Mechanisms of Ageing and Development
114 (2000) 123 – 132
www.elsevier.com/locate/mechagedev

Tissue speciﬁc expression of PKR protein
kinase in aging B6D2F1 mice
Warren Ladiges a,b,*, John Morton a, Collin Blakely c,
Michael Gale c,1
a

The Nathan Shock Center for Excellence in the Biology of Aging, School of Medicine,
Uni6ersity of Washington, Seattle, WA, USA
b
Department of Comparati6e Medicine (357190), Uni6ersity of Washington, Seattle, WA 98195, USA
c
Department of Microbiology, School of Medicine, Uni6ersity of Washington, Seattle, WA, USA
Received 5 January 2000; received in revised form 11 February 2000; accepted 11 February 2000

Abstract
A decline in the rate of protein synthesis is a common biochemical change observed with
aging in a wide variety of cells and organisms. The double stranded RNA-dependent protein
kinase PKR has been shown to phosphorylate eukaryotic initiation factor 2 alpha (eIF-2a),
a well-characterized factor for down-regulating protein synthesis, in response to environmental stress conditions. Therefore, we were interested in evaluating the role of PKR in the aging
process. Tissues from 2- and 20-month-old B6D2F1 male mice were evaluated by Western
blot analysis. PKR was detected in all tissues of aging mice conﬁrming its ubiquitous nature.
Tissues examined from young mice showed little evidence of PKR expression, suggesting an
age-associated up-regulation. P58IPK, a cellular inhibitor of PKR, was expressed in tissues
from both age groups but to a greater extent in tissues of aging mice suggesting an
up-regulation to control PKR activity. Hyperphosphorylated eIF-2a was increased in
selected tissues from older mice compared with tissues from younger mice indicating a
possible correlation between PKR expression and kinase function. The data suggest that
translational activity is slowing down in a tissue speciﬁc manner during the aging process in
mice, possibly as the result of increased levels of PKR, and could be a factor in the reduction

This work was supported by the Nathan Shock Center for Excellence in the Biology of Aging, NIA
AG13280, and the Helen Hey Whitney Foundation.
* Corresponding author. Tel.: + 1-206-6853260; fax: + 1-206-6853006.
E-mail address: wladiges@u.washington.edu (W. Ladiges)
1
Present address: Department of Microbiology, Southwestern Medical Center, University of Texas,
Dallas, TX, USA.

0047-6374/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 0 9 7 - X

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W. Ladiges et al. / Mechanisms of Ageing and De6elopment 114 (2000) 123–132

of the rate of protein synthesis during senescence seen in speciﬁc tissues of many organisms.
© 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Aging; PKR protein kinase; Mouse

1. Introduction
A gradual decrease in the rate of total protein synthesis is a common biochemical
change observed with aging in a wide variety of cells and organisms including
humans (Richardson and Semsei, 1987; Rattan, 1996). There are multiple implications and consequences of slower rates of protein synthesis including a decrease in
production and secretion of hormones, neurotransmitters, antibodies, and components of the extracellular matrix, an increased inefﬁciency of intracellular and
intercellular signaling pathway mechanisms and a decline in the concentration of
enzymes available for cellular maintenance, repair, and normal metabolic functioning. The molecular mechanisms responsible for such alterations are still poorly
understood. Protein kinases, which catalyze phosphorylation in order to regulate
several biological processes, including protein synthesis, are receiving attention as
factors in age-related alterations of gene expression (Stein et al., 1990; Richter et al.,
1991; Dulic et al., 1993). Phosphorylation of the alpha subunit of eukaryotic
initiation factor 2 (eIF-2a) is one of the best characterized mechanisms for
down-regulating protein synthesis in higher eukaryotes in response to various stress
conditions (Merrick, 1992). The phosphorylation of eIF-2a results in the shutdown
of protein synthesis by regulating both global as well as speciﬁc mRNA translation
(Sonenberg, 1996). It has been shown that conditions like starvation, heat shock,
and viral infections can induce the phosphorylation of eIF-2a through a protein
kinase known as double stranded RNA-dependent protein kinase (PKR) (Brostrom
et al., 1996).
PKR is a serine/threonine protein kinase and one of the best characterized of the
many proteins induced by type I interferon (IFN). It was proposed early on that
PKR plays an important role in the cellular antiviral response, and indeed it
inhibits the replication of several viruses in transfected cell lines over-expressing the
gene product (Katze, 1995). Recent studies have also shown that PKR may play an
important role in mediating the response of uninfected cells to physiologic stress
(Hinnebusch, 1994). Conditions that damage proteins, cause protein misfolding, or
inhibit protein processing, trigger the onset of protective homeostatic mechanisms
resulting in stress responses in mammalian cells. These responses are manifested in
a number of ways including an acute inhibition of mRNA translation, a subsequent
induction of various protein chaperones, and the recovery of mRNA translation.
The production of heat shock proteins (hsp) has been described as part of a
cytoplasmic stress response system (Welch, 1992). Induction of hsp occurs in
response to elevated temperature, oxidative free radicals, and heavy metals, or from
the synthesis of aberrant cytoplasmic proteins in response to amino acid analogs. A
recent report indicates that physiologic insults, such as treatment with sodium

