Báo cáo Y học: Double-stranded RNA-activated protein kinase interacts with apoptosis signal-regulating kinase 1 Implications for apoptosis signaling pathways docx

Double-stranded RNA-activated protein kinase interacts with
apoptosis signal-regulating kinase 1
Implications for apoptosis signaling pathways
Takenori Takizawa
1
, Chizuru Tatematsu
1
and Yoshinobu Nakanishi
2
1
Department of Biochemistry, Institute for Developmental Research, Aichi Human Service Center, Kasugai, Aichi, Japan;
2
Graduate school of Medical Science, Kanazawa University, Kanazawa, Ishikawa, Japan
Double-stranded RNA-activated protein kinase (PKR), a
serine/threonine kinase, is activated in virus-infected cells
and acts as an antiviral machinery of type I interferons.
PKR controls several stress response pathways induced by
double-stranded RNA, tumor necrosis factor-a or lipo-
polysaccharide, which result in the activation of stress-acti-
vated protein kinase/c-Jun NH
2
-terminal kinase and p38 of
the mitogen-activated protein kinase family. Here we
showed a novel interaction between PKR and apoptosis
signal-regulating kinase 1 (ASK1), one of the members of
the mitogen-activated protein kinase kinase kinase family,
which is activated in response to a variety of apoptosis-
inducing stimuli. PKR and ASK1 showed predominant
cytoplasmic localization in COS-1 cells transfected with
both cDNAs, and coimmunoprecipitated from the cell
extracts. A dominant negative mutant of PKR (PKR-KR)

inhibited both the apoptosis and p38 activation induced by
ASK1 in vivo. Consistently, PKR-KR inhibited the auto-
phosphorylation of ASK1 in vitro, and exposure to poly(I)–
poly(C) increased the phosphorylation of ASK1 in vivo.
These results indicate the existence of a link between PKR
and ASK1, which modifies downstream MAPK.
Keywords: ASK1; apoptosis; MAPK; PKR; signal trans-
duction.
The interferon-inducible, double-stranded RNA (dsRNA)-
activated protein kinase (PKR) is a serine/threonine kinase
ubiquitously expressed in mammalian cells [1,2]. PKR is
activated by a variety of dsRNA molecules generated
during viral infection [3]. Upon its activation, PKR
autophosphorylates and then phosphorylates eukaryotic
translational initiation factor 2 (eIF-2a) [4], thereby inhi-
biting cell growth or viral replication [5,6]. Thus PKR
mediates the antiviral and antiproliferative actions of type I
interferons [6]. On the other hand, catalytically inactive
mutants of PKR transform NIH-3T3 cells [7,8], while
overexpression of wild-type PKR induces apoptosis of
HeLa cells [9,10]. PKR appears to up-regulate expression of
the apoptotic receptor Fas induced by viral infection [11,12].
Moreover, mouse embryonic fibroblasts deleted of the PKR
gene have been shown to resist apoptosis in response to
dsRNA, tumor necrosis factor-a (TNF-a) or lipopolysac-
charide (LPS) [13]. PKR has been shown to play some role
in the activation of p38 mitogen activated protein kinases
(MAPKs) and the stress-activated protein kinase (SAPK)/
c-Jun amino-terminal kinases (JNKs) that are strongly
activated in response to TNF-a,dsRNAorLPS[13].

