(Received for publication, September 5, 1995; and in revised form, November 28, 1995)
From the
Regulation of protein synthesis by eukaryotic initiation
factor-2 (eIF-2
) phosphorylation is a highly conserved
phenomenon in eukaryotes that occurs in response to various stress
conditions. Protein kinases capable of phosphorylating eIF-2
have
been characterized from mammals and yeast. However, the phenomenon of
eIF2-
-mediated regulation of protein synthesis and the presence of
an eIF-2
kinase has not been demonstrated in higher plants. We
show that plant eIF-2
(peIF-2
) and mammalian eIF-2
(meIF-2
) are phosphorylated similarly by both the double-stranded
RNA-binding kinase, pPKR, present in plant ribosome salt wash fractions
and the meIF-2
kinase, PKR. By several criteria, phosphorylation
of peIF-2
is directly correlated with pPKR protein and
autophosphorylation levels. Significantly, pPKR is capable of
specifically phosphorylating Ser
in a synthetic eIF-2
peptide, a key characteristic of the eIF-2
kinase family. Taken
together, these data support the concept that pPKR is a member of the
eIF-2
kinase family. In addition, the inhibition of brome mosaic
virus RNA in vitro translation in wheat germ lysates by the
addition of double-stranded RNA, phosphorylated peIF-2
,
meIF-2
, or activated human PKR suggests that plant protein
synthesis may be regulated via phosphorylation of eIF-2
.
The initiation phase in eukaryotic protein synthesis is
characterized by complex interactions between numerous initiation
factors, ribosomal subunits, nucleotides, and
Met-tRNA. Framework events in
initiation-reinitiation involve ternary complex
(eIF-2
GTP
tRNA
) binding to
free 40 S ribosomal subunits to form a 48 S preinitiation complex. The
subsequent binding of mRNA and 60 S ribosomal subunits is dependent
upon GTP hydrolysis, yielding a viable initiation complex and releasing
eIF-2
GDP(
)(1, 2) . In order for
another round of initiation to begin, GDP must be exchanged with GTP.
This guanine nucleotide exchange is catalyzed by eIF-2B. It is
generally believed that phosphorylation of the
subunit of eIF-2
on Ser
stabilizes the eIF-2
GDP
eIF-2B complex,
effectively sequestering eIF-2B. This elegant regulatory mechanism is
induced by various stresses including virus infection(3) , heat
shock (4) , and deprivation of amino acids (5) or
heme(6) .
The eIF-2 kinases comprise a specific protein
kinase subfamily(7) . They include the heme-deficient kinase,
HRI(6, 8) ; the interferon-induced, double-stranded
RNA (dsRNA)dependent kinase, mPKR(9, 10) ; and yeast
GCN2(11, 12) . Although their noncatalytic regions
differ significantly, all contain inviolate amino acid domains
associated with Ser/Thr kinase activity and eIF-2
phosphorylation (7) . Collectively, the mammalian interferon-induced
dsRNA-dependent protein kinases are termed PKR or mPKR. Low levels
(1-10 µg/ml) of dsRNA or appropriately structured
single-stranded RNA (ssRNA) bind to and activate mPKR via
autophosphorylation, whereas higher levels have an inhibitory effect (7, 13) . Recently a plant-encoded analog of mPKR has
been identified and characterized from monocot and dicot
tissues(14, 15) . Plant PKR, pPKR, is an M
68,000-70,000 Ser/Thr kinase present in
both cytosolic and ribosome-associated fractions. Although it is
constitutively expressed in healthy cells, pPKR protein and
phosphorylation levels are significantly stimulated by dsRNA, select
polyanions, or virus/viroid infection(16, 17) . (
)
The regulation of plant protein translation by
eIF-2 phosphorylation has not been demonstrated. Indeed, there is
considerable confusion in the literature regarding the phosphorylation
of peIF-2, in large part due to discrepancies in subunit
identification(18, 19, 20, 21) .
meIF-2 is composed of three subunits, the M
36,000
subunit, the M
38,000
subunit, and the M
52,000
subunit(22) . Plant eIF-2
(peIF-2) has been purified and is also composed of three subunits of M
38,000, M
42,000 (doublet),
and M
50,000(18, 19, 20, 23, 24, 25, 26) .
