(Received for publication, July 21, 1995; and in revised form, October 13, 1995)
From the
Upon binding to double-stranded (ds) RNA, the dsRNA-dependent
protein kinase (PKR) sequentially undergoes autophosphorylation and
activation. Activated PKR may exist as a dimer and phosphorylates the
eukaryotic translation initiation factor 2 subunit (eIF-2
)
to inhibit polypeptide chain initiation. Transfection of COS-1 cells
with a plasmid cDNA expression vector encoding a marker gene, activates
endogenous PKR, and selectively inhibits translation of the marker
mRNA, dihydrofolate reductase (DHFR). This system was used to study the
dsRNA binding and dimerization requirements for overexpressed PKR
mutants and subdomains to affect DHFR translation. DHFR translation was
rescued by expression of either an ATP hydrolysis defective mutant PKR
K296P, the amino-terminal 1-243 fragment containing two dsRNA
binding motifs, or the isolated first RNA binding motif (amino acids
1-123). Mutation of K64E within the dsRNA binding motif 1
destroyed dsRNA binding and the ability to rescue DHFR translation.
Immunoprecipitation of T7 epitope-tagged PKR derivatives from cell
lysates detected interaction between intact PKR and the amino-terminal
1-243 fragment as well as a 1-243 fragment harboring the
K64E mutation. Expression of adenovirus VAI RNA, a potent inhibitor of
PKR activity, did not disrupt this interaction. In contrast, intact PKR
did not interact with fragments containing the first dsRNA binding
motif (1-123), the second dsRNA binding motif (98-243), or
the isolated PKR kinase catalytic domain (228-551). These results
demonstrate that the translational stimulation mediated by the dominant
negative PKR mutant does not require dimerization, but requires the
ability to bind dsRNA and indicate these mutants act by competition for
binding to activators.
Phosphorylation of translation initiation and elongation factors
is a fundamental mechanism that regulates the rate of protein synthesis
as cells respond to their external environment(1) . The best
well characterized mechanism that regulates the rate of polypeptide
chain initiation is phosphorylation of the subunit of the
translation initiation factor 2 (eIF-2
). (
)eIF-2 is a
heterotrimer of
,
, and
subunits that is essential to
transfer initiator tRNA (Met-tRNAi) in a ternary complex with GTP to
the 40 S ribosomal subunit in the first step of polypeptide chain
initiation. Upon 60 S ribosomal subunit joining, GTP is hydrolyzed and
the eIF-2
GDP complex is released. In order for eIF-2 to promote
another round of initiation, the GDP must be exchanged for GTP. This
reaction is catalyzed by the guanine nucleotide exchange factor
(eIF-2B)(2, 3). Control of eIF-2 utilization is mediated by
phosphorylation of eIF-2
on serine residue 51. Phosphorylated
eIF-2 cannot undergo GDP/GTP exchange and forms a non-dissociable
complex between eIF-2B and eIF-2
GDP(4, 5) .
Since eIF-2B is present at a lower concentration than eIF-2, eIF-2B
becomes sequestered by small increases in eIF-2
phosphorylation
and prevents further initiation events(6) .
Three protein
kinases are known to control protein synthesis through eIF-2
phosphorylation(7, 8, 9) . Most mammalian
cells express the dsRNA-dependent protein kinase (PKR)(10) .
PKR expression is induced by interferon and its activation is dependent
upon dsRNA. Many of the anti-viral activities of interferon are
mediated by PKR. Interferon-resistant viruses have evolved specific
mechanisms to inactivate PKR, such as the RNA polymerase III gene
product VAI RNA from adenovirus(10, 11) .
Overexpression of wild-type PKR inhibits protein synthesis and cell
growth(12, 13, 14, 15) , whereas
overexpression of mutant PKR inhibits endogenous PKR activity and
causes cell transformation(16, 17) .
The primary
structure of PKR deduced from its cDNA sequence (18) identified
the presence of 11 conserved Ser/Thr kinase subdomains(19) .