However, the precise pathway linking PKR and the MAPK
family remains to be elucidated.
Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK
kinase kinase (MAPKKK) that acts upstream of JNK and
p38 MAPKs [14,15]. ASK1 phosphorylates SEK1/MKK4
or MKK3/MKK6, one of the members of the MAPK
kinase family, which in turn activates JNK or p38 MAPK,
respectively [15]. A wide variety of stress-related stimuli
activate ASK1, including serum withdrawal, TNF-a, react-
ive oxygen species, microtubule-interfering agents, genoto-
xic stress, and possibly Fas ligand [16]. Overexpression of
the wild type or constitutively active form of ASK1 induces
cell death through signals involving the mitochondrial cell
death pathway [17]. ASK1 binds proteins associated with
death receptors as TNF-receptor-associated proteins
(TRAFs) or Daxx, which also results in MAPK activation
[18,19]. In the present study, we show that PKR interacts
with ASK1 and modifies the ASK1 signaling pathway both
in vivo and in vitro. These results suggest that PKR acts as a
signal transducer by interacting with MAPKKKs, which
then modifies the downstream MAPK cascade.
MATERIALS AND METHODS
Plasmids and DNA transfection
Human PKR cDNA was kindly provided by A. Hovanes-
sian (Institut Pasteur, France). A mutant PKR cDNA
Correspondence to T. Takizawa, Department of Biochemistry,
Institute for Developmental Research, Aichi Human
Service Center, Kasugai, Aichi 480-0392, Japan.
Fax: + 81 568 88 0829, Tel.: + 81 568 88 0829,
E-mail:

Abbreviations: ASK1, apoptosis signal-regulating kinase 1; dsRNA,
double-stranded RNA; eIF-2a, eukaryotic translational initiation
factor 2a; EGFP, enhanced green fluorescence protein; FITC,
fluorescein isothiocyanate; LPS, lipopolysaccharide; MAPK,
mitogen activated protein kinase; PKR, double-stranded RNA-acti-
vated protein kinase; RITC, tetaramethyl rhodamine isothiocyanate;
DMEM, Dulbecco’s modified Eagle’s medium.
(Received 29 June 2002, revised 4 September 2002,
accepted 22 October 2002)
Eur. J. Biochem. 269, 6126–6132 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03325.x
carrying a point mutation of K to R at position 296
(PKR-KR) was constructed as described [20]. cDNAs for
human ASK1 and dominant negative mutant of ASK1
carrying a point mutation of K to M at position 709 (ASK-
KM) were kindly provided by H. Ichijo (Laboratory of Cell
Signaling, Tokyo Medical and Dental University). The
plasmid encoding PKR or PKR-KR fused to enhanced
green fluorescent protein (EGFP) (Clontech Laboratories,
Inc., Palo Alto, CA, USA) was described previously [21].
Human embryonic kidney 293 (HEK293), COS-1 and
NIH-3T3 cells were cultured in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% fetal bovine
serum, and maintained under 5% CO
2
at 37 °C. Cells were
transfected with 2 lg of plasmid DNA using 6 lLof
Lipofectamine-plus and 4 lL of Lipofectamine (Gib-
coBRL) according to the manufacturer’s instructions. To
establish permanent transfectants, HEK293 cells were
diluted about 10-fold and replated in medium containing

750 lgÆmL
)1
of G418 two days after transfection, and then
drug-resistant colonies were isolated. For microscopic
observation, cells were seeded on a cover glass and DNAs
were transfected as described above. For in vivo labeling of
ASK1 or PKR, cells were washed with phosphate-free
DMEM, and then incubated in phosphate-free DMEM in
the presence of [
32
P]-orthophosphate (400 lCiÆmL
)1
)(ICN
Biomedicals, CA, USA) for the indicated period as
described in the figure legend. The phosphorylation reaction
was resolved by SDS/PAGE and visualized by autoradio-
graphy. Autoradiogram was scanned and densitometric
analysis was performed with Kodak
DIGITAL SCIENCE
1
D
software (Eastman Kodak).
Indirect immunofluorescence
The day after transfection, cells were fixed with 4%
paraformaldehyde containing 0.2% Triton X-100 in
NaCl/P
i
for 30 min and washed with NaCl/P
i
. Subse-

