
required for PKR kinase activity and also contributes to its
interaction with eIF2
.
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EXPERIMENTAL PROCEDURES |
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PKR Mutagenesis and Plasmid Constructions-- PKR mutants were generated as described (24) using the Transformer site-directed mutagenesis kit (CLONTECH). PKR cDNA was cloned into the HindIII and PstI sites of pBluescript KS. After mutagenesis, transformants were selected with ampicillin and confirmed by DNA sequencing.
The bacterial expression vector pGEX-PKR was constructed by digestion of pBS-PKR with HindIII, end-filling of the PKR fragment, and then ligation into the Escherichia coli expression vector pGEX-1 (Pharmacia Biotech Inc.) at the SmaI site. The yeast expression vector pYex-PKR was created by inserting the 1.8-kilobase XbaI-PstI PKR cDNA fragment from pBS-PKR into pEMBLyex4 (40). The mammalian expression vectors were constructed by insertion of PKR/HindIII fragment from pBS-PKR into pRC/CMV (Invitrogen). The mutant yeast eIF2Purification of Bacteria-expressed PKR--
E. coli
BL21(DE3)pLysS cells harboring the pGEX-PKR expression vector were
grown overnight in 50 ml of Luria-Bertani broth containing 50 µg/ml
ampicillin. Following 1:10 dilution in fresh medium, cells were grown
for 2 h to an optical density of approximately 0.9, induced with
0.4 mM isopropyl-1-thio--D-galactopyranoside and incubated for an additional 3-4 h. Bacteria were pelleted and
resuspended in modified NETN (20 mM Tris-HCl, pH7.6, 150 mM NaCl, 1 mM EDTA, 0.2 mM
phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, 10 µg/ml
leupeptin). Cells were lysed by adding Triton X-100 to 1% (w/v) and
sonicating, and the lysate was centrifuged at 27,000 × g at 4 °C for 20 min. The supernatant was incubated with
glutathione-Sepharose beads (Pharmacia) for 30 min at 4 °C with
agitation to isolate the PKR fusion protein. After low speed centrifugation, the bound protein was washed four times with cold modified NETN. Bound protein was eluted with elution buffer (50 mM Tris, pH 8.0, 1 mM EDTA, 10 µg/ml reduced
glutathione).
In Vitro Phosphorylation Assays--
The PKR autophosphorylation
assay was performed as follows. PKR protein (~100 ng) was brought up
to a volume of 15 µl in buffer DBGA (10 mM Tris-HCl, pH
7.6, 50 mM KCl, 2 mM magnesium acetate, 20%
glycerol, 7 mM 2-mercaptoethanol) before addition of 10 µl of DBGB (DBGA containing 2.5 mM MnCl2) and
5 µCi of [-32P]ATP (50 Ci/mmol) in a final volume of
30 µl. The reactions were incubated at room temperature for 20 min
before the addition of SDS loading buffer and boiled for 3 min prior to
loading on 10% SDS-PAGE gels and autoradiography. For the eIF2
phosphorylation assay, 200 ng of eIF2
was added into the above
kinase reaction.
PKR-eIF2 Binding Assays--
To produce recombinant
histidine-tagged eIF2
, an overnight culture of pQE-eIF2
containing E. coli M15[pREP4] cells (QIAGEN) was diluted
1:10 in fresh medium and grown for 1 h at 37 °C.
Isopropyl-1-thio-
-D-galactopyranoside was added to a
final concentration of 1 mM, and the culture was incubated
for an additional 2 h at 37 °C prior to harvesting by centrifugation. The cells were resuspend in phosphate-buffered saline
(137 mM NaCl, 2.7 mM KCl, 10 mM
Na2HPO4, 1.84 mM
KH2PO4, pH 7.4) containing 0.1% (v/v) Triton X-100 and
10% (v/v) glycerol, lysed by sonication for 3 × 20 s, and
centrifuged. The supernatant was aliquoted and stored at
80 °C.
Expression of the Human PKR in Saccharomyces cerevisiae-- Expression plasmids were introduced into the haploid yeast strain, W303-la (MATa, can1-100, his3-11, 15, leu2-3, 112, trp1-1, ura3-1, ade2-1) using the lithium acetate procedure (25). Transformed yeast cells were grown to stationary phase in synthetic medium (26) containing 2% glucose and lacking uracil or leucine. 10 µl of culture was dropped on agar containing medium lacking uracil or leucine with 2% glucose or 2% galactose as carbon sources, and yeast growth on the agar plates was monitored.