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125

arsenite, dithiothreitol, or t-butylhydroperoxide, that induce cytoplasmic hsp responses in cells, also cause inhibition of protein synthesis through activation of
PKR (Brostrom et al., 1996). The mechanism of this response may involve
inactivation of P58IPK, a cellular inhibitor of PKR (Lee et al., 1994) by another
cellular protein recently identiﬁed as the molecular chaperone hsp40 (Melville et al.,
1997a). Hsp40 is involved in mediating the correct folding and assembly of
polypeptide chains and may provide a link between hsp-type signaling mechanisms
and the regulation of protein synthesis via P58IPK and PKR.
Because phosphorylation of regulatory and structural proteins is the major
mechanism by which intracellular control mechanisms operate in eukaryotes, we
were interested in evaluating the role of the PKR system in aging. Oxidative
damage to subcellular components is a reported mechanism of aging (Harmon,
1992; Sohal and Brink, 1992), and a purported factor in the development of chronic
degenerative diseases (Ames et al., 1993). Such damage is a constant occurrence at
the cellular level and accumulates over time, especially if the body’s defenses against
oxidative stress are diminished. We are suggesting that the PKR regulatory
pathway is a stress response system which operates to protect cellular resources
against environmental stress, including oxidative stress, at the pretranslational and
possibly transcriptional levels. This report describes our preliminary ﬁndings of
expression of PKR in tissues of aging mice but not in tissues of young mice. The
expression of P58IPK was seen in tissues from both age groups but generally to a
greater extent in tissues of aging mice. The alpha subunit of eIF2 was generally
expressed to a lesser degree in all tissues tested, except for liver and lung, of aging
mice compared with tissues in younger mice.

2. Methods

2.1. Animals
Five of each of 2- and 20-month-old male B6D2F1 mice were obtained from a
contract vendor for the National Institute on Aging. Mice were housed under
speciﬁc pathogen free conditions for 3 weeks, and then euthanitized by cervical
dislocation and tissues immediately collected. Colon, liver, kidney, testes, pancreas,
lungs, heart, and brain were collected from ﬁve mice of each age group and quickly
placed in ice-cold saline, blotted dry, weighed, and homogenized in 4 volumes of
ice-cold buffer (20 mM Tris, pH7.5, 1 mM dithiothreitrol, 35 mM sucrose, 0.1 mM
EDTA containing 125 mM KCl). The homogenates were then quick frozen in
liquid nitrogen and stored at −72°C until analyzed. There was no evidence of
clinical, serological, or pathological disease or infection in any of the mice. All
procedures relevant to the study were approved by the University of Washington
Animal Care and Use Committee. The University of Washington is fully accredited
by the Association for Assessment and Accreditation of Laboratory Animal Care
International.

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2.2. Determination of PKR, P58 IPK, and eIF-2h protein expression
Protein concentrations of tissue extracts were determined by using a protein assay
as described by the manufacturer (Bio-Rad Hercules, CA). Fifty ug of protein from
indicated tissue extracts were separated by SDS-polyacrylamide gel electrophoresis
and transferred to nitrocellulose membranes. Membranes were incubated for one
hour in blocking buffer and subsequently probed with the indicated primary
monoclonal antibodies. After a washing step, membranes were probed with a
horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch
Lab, Inc, West Grove, PA). Bound antibodies were detected by enhanced chemiluminescence (NEN Life Science Products, Boston, MA). Detection of mouse PKR,
P58IPK and eIF-2a, respectively, utilized antibody B10 (Santa Cruz Biotechnology,
Santa Cruz, CA), antibody 9F10 (Barber et al., 1994), and an antibody received
from Dr Michael Clemons. To control for errors in gel loading or protein
concentration assays, all blots were also probed with an antibody speciﬁc for actin.
Band intensities were quantitated from autoradiograms using a Bio-Rad GS 700
imaging densitometer and computer software supplied by the manufacturer.