quently, cells were incubated with anti-HA mAb (12CA5,
Boehringer Mannheim, Germany) or anti-PKR polyclonal
antibody (N-18, Santa Cruz, CA, USA) at a dilution of 200
or 100, respectively, for 60 min. Cells were then stained with
anti-(mouse IgG) conjugated with fluorescein isothiocya-
nate (FITC) (MBL, Nagoya, Japan) or anti-(rabbit IgG)
conjugated with tetaramethyl rhodamine isothiocyanate
(RITC) (Jackson Immunoresearch Laboratory, West
Grove, PA, USA) at a dilution of 200 for 60 min, and
observed under a fluorescence microscope at a magnifica-
tion of 270 (Olympus BX-60, Tokyo, Japan). For the
expression of EGFP, cells were fixed with 4% paraformal-
dehyde, and observed under a fluorescence microscope as
described above.
In vitro
kinase assay
Cell extracts were prepared with PKR buffer I (20 m
M
Tris/
HClpH7.6,50m
M
KCl, 400 m
M
NaCl, 1 m
M
EDTA,
5m
M
2-mercaptoethanol, 1% Triton X-100, 0.2 m
M

phe-
nylmethane sulfonyl fluoride, 100 UÆmL
)1
aprotinin, and
20% glycerol) and cleared by centrifugation. Then ASK1 in
the cell extract was precipitated by incubation with anti-HA
mAb (5 lL) for 1 h at 4 °C followed by 50 lLofa1:1
slurry of protein–G Sepharose 4FF (Pharmacia, Piscata-
way, NJ, USA). The immune complex on the beads was
washed four times with PKR buffer I and then once with
PKR buffer III (20 m
M
Tris/HCl pH 7.6, 80 m
M
KCl,
5m
M
b-mercaptoethanol, 2 m
M
MgCl
2
,2m
M
MnCl
2
,and
20% glycerol). The beads were then resuspended in PKR
buffer III containing 2 l
M
[c-

32
P]ATP (5 lCi) (ICN
Biomedicals) in the presence or absence of poly(I)–poly(C)
at the concentration indicated in the legends for 15 min at
30 °C. The phosphorylation reaction was resolved by SDS/
PAGE and visualized by autoradiography. PKR activity
was measured as described [11].
Immunoprecipitation and Immunoblot analyses
Cell extracts were prepared with PKR buffer I and
incubated with anti-PKR mAb (2 lL) or anti-HA antibody
(5 lL) for 1 h at 4 °C followed by 50 lL of a 1 : 1 slurry of
protein-G Sepharose 4FF for another 1 h at 4 °C. The
immune complex on the beads was washed four times with
PKR buffer I. The beads were then boiled in Laemmli’s
sample buffer [22] and resolved by SDS/PAGE. Proteins
were transferred onto nitrocellulose filters (Bio-Rad Labor-
atory, Hercules, CA, USA) and were incubated with
polyclonal anti-PKR Ig or anti-HA Ig followed by anti-
(rabbit IgG) or anti-(mouse IgG) conjugated with
peroxidase. Signals were visualized using an enhanced
chemiluminescence (ECL) detection system (Amersham,
Boston, MA, USA). Protein was measured by Bradford
reagent (Bio-Rad Laboratory).
RESULTS
PKR interacts with ASK1
To investigate whether PKR and ASK1 interact with each
other, the localization of PKR and ASK1 was first
examined by indirect immunofluorescence. As the wild type
of PKR is hardly expressed at all by transfection due to
translational inhibition [23], we used the kinase negative