PKR Expression in Mammalian Cells-- PKR mutants were subcloned into the mammalian expression vector pRc/CMV. COS-1 cells were transiently transfected with 400 ng of pRc-PKR and 2 µg of reporter plasmid pGL2-Control (Promega) by the lipofectamine procedure (Life Technologies, Inc.). Cells were harvested 48 h after transfection and assayed for luciferase activity after normalizing for transfection efficiency by measuring the total protein (27).
Immunopurification of PKR from COS-1 Cells-- COS-1 cells transiently transfected with PKR were harvested 48 h after transfection, and the cells lysed in buffer I (400 mM NaCl, 50 mM KCl, 20 mM Tris-HCl, pH 7.6, 1 mM EDTA, 1 mM dithiothreitol, 100 µg/ml aprotinin, 20% (v/v) glycerol, 1% (v/v) Triton X-100). PKR was immunoprecipitated from lysate containing 2 mg of total protein using anti-PKR monoclonal antibody (28) and detected by Western blot with anti-PKR polyclonal antibody.
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RESULTS |
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Site-directed Mutation of PKR--
The eIF2 kinases (HRI, GCN2,
and PKR) are distinguished in the family of the serine/threonine
protein kinases by possessing a kinase insert between subdomains IV and
V. Sequence alignment shows that the amino acid residues flanking the
insert regions are highly conserved, including two amino acids in
subdomain IV (Tyr-232 and Trp-237 in PKR) and a nine-amino acid motif
in subdomain V (LFIQMEFCD) (Fig.
1A). It has been proposed that
these amino acids may contribute to substrate recognition (7, 8). To investigate the function of these residues, we designed and constructed 13 mutated forms of PKR, including a deletion of the conserved motif
(M12), a deletion of the subdomain V together with part of the kinase
insert (M13), and single point mutations of different conserved
residues (Fig. 1B). Site-directed mutagenesis was performed in pBluescript, confirmed by sequencing, and the mutated cDNAs were
subcloned into expression vectors for in vitro and in
vivo functional analysis.
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Expression and Kinase Analysis of PKR Mutants in Vitro-- Wild-type and PKR mutant cDNAs were expressed in E. coli as GST fusion proteins in the bacterial expression vector pGEX (Fig. 2). The GST fusion proteins were purified by glutathione-Sepharose affinity chromatography, and their expression was monitored by SDS-PAGE with Coomassie Blue staining (data not shown). All mutants were well expressed except M5, which may be intrinsically unstable. The GST fusion proteins showed different mobilities on SDS-PAGE, which were likely due to different phosphorylation levels since recombinant PKR can be partially phosphorylated in bacteria (29).
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Analysis of eIF2 Binding--
To study the interaction of PKR
with eIF2
, the bacterially expressed GST-PKR proteins were reacted
with histidine-tagged eIF2
. Glutathione-Sepharose beads were used to
immobilize the GST-PKR/eIF2
complex, and PKR and eIF2
were
detected in Western blots probed with monoclonal antibodies against PKR
and eIF2
, respectively (Fig. 2B). The interaction between
PKR and eIF2
was specific because GST alone was not able to bind
histidine-tagged eIF2
(Fig. 2B, lower panel),
and wild-type GST-PKR was not able to bind an unrelated
histidine-tagged protein His-WT1 (data not shown). All the PKR mutants
were able to bind to eIF2
but with different affinities (Fig.
2B). The relative affinity of different mutants for eIF2
were calculated by counting the ratios of how much eIF2
bound to
certain amounts of PKR (Fig. 2D). Interestingly, there was
no correlation between the affinity of the PKR mutants for eIF2
and
their kinase activities (Fig. 2, C and D). For
example, M4 has no kinase activity yet has almost twice the affinity of wild-type PKR for eIF2
. In contrast, mutant M11 has only 20% of
wild-type affinity for eIF2
but retains 90% of wild-type kinase activity. Furthermore, M11 can phosphorylate eIF2
very well, suggesting that the high affinity of PKR for eIF2
might not be a
prerequisite for PKR function.
Effects of PKR Mutants on Yeast Cell Growth--
To analyze the
activities of the PKR mutants in vivo, we expressed PKR
mutants in yeast cells (7, 24, 30). There is 80% homology between
yeast and human eIF2 when conservative substitutions are considered
(31), and yeast eIF2
can be phosphorylated by human PKR, leading to
growth inhibition (7). Accordingly, the different PKR mutants were
subcloned into the vector pEMBLyex4 and transformed into S. cerevisiae strain W303a. PKR protein expression was controlled via
the GAL10-Cyc1 hybrid promoter, whereby growth on galactose as the sole
carbon source activates expression. In glucose medium, yeast
transformed with M1, M2, M6, M7, or wild-type PKR grew very slowly
(Fig. 3A). This was likely
caused by a low basal level expression of PKR. When PKR expression was
induced in galactose medium (Fig. 3B), the growth of yeast
transformed with M1, M2, M4, M6, M7, M9, M10, M11, and wild-type PKR
was completely inhibited. Significantly, all of these mutants showed
kinase activities in vitro except M4 (Fig. 2A).