Several studies have indicated that kinases that phosphorylate the
-subunit of meIF-2 also phosphorylate the M
42,000 subunit of peIF-2, although often this subunit has been
designated as
eIF-2
(18, 19, 21, 27) . It has
not been conclusively shown that phosphorylation of the M
42,000 subunit affects translational activity in
wheat germ systems. Previous failure to detect an effect of
phosphorylation in vitro(28) or in
vivo(29) , the lower dissociation constant of eIF-2 for
GDP in plants (20) compared with mammals, and the apparent
absence of plant recycling factor activity (eIF-2B) have led to the
general assumption that plants do not use phosphorylation of eIF-2
as a means to regulate protein translation(20) . However, the
wheat germ M
42,000 subunit has now been
conclusively identified as the
-subunit and the M
38,000 as the
-subunit by molecular cloning and cDNA
sequencing. (
)Further, Arabidopsis
thaliana-expressed sequence tags (EST, GenBank
accession numbers T45955 and T22066) that encode functional
equivalents of meIF-2
contain the inviolate phosphorylation sites
corresponding to Ser
. In addition, an A. thaliana EST (GenBank
accession number T44879) and a rice EST
(GenBank
accession number D25052) appear to encode the
functional equivalents of yeast GCN3 and GCD2, respectively, that are
subunits of recycling factor eIF-2B. Thus, it appears highly likely
that plants contain the necessary factors for an eIF-2
phosphorylation pathway.
Here we show that the plant-encoded pPKR is
capable of phosphorylating the subunit of both plant and
mammalian eIF-2. In addition, in vitro protein synthesis in
wheat germ lysates is specifically inhibited by phosphorylated eIF-2,
dsRNA, an inducer of pPKR activity, or activated mPKR.
Barley (Hordeum
vulgare L. ``Steptoe'') was grown in the dark for 7 days
at 25 °C. Leaves were homogenized in 150 mM PIPES, pH 7.5,
50 mM EDTA, 5 mM dithiothreitol, 100 mM KCl,
1 µg/ml aprotinin, 1 µg/ml leupeptin, and 2 mM phenylmethylsulfonyl fluoride. Raw wheat germ was purchased
locally. Plant extracts were prepared by brief homogenization at 4
°C in a Waring blendor followed by filtering through four layers of
cheesecloth. Following centrifugation at 30,000 g for
30 min, the supernatant was used to prepare S-100 (cytosolic) and
ribosomal salt wash fractions(15) . Fractions were dialyzed
overnight in 20 mM HEPES, pH 7.5, 100 mM NaCl, 5
mM dithiothreitol, and 1 mM phenylmethylsulfonyl
fluoride. Extracts from HeLa cells were prepared as described
previously(34) . Ribosome salt wash-associated mammalian PKR
was partially purified using previously described fast protein liquid
chromatography Mono S and Mono Q chromatographic methods(34) .
In vitro translation assays to
evaluate the effect of phosphorylated eIF-2 on protein synthesis were
modified as follows: mPKR or buffer (20 mM HEPES, pH 7.5, 10%
glycerol, 100 mM KCl, 5 mM MnCl, 5 mM MgOAc, 14 mM
-mercaptoethanol) was incubated with
poly(rI)
poly(rC)-agarose and thoroughly washed in 1 M KCl buffer followed by a 10 mM KCl wash (31) prior to the addition of phosphorylation mix alone or
plant or mammalian eIF-2 (3 µg) in phosphorylation mix.
Phosphorylation mix contained 100 µM unlabeled ATP, 5
mM MnCl
, 5 mM MgOAc, 20 mM HEPES, pH 7.5, and 10 mM KCl. After incubation for 20 min
at 30 °C, the resin containing the bound mPKR or the resin
incubated with buffer was pelleted, and the supernatant was removed and
brought to 10 mM EDTA. This solution was added to a wheat germ
translation mix manufactured by Ambion, and BMV RNA translation assays
were performed according to the manufacturer's directions.
Specific kinases present in animals and yeast phosphorylate
eIF-2, resulting in translational regulation(3) . A
corresponding eIF-2
phosphorylation pathway has not been
identified in plants, although a plant analog of the mammalian
eIF-2
kinase, PKR, has been
characterized(14, 15, 16) . Fig. 1A demonstrates that a kinase present in the ribosomal
salt wash fractions from barley leaves is capable of specific in vitro phosphorylation of the M
36,000
subunit
of meIF-2 and the M
42,000 doublet of peIF-2, now
termed peIF-2
(lanes B and C, respectively).