mutant of PKR (PKR-KR), which shows the same
localization pattern as the wild type as previously reported
[24]. The signal for PKR-KR was predominantly localized
in the cytoplasm, whereas that for ASK1 distributed
diffusely with relatively intense staining at the periphery of
the cells (Fig. 1A, a and b). When PKR-KR and ASK1
were cotransfected into COS-1, both proteins showed
predominant cytoplasmic localization (Fig. 1A, c and d),
indicating colocalization of PKR and ASK1.
We next used a coimmunoprecipitation assay to
define the interaction between PKR and ASK1. They were
transfected into COS-1 cells, and immunoprecipitated with
antibody against either PKR or ASK1. When the immune-
complexes were precipitated with anti-PKR Ig and analyzed
by Western blotting with anti-HA Ig, the signal of ASK1
was detected only in the cell extracts transfected with both
cDNAs (lane 2 in Fig. 1B). PKR-KR was also coimmuno-
precipitated with anti-HA Ig (lane 4 in Fig. 1B). Expression
of these proteins was verified by Western blotting (Fig. 1B,
right panel). To examine whether endogenous PKR is
coimmunoprecipitated with ASK1, we established two
HEK 293 cells permanently expressing ASK1 (ASK-4 and
ASK-8 cells) and control cells containing empty plasmid
(pcDNA). The expressions of ASK1 and endogenous PKR
in these cells were confirmed by Western blotting (Fig. 1C,
Ó FEBS 2002 PKR interacts with ASK1 (Eur. J. Biochem. 269) 6127
right panel). ASK1 or endogenous PKR was immunopre-
cipitated with anti-PKR or anti-HA Ig, respectively
(Fig. 1C, left panel). All these results indicate that PKR
directly interacts with ASK1. Alternatively, the interaction

might be bridged by RNA. However, as all these coimmu-
noprecipitation assays were conducted in the presence of
high salt (0.45
M
), and immunecomplexes were resistant to
RNase treatment (data not shown), direct protein–protein
interaction seems to be likely.
Dominant negative mutant of PKR inhibits
ASK1 activity
To explore the potential influence of PKR on ASK1 in vivo,
the effect of PKR-KR on the ASK1-induced apoptosis was
investigated. We used constructs of PKR fused with EGFP
to directly visualize cell morphology. We have shown that
EGFP-PKR induced apoptosis without poly(I)–poly(C),
whereas EGFP-PKR-KR inhibited Fas-induced apoptosis
[21]. NIH-3T3 cells were transfected with ASK1 and either
pEGFP-PKR-KR or pEGFP. Serum was removed from
the medium 24 h after transfection, and the cells were
incubated for another 24 h. The cells were then fixed and
ASK1 was stained with anti-HA Ig followed by RITC-
labeled secondary antibody. ASK1 and EGFP-expressing
cells exhibited a round shrunken morphology indicating an
induction of apoptosis (Fig. 2B, arrow heads in upper
panel), whereas the cells expressing both ASK1 and
pEGFP-PKR-KR exhibited a flat spread shape (Fig. 2B,
arrow heads in lower panel). EGFP-expressing cells without
ASK1 expression were a flat shape as well (Fig. 2B, upper
panel). The expression of these proteins was verified by
Western blotting (Fig. 2A). The number of cells exhibiting a
shrunken morphology was counted in several fields and

summarized (Fig. 2C). Transfection of ASK1 and subse-
quent serum deprivation caused about 60% of cells to die,
whereas cotransfection of pEGFP-PKR-KR suppressed the
cell death to almost the control level (Fig. 2C). Transfection
of either empty vector or PKR-KR alone did not cause
significant cell death.
As PKR-KR inhibited the ASK1-induced apoptosis, it
seems reasonable to speculate that PKR-KR inhibited
ASK1 signaling. As ASK1 has been shown to activate
stress-activated MAPKs, the effect of PKR-KR on the
activation of p38 by ASK1 was examined. COS-1 cells
were transfected with ASK1 and/or PKR-KR, and p38
activation was examined by Western blotting using
antibody against the phosphorylated form of p38
(Fig. 3). ASK1 increased p38 phosphorylation 24 h after
transfection (lane 6 in Fig. 3) (an average of 5.4-fold
increase in the intensity from three independent experi-
ments compared with that of pcDNA at 24 h), whereas
cotransfection of PKR-KR inhibited its increase to about
60% level of ASK1 (lane 8 in Fig. 3) (an average of 3.4-
fold increase).
Fig. 1. PKR interacts with ASK1. (A) Cytoplasmic localization of
ASK1 with PKR. COS-1 cells were transfected with pcDNA-PKR-
KR (a), pcDNA-ASK1-HA (b), or both (c and d). Cells were fixed
18 h after transfection and then incubated with anti-PKR Ig (a) or
anti-HA Ig (b) followed by FITC-labeled anti-rabbit or anti-mouse
immunoglobulin, respectively. For double staining of PKR-KR (c)
and ASK1 (d), cells were incubated with both antibodies, followed by
RITC-labeled and FITC-labeled secondary antibodies. Cells were
observed under a fluorescent microscope at a magnification of 270. (B)