The NH-terminal 171 amino acids of the protein comprise two
copies of a 67-amino acid RNA-binding motif found in a number of
different RNA-binding proteins such as the TAR RNA-binding protein 1,
vaccinia virus E3L protein, Drosophila staufen protein, Xenopus proteins Xrlbpa and 4F, and Escherichia coli RNase III(20, 21, 22, 23) . The
consensus RNA binding sequence contains a positively charged
-helical region in the COOH-terminal third of the motif. Mutations
of the positively charged residues within this motif destroy RNA
binding(24, 25) .
At present, mutagenesis experiments have identified residues important for RNA binding. Both dsRNA binding domains apparently contribute to the stability of the RNA-protein complex. However, the first domain is more important for binding(21, 22, 24, 25, 26) . In addition, mutation of the conserved lysine 64 to glutamic acid (mutant K64E) significantly reduced RNA binding(21) . The PKR amino acid requirements for dsRNA binding appear to be the same or overlap the requirements for dsRNA-dependent activation, however, very little is known about how dsRNA binding leads to PKR activation. RNA molecules that activate apparently bind the same sites as RNA molecules that inhibit activation(27, 28) . PKR activation requires critical concentrations of dsRNA. High concentrations of dsRNA inhibit PKR activation. dsRNA binding to PKR induces autophosphorylation(29, 30) . PKR is detected in the cytosol as a partially phosphorylated dimer(31) . Although autophosphorylation can occur in trans(32, 33) , it is not known if dimerization is required for dsRNA-dependent activation. Two models proposed to explain the dsRNA dependent activation of PKR differ in their requirement for dimerization. One model proposes that each PKR molecule has one dsRNA binding site and activation results from bridging of two PKR molecules to elicit intermolecular autophosphorylation(32) . Another model proposes that activation involves intramolecular autophosphorylation that is dependent upon proper occupancy of two dsRNA binding sites(34) . Similarly, two models based on whether PKR dimerizes have been proposed to explain the capability of mutant PKR to dominantly down-regulate wild-type PKR. One model proposes that mutant PKR forms mixed heterodimers with the wild-type PKR and prevents intermolecular phosphorylation and activation(16, 35) . The second model proposes that overexpression of the mutant PKR sequesters PKR activators such as specific dsRNA sequences(12, 36) . We have evaluated the validity of these models through analysis of the functional activity and dimerization of PKR mutants and subdomains upon expression in COS-1 cells. Our results support the hypotheses that dsRNA activation does not require dimerization and that mutant PKR acts as a dominant negative through sequestering activators.
Direct studies on the activation of PKR in vivo have been limited due to the inhibition of protein synthesis observed in the presence of active PKR(12, 13, 14, 15, 36) . Transfection of selective plasmids into COS-1 cells activates PKR to selectively inhibit in a cis-acting manner the translation of reporter mRNAs derived from the plasmid DNA(37, 38, 39, 40) . In this study we show that expression of a dominant negative mutant K296P PKR can rescue translation of the reporter mRNA in this system. This system was used to study the PKR structural requirements for PKR dimerization and dsRNA binding to down-regulate endogenous PKR activity. By immunoprecipitation of T7 epitope-tagged proteins transiently expressed in COS-1 cells, we demonstrated that dsRNA-independent dimerization of PKR is mediated through the intact dsRNA binding domain (residues 1-243). In addition, expression of a 1-243 fragment harboring a K64E mutation that is defective in dsRNA binding did not rescue protein synthesis although it dimerized with intact PKR. These results support the hypothesis that the PKR dominant negative phenotype observed by mutant PKR overexpression results from competition for binding to potential dsRNA activators and is not due to formation of inactive heterodimers.