Phosphorylation of peIF-2
by the plant kinase appears better than
phosphorylation of meIF-2
, given that equal protein amounts were
used. Purified human PKR likewise phosphorylates meIF-2
and
peIF-2
(Fig. 1B, lanes B and C) (36) . These results suggest that peIF-2
may contain a
domain similar to the phosphorylation domain of meIF-2
. Supporting
this conclusion is the existence of an Arabidopsis EST
(T45955) having 83% identity at the amino acid level with the
phosphorylation domain of human eIF-2
corresponding to residues
45-56. Further this plant EST has 100% identity with a domain
(residues 72-90) that is highly conserved between eIF-2
and
K3L. K3L is a vaccinia virus-encoded protein that is a competitive
inhibitor of PKR-mediated phosphorylation of eIF-2
(37) .
The labeled bands at M
48,000 and 55,000 in lane A are degradation products of pPKR based upon peptide
mapping (data not shown), and it is likely that proteolytic activity
not controllable by the inhibitors used in extract preparation is also
responsible for the apparent degradation in the eIF-2 preparations
observed in Fig. 1A, lanes B and C.
Figure 1:
Phosphorylation of
plant and mammalian eIF-2. Panel A, ribosome salt wash
fractions were prepared from barley tissue followed by incubation with
poly(rI)poly(rC)-agarose and in vitro phosphorylation in
the presence of
P-ATP. Bound proteins were eluted,
separated by SDS-PAGE, and visualized by autoradiography. In lane
A, there were no further additions. In lanes B and C, 3 µg of mammalian eIF-2 and plant eIF-2, respectively,
were added prior to the addition of
[
-
P]ATP. Panel B, similar assays
to those in panel A were performed, only the source of kinase
was partially purified mPKR from HeLa cells. The positions of molecular
mass markers (in kDa), pPKR, mPKR, peIF-2, and meIF-2 are
shown.
The fact that plant kinase fractions were incubated with
dsRNA-agarose and thoroughly washed prior to in vitro phosphorylation assays suggests that the responsible kinase is a
dsRNA binding protein. In order to establish the dsRNAdependent
phosphorylation of peIF-2 and meIF-2
, excess soluble dsRNA
was added to plant extracts prior to binding to dsRNA-agarose.
Phosphorylation of peIF-2
and meIF-2
was specifically
decreased in the presence of competitor dsRNA (Fig. 2, lanes
B and F, respectively). The only dsRNA-binding protein
kinase yet identified from plants is pPKR(17) . As determined
by immunoblotting according to (15) , dsRNA competition
decreased pPKR protein levels binding to dsRNA-agarose (data not
shown). This decrease in pPKR protein corresponded to a decrease in
phosphorylation of the M
68,000-70,000 pPKR (lanes B and F) and was directly correlated with
eIF-2
phosphorylation levels. Preincubation with 500 µg of
soluble ssRNA as poly(rA) or poly(rU) had no effect on the
phosphorylation levels of either pPKR or eIF-2
(data not shown).
Similarly, dsRNA competition specifically decreased mPKR protein
binding to dsRNA-agarose, resulting in decreased phosphorylation of
mPKR, peIF-2
(lane D) and meIF-2
(lane H).
These data are consistent with the demonstration that mPKR binding of
dsRNA induces two phosphorylation activities, autophosphorylation and
eIF-2
phosphorylation(7, 13) , and indicate a
similar mechanism for pPKR. Several phosphorylated bands are noted in Fig. 2that do not decrease with dsRNA competition, suggesting
that a contaminating dsRNA-independent kinase may be present in the
plant extract. Therefore, the role of pPKR in eIF-2
phosphorylation was further evaluated by immunoclearing experiments.
Ribosome salt wash extracts from barley leaves were incubated with
preimmune serum or anti-PKR sera (consisting of a mixture of anti-pPKR
and anti-dsRNA domain polyclonal sera and monoclonal antiserum to mPKR)
followed by immunoprecipitation. Supernatants were then subjected to
dsRNA-agarose purification and in vitro phosphorylation in the
presence or absence of peIF-2 or meIF-2. pPKR protein levels (data not
shown) and pPKR phosphorylation levels decreased in pPKR-immunocleared
extracts compared with fractions incubated with preimmune serum (Fig. 3, compare lanes B and C). The observed
decrease in both peIF-2
and meIF-2
phosphorylation levels is
directly correlated with decreased pPKR levels from immunoclearing (lanes B and C).