PKR and ASK1 were coimmunoprecipitated. COS-1 cells (approxi-
mately 4 · 10
5
cells) were transfected with the plasmid DNAs indi-
cated above the lanes, and lysed in a lysis buffer 48 h after transfection.
Lysates (200 lg of total protein) were incubated with anti-PKR (lanes
1 and 2) or anti-HA (lanes 3 and 4) antibody, followed by protein
G–Sepharose. Immunecomplexes were then analyzed by Western
blotting with anti-HA (lanes 1 and 2) or anti-PKR (lanes 3 and 4) Ig.
Expression of ASK1 and PKR-KR in a total lysate (100 lgoftotal
protein) was examined by Western blotting using anti-HA (ASK1) or
anti-PKR Ig (PKR) (right panel). Signals were visualized by ECL. Ig
denotes immunoglobulin. (C) ASK1 and endogenous PKR were
coimmunoprecipitated. HEK293 cell lines permanently transfected
with pcDNA (pcDNA) or ASK1 (ASK-4, ASK-8) were established.
Approximately 2 · 10
7
celles were lysed in a lysis buffer, and ASK1 or
PKR in a lysate (12 mg of total protein) was immunoprecipitated with
anti-HA or anti-PKR Ig (immppt) as described in (B), and Immune-
complexes were analyzed by Western blotting with anti-HA or anti-
PKR Ig (WB). Expression of ASK1 or endogenous PKR in a total
lysate (100 lg of total protein) was examined by Western blotting
(right panel). Signals were visualized by ECL.
6128 T. Takizawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Dominant negative mutant of PKR inhibits
ASK1 activity
in vitro
The above results suggested that PKR modifies ASK1
activity. We therefore examined by means of an in vitro

kinase assay whether PKR directly affects ASK1 activity.
ASK1 was immunoprecipitated with anti-HA Ig from the
extract of HEK293 cells permanently expressing ASK1, and
an autophosphorylation reaction was induced in the
presence or absence of poly(I)–poly(C). Poly(I)–poly(C),
however, did not affect ASK1 activity at all (Fig. 4A). This
might be due to the amount of PKR coimmunoprecipitated,
which was so small that its effect could not be detected.
Therefore, PKR-KR was further transfected into ASK-8
cells and ASK1 activity was examined. An increase in the
intensity of the PKR signal was observed in PKR-KR-
transfected cells by Western blotting (lanes 2 and 4 in
Fig. 2. Effect of dominant negative mutant of PKR (PKR-KR) on
ASK1-induced apoptosis. (A) Western blotting of PKR-KR or ASK1.
NIH-3T3 cells were transfected with empty vector of pEGFP (GFP),
pEGFP-PKR-KR (PKR-KR), pEGFP and ASK1 (ASK + GFP) or
pEGFP-PKR-KR and ASK1 (ASK + PKR-KR) as indicated above
the lanes. Cell lysates were prepared 48 h after transfection and then
examined by Western blotting using anti-GFP, anti-PKR, or anti-HA
Ig. Signals were visualized by ECL. (B) Effect of PKR-KR on the
ASK1-induced apoptosis. NIH-3T3 cells were transfected with 1.5 lg
of pcDNA-ASK1-HA with either 0.5 lg of pEGFP or pEGFP-PKR-
KR. Serum was removed from the medium 24 h after transfection, and
the cells were incubated for another 24 h. They were then fixed with
4% PFA and stained with anti-HA Ig followed by anti-(mouse IgG)
conjugated with RITC. Arrowheads indicate the cells expressing
ASK1 and either EGFP or PKR-KR fusion protein. (C) Rounded and
shrunken cells were counted as apoptotic. The percentages of apop-
totic cells are shown as an average of three independent experiments
±S.D.