Figure 1:
Expression vectors and
PKR mutants and fragments used in this study. The construction of the
vectors (A) and PKR fragments (B) are described under
``Experimental Procedures.'' The pETFVA expression vector contains the SV40 origin of replication (SV40 ori), the adenovirus major late promoter (AdMLP), the adenovirus tripartite leader (TPL), a
small intron (IVS), a polycloning site for insertion of
foreign DNA, the encephamolomyocarditis internal ribosomal entry site (EMC), tissue factor cDNA (TF), and the SV40 early
polyadenylation signal (SV40 poly(A)). pD61 contains the DHFR
coding sequence and p9A contains the adenosine deaminase coding
sequence. pETFVA
differs from pD61 and p9A in the
plasmid backbone where pUC18 is in place of
pBR322.
At 48 h post-transfection, total RNAs were
prepared using the Trizol method (Life Technologies, Inc.) and analyzed
by RNA Northern blot hybridization (44, 45) using
probes (DHFR, PKR, and -actin) prepared by
[
P]dCTP labeling with random priming (Pharmacia
Inc., Piscataway, NJ).
COS-1 cells were
co-transfected with pD61 and the vector pETFVA containing wild-type, mutant, or fragments of PKR. Since the mRNA
derived from pETFVA
does not activate PKR and is
efficiently translated(39) , it is possible to measure the
ability of the protein expressed from pETFVA
to
rescue translation of DHFR mRNA derived from pD61. DHFR protein
synthesis was quantitated by
[
S]methionine/cysteine pulse labeling of cells
and analyses of harvested cell extracts by SDS-PAGE. Results were
quantitated by PhosphorImage scanning (Table 1A). In parallel
cells were harvested for quantitation of mRNA by Northern blot
hybridization analysis. Transfection of pD61 with pETFVA
vector alone into COS-1 cells detected a low level of DHFR
synthesis (Fig. 2A, lane 2) that was significantly
above the background observed in cells that did not receive pD61 DNA (Fig. 2A, lane 1). Co-transfection of pD61 with
wild-type PKR reduced DHFR synthesis to a non-detectable level above
background (Fig. 2A, lane 3). Co-transfection of pD61
with PKR mutant K296P increased DHFR synthesis by 7.0-fold compared to
co-transfection with the pETFVA
vector alone (Fig. 2A, lanes 2 and 5). Whereas wild-type
PKR synthesis was not detected, K296P PKR synthesis was observed as a
polypeptide migrating at 69 kDa. Co-transfection with the mutant K64E
PKR slightly reduced DHFR synthesis by 10% and K64E mutant PKR
synthesis was significantly reduced compared to the K296P mutant PKR (Fig. 2A, lanes 4 and 5). These results
indicate that K64E mutant PKR displays intermediate activity between
the K296P mutant and wild-type PKR. Co-transfection with KD yielded a
low level of KD synthesis, likely resulting from its constitutive
activation and eIF-2
phosphorylation ((46) ; see below).
Expression of KD inhibited DHFR synthesis by 30% (Fig. 2B,
lane 11). In contrast, expression of either PKR fragments BD or D1
rescued DHFR synthesis (Fig. 2A, lanes 6 and 8), whereas expression of fragment D2 did not alter DHFR
synthesis (Fig. 2A, lane 10). In addition, D1 harboring
the K64E mutation did not rescue DHFR synthesis (Fig. 2A,
lanes 7 and 9). Co-transfection with BD harboring the
K64E mutation increased DHFR synthesis 1.8-fold, possibly due to
residual dsRNA binding of the BD K64E mutant. Northern blot analysis
demonstrated that the DHFR mRNA level did not significantly change upon
co-transfection with wild-type or mutant PKR (Fig. 2B, lanes
2-11), demonstrating the changes in DHFR synthesis were due
to changes in mRNA translational efficiency. The different PKR
fragments were detected as polypeptides migrating at the expected sizes
(identified in Fig. 2A) and were all expressed at high
levels. Northern blot analysis of the PKR mRNA demonstrated the
translational efficiency of the mutant PKRs, for example, BD and D1,
compared to K64E-BD or K64E-D1, were less affected than the
translational efficiencies of DHFR. These results demonstrated that
expression of either BD or D1 could rescue DHFR translation and that
the K64E mutation destroyed this capability.