Figure 2:
Phosphorylation of eIF-2 by a
dsRNA-binding protein kinase. Barley ribosome salt wash fractions (lanes A, B, E, and F) or partially
purified HeLa mPKR (lanes C, D, G, and H) were incubated with poly(rI)
poly(rC)-agarose followed
by the addition of [
-
P]ATP and plant eIF-2 (lanes A-D) or mammalian eIF-2 (lanes
E-H). In lanes B, D, F, and H, extracts were incubated with 500 µg of soluble
poly(rI)
poly(rC) prior to incubation with
poly(rI)
poly(rC)-agarose. Radiolabeled proteins were eluted,
separated by SDS-PAGE, and visualized by autoradiography. The positions
of molecular mass markers (in kDa), PKR, peIF-2, and meIF-2 are
shown.
Figure 3:
Decreased eIF-2 phosphorylation in
pPKR immunocleared extracts. Barley ribosome salt wash fractions (lanes B and C) were incubated with preimmune serum (lane B) or anti-PKR sera (lane C). S. aureus cells were then added, and incubation continued. The bacterial
cells were pelleted, and the supernatant was used in standard
poly(rI)
poly(rC)-agarose binding assays. After thorough washing
of the resin, [
-
P]ATP and plant eIF-2 (panel A) or mammalian eIF-2 (panel B) were added. In lane A, partially purified HeLa mPKR was incubated with
poly(rI)
poly(rC)-agarose followed by the addition of
[
-
P]ATP and plant eIF-2 (panel A)
or mammalian eIF-2 (panel B). Radiolabeled proteins were
eluted, separated by SDS-PAGE, and visualized by autoradiography. The
position of molecular mass markers (in kDa), PKR, and eIF-2 are
shown.
To further define the role of
pPKR in eIF-2 phosphorylation, dsRNA-agarose-purified ribosomal
salt wash fractions from barley containing pPKR were incubated with
2-aminopurine, a potent inhibitor of PKR(38) . The inhibition
of meIF-2
and peIF-2
phosphorylation by barley and HeLa cell
ribosomal salt wash fractions in the presence of 2-aminopurine was
directly correlated with inhibition of pPKR and mPKR phosphorylation
(data not shown).
The eIF-2 kinases specifically phosphorylate
Ser
of meIF-2
(1, 2, 7) . To
confirm pPKR as a member of the eIF-2
kinase family, the ability
of pPKR to specifically phosphorylate Ser
of eIF-2
was evaluated in in vitro phosphorylation reactions using a
synthetic eIF-2
peptide substrate. A peptide substrate with the
sequence ILLSELSRRRIR corresponding to residues 45-56 of
meIF-2
(33) was synthesized and subjected to in vitro phosphorylation in the presence of either purified mPKR or
partially purified pPKR. Mellor and Proud (33) reported that
only eIF-2
kinases are capable of phosphorylating the residue
corresponding to Ser
in this peptide in the absence of
Ca
and phosphatidylserine. Both mPKR and a
dsRNA-binding kinase present in the partially purified plant extract
from barley tissues phosphorylated the peptide substrate (Fig. 4A, lanes A and C, respectively). The M
68,000-70,000 pPKR band was visible in lane C only after prolonged exposure. The level of peptide
phosphorylation decreased when purified mPKR or plant extracts were
incubated with soluble dsRNA (lanes B and D) but not
ssRNA (lanes A and C) prior to dsRNA-agarose binding
and in vitro phosphorylation. The identity of pPKR as the
kinase responsible for peptide phosphorylation was also indicated by
immunoclearing experiments. Immunoabsorption of plant extracts with
specific anti-pPKR sera prior to dsRNA-agarose binding resulted in
decreased pPKR phosphorylation levels and a corresponding decrease in
peptide phosphorylation (Fig. 4B, lanes A and B).
Figure 4:
pPKR
phosphorylation of a synthetic eIF-2 substrate. Panel A,
partially purified HeLa mPKR (lanes A and B) or
barley ribosome salt wash fractions (lanes C and D)
were incubated with poly(rI)
poly(rC)-agarose followed by the
addition of [
-
P]ATP and the eIF-2 peptide
(3 µg). Extracts were incubated with 500 µg of soluble
poly(rI)
poly(rC) (lanes B and D) or 500 µg
of soluble poly(rA) (lanes A and C) prior to
incubation with poly(rI)
poly(rC)-agarose. Radiolabeled proteins
were eluted, separated on a Tricine gel, and visualized by
autoradiography. Panel B, barley ribosome salt wash fractions
were incubated with preimmune serum (lane A) or anti-PKR sera (lane B). S. aureus cells were then added, and
incubation continued. The bacterial cells were pelleted, and the
supernatant was used in standard poly(rI)
poly(rC)-agarose binding
assays. After thorough washing of the resin,
[
-
P]ATP and the eIF-2 peptide (3 µg)
were added. Radiolabeled proteins were eluted, separated on a Tricine
gel, and visualized by autoradiography. The positions of molecular mass
markers (in kDa), PKR, and the eIF-2 peptide are
shown.