Because M4 has a high affinity for eIF2
in vitro (Fig.
2), we speculated that the yeast slow growth phenotype might be caused
by the direct binding of M4 to eIF2
. To prove this hypothesis, we
co-expressed M4 with sui2M, a yeast eIF2
allele that contains a
mutation (Ser-51
Ala) that completely abolishes phosphorylation by
PKR. We have previously shown that sui2M is able to rescue the slow
growth phenotype when co-expressed with wild-type PKR (7). We found
that sui2M also reverted the slow-growth phenotype caused by M4 (Fig.
4B). This suggests that M4
inhibits yeast growth by direct interaction with eIF2
independent of
auto- or substrate phosphorylation.
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Expression of PKR Mutants in Mammalian Cells--
Transient
transfection of COS-1 cells with expression plasmids encoding a
reporter gene such as DHFR has been shown to result in inefficient
translation due to PKR activation and eIF2 phosphorylation (16). A
variety of agents can stimulate the synthesis of the reporter protein,
including vaccinia virus gene products E3L and K3L, the catalytically
inactive PKR mutant K296R, and the dsRNA binding-deficient mutant K64E
(32, 33). The luciferase reporter plasmid pGL2-control was also
successfully used in this system (27). Therefore, we used the COS-1
expression system to analyze the function of some of our PKR mutants in
mammalian cells. Three PKR mutants (M3, M4, M11) together with
wild-type PKR and the K296R mutant were subcloned into the mammalian
expression vector pRc/CMV and transiently transfected into COS-1 cells
together with the luciferase reporter vector. Transfection of wild-type PKR almost completely inhibited luciferase translation, whereas the
K296R mutant had a dominant-negative effect, resulting in enhanced
luciferase activity (Fig. 5A).
Mutant M3, which has very little autophosphorylation activity and no
eIF2
phosphorylation activity in vitro, was not able to
inhibit translation in COS cells, consistent with its function in
yeast. However, mutant M4, which inhibited yeast growth, also enhanced
luciferase activity, suggesting that it is inactive in COS cells. As
with wild-type PKR, mutant M11 inhibited luciferase gene translation,
consistent with its function in yeast. As shown previously, although
M11 has a low eIF2
affinity for eIF2
, it can phosphorylate
eIF2
almost as well as wild-type PKR in vitro (Fig. 2).
In vivo experiments demonstrate that M11 is fully functional
in both yeast and COS cells.
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DISCUSSION |
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The anti-viral effect of PKR is an important mechanism for host
defense against viral infection and is mediated through the phosphorylation of eIF2 and inhibition of viral protein synthesis. In addition, PKR has been shown to have an antiproliferative function and to regulate cell growth (7, 23, 35), which is at least in part
linked to eIF2
phosphorylation. In this study, we have examined the
effects of mutations of the conserved amino acids around the PKR kinase
insert region in vitro and in vivo. By using bacterially expressed PKR and eIF2
, we were able to study their direct interaction in vitro. We found that amino acids in
the conserved sequence LFIQMEFCD (amino acids 362-370) in subdomain V
are important for PKR kinase activity, as the single point mutations L362Q (M3), I364N (M4), and F363S (M8) resulted in a loss of both kinase activity in vitro and growth and translation
suppression function in vivo. However, these amino acids may
not be directly involved in substrate recognition because all mutants
were able to bind to eIF2
, including the two deletion mutants,
although some mutants bound eIF2
at a relatively low level compared
with wild-type PKR. For example, M11 (E367R), which has kinase activity and is fully functional in vivo, showed only about 20% of
normal (wild-type) eIF2
binding activity.
Because of the homologous nature of protein kinase domains, they all
fold into topologically similar three-dimensional core structures and
carry out phosphotransfer by a common mechanism (6). The crystal
structures of more than ten eukaryotic protein kinases have been
reported, and their kinase domains all fold into a two-lobed structure
(6). The smaller lobe contains subdomains I-IV, and the larger lobe
includes subdomains VI-XI. Subdomain V residues link the two lobes.
The catalytic domain of PKR may also have the same general structure.
According to sequence alignment with PKA-C, the N-terminal part of
the conserved motif (LFIQM) in PKR kinase subdomain V is predicted to
form a very hydrophobic
-strand (
5) in the small lobe (Fig.