Figure 2:
Rescue of DHFR translation by expression
of specific mutants and fragments of PKR. Panel A, COS-1 cells
were co-transfected with the DHFR expression vector pD61 in the
presence of the pETFVA expression vector alone (lane 2) or of pETFVA
containing the
indicated PKR inserts depicted in Fig. 1. Lane 1 received only pETFVA
DNA. Equal amounts (4
µg/10-cm plate) of the two plasmid DNAs were used. The transfected
cells were labeled with [
S]methionine/cysteine
and analyzed as described under ``Experimental Procedures.''
The migration of DHFR and the different PKR fragments is indicated. Panel B, total RNA from cells transfected in parallel was
isolated and the mRNA levels for DHFR, PKR, and
-actin were
analyzed by Northern blot hybridization.
Figure 3:
In vivo phosphorylation of
co-expressed eIF-2 subunit. COS-1 cells were co-transfected with
the eIF-2
-subunit expression vector
pETFVA
-2
wt in the presence of pETFVA
vector alone (lane 2) or pETFVA
containing the indicated PKR inserts. Lane 1 represents
cells that did not receive the eIF-2
expression vector. After 48
h, cells were labeled with [
P]phosphoric acid
for 4 h and lysed using Nonidet P-40 buffer. The labeled eIF-2
was
immunoprecipitated with anti-eIF-2
monoclonal antibody. The
immunoprecipitates were analyzed by SDS-PAGE and Western blot analysis
using anti-eIF-2
monoclonal antibody. The same membrane was
autoradiographed to measure the incorporation of
PO
into the eIF-2
subunit.
Figure 4:
Co-immunoprecipitation of intact PKR and
BD with different PKR fragments. Panel A, COS-1 cells were
co-transfected with untagged intact K296P PKR (lanes
1-7) or BD (lanes 8-15) contained in the
pETFVA vector in the presence of T7-tagged versions
of BD (lanes 1 and 8), K64E-BD (lanes 2 and 9), KD (lanes 3 and 10), K296P-KD (lanes
4 and 11), D1 (lanes 5 and 12), K64E-D1 (lanes 6 and 13), or D2 (lanes 7 and 14) contained in the same vector. Co-transfection of untagged
intact K296P PKR with untagged BD is shown in lane 15. Protein
synthesis was analyzed as described under ``Experimental
Procedures.'' Panel B, T7-tagged proteins were
immunoprecipitated using anti-T7 monoclonal antibody and the
immunoprecipitates were analyzed by SDS-PAGE followed by
autoradiography. The identity of the different PKR polypeptides is
indicated on the right. Panel C, cell extracts were
immunoprecipitated with anti-PKR polyclonal antibody and analyzed as
described under ``Experimental
Procedures.''
Immunoprecipitation of the cell extracts with anti-T7 antibody specifically adsorbed each T7-tagged fragment (Fig. 4B, lanes 1-14) that was not detected in the absence of the T7-epitope tag (Fig. 4B, lane 15). Immunoprecipitation of T7-tagged BD yielded the expected polypeptide at 33 kDa as well as some 69-kDa polypeptide that represented the K296P intact PKR (Fig. 4B, lane 1). Immunoprecipitation of T7-tagged BD also co-immunoprecipitated untagged BD in co-transfected cells (Fig. 4B, lane 8). The K64E mutation contained in BD did not reduce the amount of co-immunoprecipitation detected with K296P PKR or BD (Fig. 4B, lanes 2 and 9). In addition, co-immunoprecipitation of K296P PKR or BD was not detected upon co-expression with D1, D2, or KD (Fig. 4B, lanes 3-7 and 10-14).