The synthetic eIF-2 peptide contains two Ser
residues corresponding to positions 48 and 51 of meIF-2
. These are
separated by a single cleavage site for V8 protease between residues
corresponding to Glu
and Leu
. The positively
charged fragment (LSRRRIR) containing Ser
can be separated
from the negatively charged fragment containing Ser
(ILLSE) by electrophoretic thin layer
chromatography(33) . When the peptide was in vitro phosphorylated in the presence of
[
-
]P)ATP and mPKR or the plant
dsRNA-binding kinase followed by V8 digestion and peptide separation,
results show that the residue corresponding to Ser
of the
eIF-2
was specifically phosphorylated by mPKR and the plant dsRNA
binding kinase (data not shown).
Taken together, these results
demonstrate that the M 42,000 doublet that is the
subunit of plant eIF-2 and the M
36,000
subunit of meIF-2 can be specifically phosphorylated in vitro by the M
68,000-70,000 plant-encoded
dsRNAdependent protein kinase, pPKR, as well as the eIF-2
kinase,
mPKR.
Since phosphorylation of eIF-2 by mPKR inhibits protein
synthesis and pPKR is capable of phosphorylating plant eIF-2
, we
were interested in determining if plant protein synthesis is inhibited
in the presence of phosphorylated eIF-2. Phosphorylation of meIF-2 and
peIF-2 was accomplished by incubation with mPKR bound to
poly(rI)
poly(rC)-agarose in the presence of unlabeled ATP and
phosphorylation buffer. Following incubation, mPKR bound to
poly(rI)
poly(rC)-agarose was pelleted, and the supernatant
containing the phosphorylated meIF-2 or peIF-2 was added to translation
reactions. Controls consisted of 1) phosphorylation mix (containing
only buffer and ATP), 2) phosphorylation mix containing
unphosphorylated peIF-2, 3) phosphorylation mix containing
unphosphorylated meIF-2, and 4) phosphorylation mix alone incubated
previously with mPKR bound to poly(rI)
poly(rC)-agarose. Controls
1-3 were incubated with poly(rI)
poly(rC)-agarose in the
absence of mPKR. Fig. 5shows that in vitro translation
of BMV RNA is unaffected in the presence of phosphorylation mix alone (lane A), unphosphorylated meIF-2
(lane B),
unphosphorylated peIF-2
(lane C) or phosphorylation mix
supernatant that was incubated with mPKR bound to
poly(rI)
poly(rC)-agarose (lane D). However, the addition
of meIF-2 or peIF-2 that was phosphorylated by mPKR dramatically
inhibited BMV RNA translation in wheat germ lysates (lanes E and F, respectively).
Figure 5:
Inhibition of BMV RNA in vitro translation in wheat germ lysates by phosphorylated eIF-2. Buffer (lanes A-C) or mPKR (lanes D-F) were
incubated with poly(rI)poly(rC)-agarose, washed extensively with
1 M KCl buffer and then 10 mM KCl buffer, and
incubated with phosphorylation mix (lanes A and D) or
phosphorylation mix containing meIF-2 (lanes B and E)
or peIF-2 (lanes C and F). The supernatant containing
phosphorylation mix alone (lanes A and D), mix and
unphosphorylated eIF-2 (lanes B and C), or mix and
phosphorylated eIF-2 (lanes E and F) was removed and
added to standard wheat germ lysate in vitro translation
reactions in the presence of BMV RNA and
[
S]methionine. Translated proteins were
separated by SDS-PAGE and visualized by autoradiography. The positions
of the molecular mass markers (in kDa) are shown. Asterisks indicate the positions of BMV translation products of M
94,000, 35,000, 20,000, and
15,000.