1A), and the hydrophobicity of these residues may be
important for the maintenance of the two-lobed structure. This
prediction is supported by our results showing that the deletion of
this region (M12, M13) along with single point mutations that replace
hydrophobic with hydrophilic residues (M3-L362Q, M4-I364N, M8-F363S)
result in a loss of kinase activity. The C-terminal amino acids of the
conserved motif (EFCD) form part of the extended chain that connects
the two lobes. In PKA, Glu-127 on the extended chain participates in
peptide binding by forming an ion pair with an arginine in the
pseudosubstrate site of the PKA inhibitor peptide (39). The
phosphorylation site in eIF2
also contains basic residues C-terminal
to the serine phosphorylation site
(Glu-Leu-Ser(P)-Arg-Arg) (2). PKR has no residues
corresponding to PKA Glu-127 but has two acidic amino acids a few
residues upstream, Glu-367 and Asp-370 (Fig. 1). However, the single
point mutants M10 (E367Q) and M11 (E367R) have nearly full kinase
activity, suggesting that Glu-367 has little effect on kinase
structure. Nevertheless, in GST-pull down assays, M10 exhibits 50%
eIF2
binding compared with wild-type PKR, and M11 has only 20%
eIF2
binding activity, suggesting that Glu-367 may be involved in
substrate anchoring through an ion pair with a basic residue in the
substrate, possibly an arginine. This residue may not be crucial for
substrate recognition, however, because the PKR mutants are still able
to bind eIF2
, and more important, they are fully functional in
vivo (Figs. 3 and 5). Asp-370 may also contribute to eIF2
binding, but this remains to be tested by mutagenesis.
We have found that there is a contradiction in M4 function in yeast and
in COS-1 cells. M4 is able to inhibit yeast growth but does not inhibit
translation in COS-1 cells. The mechanism by which M4 inhibits yeast
growth is unclear, although our data suggest that it may result from an
interaction with yeast eIF2. In yeast, the level of phosphorylation
of eIF2 by GCN2 during amino acid starvation is regulated precisely to
lower the efficiency of reinitiation without affecting primary
translation (35). It has been shown that low level expression of the
mammalian kinases (PKR or HRI) or activation of GCN2 under amino acid
starvation conditions leads to increased GCN4 expression, whereas yeast
cells continue to grow and divide (36). In contrast, high level
expression of the mammalian kinases or genetic activation of GCN2
severely reduces the growth rate of yeast cells (37, 38). In our yeast system, GCN4 expression or low level eIF2
phosphorylation may be
required for yeast growth because we used synthetic medium with only
essential amino acids, requiring that the yeast synthesize nonessential
amino acids. When M4 overexpression is induced by galactose, it might
sequester eIF2
from phosphorylation by GCN2 due to its high affinity
for eIF2
, thus blocking the induction of GCN4 expression and
inhibiting yeast growth. The reversal of this effect by the eIF2
mutant sui2M could then be explained by competition with endogenous
eIF2
for binding to M4, freeing eIF2
to release the suppression
of GCN4 expression. The mechanism of action of M4 in COS-1 cells is
likely independent of eIF2
binding and may involve the formation of
inactive heterodimers with endogenous PKR.
Finally, our data show that phosphorylation of PKR is not required for
binding to its substrate, eIF2. The catalytically inactive mutant
K296R has no kinase activity but is able to bind eIF2
. Moreover,
mutant M4 can bind eIF2
more readily than wild-type PKR. This
increased affinity of M4 for eIF2
may result from a conformational
change due to the replacement of the hydrophobic isoleucine residue
with a hydrophilic asparagine. In any case, high affinity of the PKR
mutants for eIF2
is not required for kinase function. M11 has only
20% of eIF2
binding activity versus wild-type PKR but is
fully functional in vitro and in vivo. Mutant M1
has less than half of the normal eIF2
binding activity but has a
higher kinase activity in vitro than wild-type PKR (Fig. 2).
The identification of the exact eIF2 recognition sites within PKR
requires further experimentation. However, as the substrate binding
sites may not be contiguous in primary sequence but scattered through
the catalytic domain and close in the ternary structure, it will be
essential to solve the structure of PKR to address this question
directly.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant AI34039-02 (to B. R. G. W.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Cancer
Biology, Lerner Research Institute, Cleveland Clinic Foundation, 9500 Euclid Ave., Cleveland, OH 44195. Tel.: 216-445-9652; Fax: 216-445-6269; E-mail: williab{at}cesmtp.ccf.org.
1 The abbreviations used are: HRI, hemin-regulated inhibitor; PKR, dsRNA-activated protein kinase; PAGE, polyacrylamide gel electrophoresis; GST, glutathione S-transferase; dsRNA, double-stranded RNA.
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REFERENCES |
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