The amount of dimerization was quantitated by PhosphorImager scanning of the amount of PKR or BD that was co-immunoprecipitated with the T7-tagged protein upon anti-T7 immunoprecipitation (Fig. 4B) and comparing it to the total amount of untagged BD or K64E BD measured by anti-PKR antibody immunoprecipitation (Fig. 4C). The amount of dimerization detected for BD and intact K296P PKR ranged from 5.1 to 11.0% (Table 1B). Similar amounts of dimer were detected for the BD as for the K64E mutant BD. These values were also reproduced in several independent transfection experiments. If the efficiency of heterodimer formation between these species was equivalent to homodimers, then only one-half of the dimers would be detected in this analysis. If one corrects for the total amount of dimer, then 10.2 to 22.0% of the expressed protein would be present as either homodimer or heterodimer. The data suggest that heterodimerization between intact PKR and the RNA binding domain is less efficient homodimerization between the PKR RNA binding domains. At present we do not know the efficiency of homodimerization of intact PKR. The ability of the K64E mutant BD to dimerize is not sufficient to reverse protein synthesis inhibition. The results indicate that PKR dimerization results from protein-protein interactions of the RNA binding domain of PKR. In addition, both dsRNA binding domains were required for dimerization and the dsRNA binding ability of the protein did not affect the dimerization.
Figure 5:
Adenovirus VAI RNA does not inhibit
dimerization. COS-1 cells were co-transfected with the adenovirus VAI
RNA expression vector pVASVOD or control vector pSVOD and the adenosine
deaminase ADA expression vector p9A (lanes 1 and 2)
or the PKR expression vectors encoding intact K296P PKR or the
T7-epitope tagged BD (lanes 3 and 4). At 48 h
post-transfection cells were labeled with
[S]methionine/cysteine, harvested, and analyzed
by SDS-PAGE. Equal amounts of cell extracts were immunoprecipitated
with anti-T7 monoclonal antibody (lanes 5 and 6).
The regulation of PKR activation is a crucial control
mechanism in protein synthesis initiation. Binding of dsRNA to PKR
requires perfect duplexes of 30 base pairs with most efficient binding
and activation occurring with duplexes of 85 base
pairs(27, 28) . However, RNA molecules with extensive
secondary structure such as VAI RNA that do not contain long stretches
of perfect dsRNA duplex do bind efficiently. There are two dsRNA
binding motifs in PKR and the first displays a higher affinity
interaction than the second(24, 25) . Mutations within
a basic -helical region of the COOH-terminal portion of the motif
(for example, K64E) disrupt dsRNA
binding(24, 25, 46, 47) . Although
dsRNA and protein structural requirements for interaction have been
intensively studied, very little is known how this interaction results
in activation of PKR. Two models for the dsRNA dependent activation of
PKR were proposed. The first suggests that dsRNA acts as a bridge to
promote dimerization and subsequent trans autophosphorylation
and activation(32) . This model is based on the second-order
kinetics of activation and that one study revealed only a single dsRNA
binding site(32) . The second model proposes that
autophosphorylation is intramolecular and that the difference between
activators and inhibitors is their ability to productively interact
with the two dsRNA binding motifs to activate autophosphorylation. This
is based on the observation that PKR has two sites of different
affinity for poly(rI):poly(rC)(34) . Both models can account
for the inhibition of activation at high concentrations of dsRNA. We
have addressed the validity of these models by directly measuring PKR
dimerization and functional activation in an in vivo COS-1
cell transfection system.
The requirements for dimerization were studied using mutants and fragments expressed in the presence and absence of a T7-epitope tag to specifically immunoprecipitate one species. The results show that the intact dsRNA binding domain of residues 1-243 could dimerize with itself and with intact K296P mutant PKR. In contrast, BD (residues 1-243) did not interact with fragment D1 (residues 1-123), fragment D2 (residues 98-243), or the intact isolated PKR kinase catalytic domain (residues 228-551). In addition, two methods were used to demonstrate dimerization was independent of dsRNA binding. First, a K64E mutant in BD which was previously shown to disrupt the ability to bind dsRNA (24, 47) did not affect dimerization with the wild-type BD or K296 mutant intact PKR. Since McMillan et al.(47) observed that the K64E mutant has 5% dsRNA binding activity compared to the wild-type, we cannot rule out that dimerization was dependent on a low affinity dsRNA interaction. In addition, we do not know if the dsRNA-independent dimerization observed with the isolated BD extends to intact PKR. As a second method, the presence of VAI RNA, under conditions known to inhibit endogenous PKR activity, did not inhibit dimerization. These results demonstrate that dimerization can be mediated by interactions between intact dsRNA binding domains and that this does not require high affinity dsRNA interactions.