Fig. 3. Effect of dominant negative mutant of PKR (PKR-KR) on
activation of p38 induced by ASK1. COS-1 cells (approximately 4 · 10
5
cells) were transfected with the plasmid DNAs described above the
lanes, and cells were harvested 12 h or 24 h after transfection. Cell
lysates (100 lg of total protein) were resolved by SDS/PAGE.
Expression of the phosphorylated form of p38, total p38, ASK1 and
PKR was examined by Western blotting using anti-(p38-P), anti-(p38-
total), anti-HA, and anti-PKR Ig, respectively. Signals were visualized
by ECL. The representative data of three independent experiments
with similar results were shown.
Ó FEBS 2002 PKR interacts with ASK1 (Eur. J. Biochem. 269) 6129
Fig. 4B), while the amount of ASK1 in ASK-8 cells did not
change (lanes 3 and 4 in Fig. 4B). Transfection of PKR-KR
revealed a 35% decrease (an average from three independ-
ent experiments) in the autophosphorylation activity of
ASK1 (lanes 7 and 8 in Fig. 4B), suggesting that PKR
affects the activity.
Effect of PKR on the activity of ASK1
in vivo
To examine in vivo the effect of PKR on ASK1 activity,
HEK293 cells permanently transfected with wild type or
dominant negative mutant of ASK1 were exposed to
either poly(I)–poly(C) or H
2
O
2
in the presence of [
32
P]

orthophosphate, and cell lysates were prepared. ASK1 was
immunoprecipitated with anti-HA Ig. Immunecomplex
was then resolved by SDS/PAGE, and phosphorylation
reaction was visualized by autoradiography. Exposure to
H
2
O
2
markedly increased the intensity of ASK1 about
2.7-fold (an average of two independent experiments)
compared with that without H
2
O
2
(AR in Fig. 5B),
indicating H
2
O
2
activates ASK1 as described [25]. Expo-
sure to poly(I)–poly(C) also increased the signal for ASK1
about 2.1-fold (an average of two independent experi-
ments) compared with that without poly(I)–poly(C) (AR
in Fig. 5A), suggesting that PKR could activate ASK1.
The amount of ASK1 in the immunecomplexes was not
changed by these exposures, which was verified by
Western blotting (WBs in Fig. 5A and B). On the other
hand exposure to poly(I)–poly(C) did not markedly
Fig. 5. Effect of poly(I)–poly(C) on ASK1 activity in vivo. (A) HEK293
cells (approximately 4 · 10

5
cells) permanently transfected with empty
vector (pcDNA), wild type (ASK-8) or dominant negative mutant of
ASK1 (ASK-KM-2) were incubated with or without poly(I)–poly(C)
(polyI–C) (100 lgÆmL
)1
) in the presence of [
32
P]-orthophosphate
(400 lCiÆmL
)1
) for 2 h and harvested. ASK1 was immunoprecipitated
with anti-HA mAb, and resolved by SDS/PAGE. Phosphorylation
reactions were visualized by autoradiography (AR). Expression of
ASK1, ASK-KM was verified by Western blotting (WB). (B) Cells
were incubated with or without H
2
O
2
(1 m
M
) in the presence of [
32
P]-
orthophosphate for 1 h and harvested. Phosphorylation reactions
(AR) and expression of ASK1, ASK-KM (WB) were examined as
described in (A). The representative data of two independent experi-
ments with similar results were shown.
Fig. 4. Effect of PKR-KR on ASK1 activity in vitro . (A) ASK1 activity
in ASK1-expressing HEK293 cells (ASK-4, ASK-8). Cells were lysed