The observations that dsRNA inhibits in vitro translation in rabbit reticulocyte lysates via a mPKR-mediated mechanism (39) and that pPKR is constitutively present in wheat germ (15) encouraged us to determine if dsRNA addition to wheat germ would similarly inhibit protein synthesis. In the absence of exogenous dsRNA, BMV RNA is efficiently translated in vitro in wheat germ lysates (Fig. 6, lane A). The addition of dsRNA to wheat germ in vitro translation lysates inhibited translation of BMV RNA in a concentration-dependent manner (lanes B-E). In rabbit reticulocyte lysates, 0.1 µg/ml dsRNA is sufficient to totally inhibit protein synthesis(39) , whereas in Fig. 6A, 10 µg/ml dsRNA was required to significantly inhibit protein synthesis in wheat germ lysates. However, this is consistent with the dsRNA levels necessary to activate pPKR in vitro and in vivo in plant protoplasts(40) . As shown in Fig. 6B, the specificity of inhibition is indicated by the fact that translation was not inhibited by ssRNA. It should be noted that we found significant variation between wheat germ lysate preparations in terms of dsRNA levels required for inhibition, with several showing no response to dsRNA (data not shown). This effect, we believe, is due to varying levels of a PKR inhibitor present in the lysates.
Figure 6:
Double-stranded RNA inhibition of wheat
germ translation. In vitro translation was performed in the
presence of BMV RNA and [S]methionine. In panel A, the indicated concentrations of
poly(rI)
poly(rC) were added prior to the addition of BMV RNA. In panel B, the indicated concentrations of poly(rA) were added
prior to the addition of BMV RNA. Translated proteins were separated by
SDS-PAGE and visualized by autoradiography. The positions of molecular
mass markers (in kDa) are shown. Asterisks indicate the
positions of expected BMV translation products of M
109,000, 94,000, 35,000, 20,000, and
15,000.
We next determined
if exogenous mPKR is capable of inhibiting in vitro translation in wheat germ lysates. In the absence of mPKR,
translation of BMV RNA was comparable with Fig. 6, lane
A. The addition of purified mPKR alone decreased in vitro translation of BMV RNA in wheat germ lysates (compare Fig. 7, lane A with Fig. 6, lane A).
Only the major BMV translation products of M 94,000 and M
20,000 were detectable. This is
likely due to the activation of mPKR that is typically observed during
the purification process. However, BMV translation in wheat germ
lysates was completely inhibited in the presence of mPKR and low dsRNA
concentrations (0.1 µg/ml) (Fig. 7, lane B). This
concentration of dsRNA was much lower than that required in Fig. 6, suggesting interaction with mPKR rather than pPKR.
Furthermore, this is the optimal dsRNA concentration required for mPKR
shutdown of rabbit reticulocyte translation. The addition of ssRNA or
buffer alone had no effect on translation (data not shown).
Figure 7:
Inhibition of wheat germ translation by
mammalian PKR. In vitro translation was performed in the
presence of BMV RNA and [S]methionine. In both lanes, partially purified HeLa mPKR was added prior to the
addition of BMV RNA. In lane B, 0.1 µg/ml
poly(rI)
poly(rC) was also added prior to the addition of BMV RNA.
Translated proteins were separated by SDS-PAGE and visualized by
autoradiography. The positions of molecular mass markers (in kDa) are
shown. Asterisks indicate the position of the major BMV
translation products of M
94,000 and
20,000.
The
findings that plant protein translation is inhibited by phosphorylated
eIF-2, activated mPKR, or dsRNA indicates that components necessary for
the in vivo regulation of protein synthesis via an eIF-2
phosphorylation mechanism are present in plants similarly to other
eukaryotes. The dsRNAdependent inhibition of protein synthesis data is
in apparent conflict with previous findings(20, 41) .
However, we believe that the existence of a plant-encoded M
67,000 (p67) inhibitor of pPKR and eIF-2
phosphorylation is, in part, responsible to the lack of a consistent
dsRNA-mediated inhibition of protein translation in wheat germ
lysates.
The mammalian analog of p67 is tightly
associated with meIF-2 and inhibits phosphorylation (and hence,
activation) of eIF-2
kinases and eIF-2
phosphorylation(42) . In separate studies, we found that
mammalian p67 inhibited meIF-2
and peIF-2
phosphorylation in
the presence of either purified mPKR or partially purified plant
fractions containing pPKR. Phosphorylation of mPKR and pPKR in the
presence of p67 was correspondingly decreased. Based upon Western
blotting using monoclonal antiserum, we determined that the
plant-encoded p67 inhibitor is temporally regulated during plant growth
and development.
Differential activation of the plant p67
analog may account for variations in dsRNA sensitivity between wheat
germ lysate preparations and the apparent difficulty in characterizing
the plant eIF-2
phosphorylation pathway. These results suggest
that plants have a previously unappreciated mechanism to regulate
cellular response to various environmental signals.