Overexpression of wild-type PKR inhibits cell growth
and inhibits protein synthesis. In addition, translation of PKR itself
is down-regulated and mutation of the kinase activity releases the
translational repression(48) . In contrast, overexpression of
catalytically inactive PKR mutants inhibits endogenous PKR activity,
stimulates protein translation, and causes cell
transformation(16, 17) . Recent observations from
expression of phosphorylation resistant eIF-2 suggest this growth
promoting activity might result from reduced phosphorylation of
eIF-2
(49) . The ability of mutant PKR to inhibit
endogenous PKR activity was proposed to occur by either the ability of
PKR to form inactive heterodimers (16, 35) or by the
ability of mutant PKR to bind and sequester potential
activators(12, 36) . We have provided evidence that
the inhibition of endogenous PKR activity by overexpressed PKR mutant
or fragments is dependent on a functional dsRNA binding site and not
upon the ability to dimerize. This is most strongly supported by two
observations. First, expression of the intact dsRNA binding domain 1
(residues 1-123) stimulated translation from a reporter mRNA but
did not dimerize with PKR. Mutation of K64E within this domain
destroyed the ability to stimulate translation. Second, expression of
the intact dsRNA binding domain fragment 1-243 containing dsRNA
binding motifs 1 and 2 also stimulated translation and was capable of
dimerizing with intact PKR. However, mutation of K64E within motif 1
significantly reduced the ability to stimulate translation without any
effect on the ability to dimerize. The results provide good evidence
that the dominant negative interference by defective PKR occurs
primarily through sequestering dsRNA activators rather than defective
heterodimer formation in this COS-1 cell system and is consistent with
recent observations by Patel et al.(46) . This is in
contrast to reports where amino-terminal deletion (50, 51) and point mutations (47) that disrupt
dsRNA binding can transform NIH 3T3 cells (50, 51) and
inhibit activation of NF
B in response to dsRNA(47) . It is
possible that PKR in these different experimental systems responds to
different activators, such as dsRNA structures or polyanions, or
phosphorylates different substrates to mediate the final response
measured. For example, a PKR mutant that does not bind dsRNA is
activable by heparin(46) . Further studies are required to
determine if multiple mechanisms are responsible for the dominant
interference by mutant PKR molecules.
Our results show that PKR can
dimerize, however, dimerization was not sufficient or essential for
activation. This was most strongly supported by the observation that
VAI RNA inhibited PKR activation but did not affect dimerization. In
addition, expression of the isolated PKR kinase domain induced
eIF-2 phosphorylation and inhibited DHFR translation, although the
expressed protein did not dimerize with intact kinase or with the dsRNA
binding domain. It is unlikely that the endogenous wild-type COS-1 cell
PKR dimerized and/or activated the isolated kinase domain because DHFR
translation was also reduced by co-transfection of the intact kinase
domain in the presence of either the intact dsRNA binding domain or the
K296P mutant PKR. (
)Both of these molecules are known to
inhibit endogenous PKR activity and stimulate DHFR translation. We
propose that the intact kinase domain is a constitutively active kinase
by deletion of the inhibitory dsRNA binding domain. Activation of
intact PKR requires productive interactions with dsRNA to relieve the
inhibition by the dsRNA binding domain. Although dimerization does not
appear to be directly involved in activation of the kinase, it may play
an important role in localizing and/or concentrating PKR at
functionally important locations in the cell.