in a lysis buffer, and ASK1 in a lysate (400 lg of total protein) was
immunoprecipitated with anti-HA monoclonal Ig followed by protein
G-sepharose. Protein G-sepharose was then suspended in PKR III
buffer with [c-
32
P]ATP in the absence or presence of poly(I)–poly(C)
(polyI–C) (1.0 lgÆmL
)1
) for 15 min at 30 °C. Reactions were resolved
by SDS/PAGE and visualized by autoradiography. (B) Effect of PKR-
KR on ASK1 activity. Control (pcDNA) or ASK-8 cells were trans-
fected with PKR-KR (+) or empty vector (–), and cell lysates were
prepared 48 h after transfection. Expression of ASK1 and PKR-KR
was verified by Western blotting (lanes 1–4). ASK1 activity in the
absence or presence of PKR-KR was examined as described in (A) and
visualized by autoradiography (lanes 5–8). The representative data of
three independent experiments with similar results were shown.
6130 T. Takizawa et al. (Eur. J. Biochem. 269) Ó FEBS 2002
increase the phosphorylation state of ASK-KM, a kinase
negative mutant of ASK1 [15,19]. This may indicate that
PKR does not directly phosphorylate ASK1 but rather
supports autophosphorylation activity of ASK1. All these
results suggest that PKR could activate ASK1, although it
remains possible that poly(I)–poly(C) directly activates
ASK1. However, the latter might be unlikely, as poly(I)–
poly(C) did not activate ASK1 in vitro (Fig. 4A).
DISCUSSION
Besides having the antiviral activity of type I interferons,
PKR has been shown to transduce signals such as dsRNA,
LPS, platelet-derived growth factor, Fas, and TNF-a

[13,26,27]. As most of these signals are capable of inducing
apoptosis, PKR seems to transduce apoptotic signals,
especially receptor-mediated stimuli. As these stimuli also
have been shown to activate protein kinases of the MAPK
family [28], cross talk between PKR and the MAPK cascade
has been proposed. However, the pathway linking PKR and
the MAPK family remains to be clarified.
In the present study, we showed that PKR colocalized
and was coimmunoprecipitated with ASK1 when cotrans-
fected into COS-1 cells. The interaction of PKR with
ASK1 does not require kinase activity of PKR, as a
kinase negative mutant as well as endogenous PKR were
coimmunoprecipitated with ASK1. This seems to be
consistent with recent reports that the interaction of
PKR with IjBkinaseb or the signal transducer and
activator of transcription1 does not require kinase activity
of PKR [29,30]. However, PKR-KR decreased the
autophosphorylation activity of ASK1 in vitro,and
inhibited both the activation of p38 MAPK and apoptosis
induced by ASK1 in vivo. Moreover, exposure to poly(I)–
poly(C) increased ASK1 phosphorylation. All these results
indicate that PKR dose not play only a structural role but
rather modulates a signaling pathway of ASK1. Therefore
the binding of PKR to ASK1 might cause a conforma-
tional change in ASK1 for activation, of which is
dependent on PKR activity, or an additional factor(s)
might be requited to activate ASK1.
It has been shown that the activation p38 by poly(I)–
poly(C) or LPS treatment was abrogated in PKR-null
fibroblasts, while generally acting stimuli such as osmotic

shock or H
2
O
2
did not require PKR to activate MAPKs
[13]. By contrast, a variety of stimuli such as TNF-a,
IL-1, Fas, ceramide, H
2
O
2
, osmotic shock, heat shock,
anticancer drugs, protein synthesis inhibitors and so on
activate ASK1 [16]. Thus, PKR seems to be a specific
transducer of inflammatory stimuli, while ASK1 is a
general transducer. Our results indicate that the signaling
pathway directed from PKR to ASK1 may define the
roles of these kinases. Determining the binding site(s) in
the PKR and ASK1 molecules will help to confirm this
speculation.
ACKNOWLEDGEMENTS
We are grateful to Dr Ara Hovanessian for providing the anti-PKR
mAb and PKR cDNA, and Dr Hidenori Ichijo for ASK1 and ASK-
KM cDNAs. This work was supported in part by a grant-in-aid for
scientific research from the Ministry of Education, Science, Sports, and
Culture, Japan.
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