2.3. Isoelectric focusing
Isolectric focusing of eIF-2a from mouse tissues was conducted as described
(Savinova and Jagus, 1997) except that tissue homogenates were supplemented with
1 mM okadaic acid, 15 mM EDTA, 1 mM PMSF, 1 mM DTT, 50 mM NaF, 35
mM b-glycerolphosphate and 10 mM 2-aminopurine. Proteins (20 mg) were separated by vertical isoelectric focusing and blotted to nitrocellulose membranes.
eIF-2a was detected by immunoblot analysis using a mouse monoclonal antibody
as described (Gale et al., 1998).

3. Results and discussion
The protein expression of PKR was seen in tissues of ﬁve aging mice but
generally not in tissues from ﬁve young mice (Table 1 and Fig. 1). Expression in all
tissues examined from aging mice conﬁrmed the ubiquitous nature of the enzyme.
The pancreas, heart, and lungs appeared to have fairly strong levels of PKR, while
levels were quite low in the liver, testes and brain. These tissue speciﬁc variations in
PKR levels in tissues from older mice may be a reﬂection of metabolic or functional
demands. Expression of P58IPK was seen in tissues of both young and older mice
but the bands were generally more intense for the tissues from the older mice. The
pancreas showed a robust expression in both age groups consistent with the
over-expression of the P58IPK mRNA in this tissue (Korth et al., 1996). Expression
of eIF-2a was seen in tissues from both age groups, with generally stronger levels
seen in tissues from the young mice, in contrast to ﬁndings with PKR and P58IPK.
An argument might be made that the difference in expression of the three
proteins is merely a reﬂection of a shift in cell population from young to old.

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127

However, the differences are consistent in the eight tissues examined suggesting the
ﬁndings are mechanistic in nature. It has recently been shown that protein kinases
are generally up-regulated in senescent fetal lung ﬁbroblast cultures (Blumenthal et
al., 1993) and in long-lived mutants of Caenorhabditis elegans (Vanﬂeteren and De
Vreese, 1997) thus supporting our ﬁndings in aging mice. However, at this point we
can only speculate on the mechanistic events that may be occurring in relation to
different levels of expression of these proteins.
If we make the assumption that increased levels of expression in aging tissues
indicate an increased function, then it is intriguing to suggest that PKR activity
increases to compensate for a continual bombardment of stress-inducing ‘hits’
during the course of aging. For example, oxidative ‘hits’ and the subsequent
oxidative damage are cumulative over the course of time as part of growing old
(Statdman, 1992). The best characterized function of PKR is an antiviral effect
based on its ability to phosphorylate eIF-2a resulting in inactivation and subsequent inhibition of protein synthesis (Katze, 1995). The anti-stress response of PKR
may occur through a similar mechanism as suggested by studies using NIH 3T3 cell
cultures exposed to sodium arsenite (Brostrom et al., 1996). Extracts from arsenitetreated cells displayed greater degrees of phosphorylation of PKR and eIF-2a than
did control extracts resulting in inhibition of translation initiation. In addition,
there was a subsequent activation of heat shock protein (hsp) chaperone activity.
Table 1
Expression of PKR, P58IPK and eIF-2a in tissues from ﬁve paired young and old B6D2 male mice,
respectivelya
Tissue

Age (months)

PKR

P58IPK

eIF-2a

Kidney

2
20
2
20
2
20
2
20
2
20
2
20
2
20
2
20

1
39+4*
6
1
20+3*
6
1
41+6*
6
1
34+8*
6
1
27+6*
6
1
222+7*
6
1
181+9*
6
2+1
6
118+5*
6

84+6
6
100+9
6
200+9
6
268+12*
6
69+7
6
79+5
6
12+7
6
41+8*
6
100+9
6
188+7*
6
705+12
6
684+27
6
8+3
6
99+4*
6
1
25+7*
6

76+4
6
11+6**
6
125+9
6
318+10**
6
140+9
6
110+11
6
114+8
6
87+7
6
166+6
6
107+8**
6
230+10
6
210+9
6
84+6
6
155+16*
6
220+6
6
49+8**
6

Liver
Colon
Brain
Testes
Pancreas
Lung
Heart

a
Values are the average of imaging densitometry readings of immunoblot analysis normalized to
percentage actin control levels for each age group.
* Signiﬁcant increase, PB0.05–0.5. Determined by  2 analysis.
** Signiﬁcant decrease, PB0.1–0.5. Determined by  2 analysis.

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Fig. 1. Representative expression of PKR regulatory pathway components in tissues from young and old
mice. Paired tissue homogenates prepared from ﬁve 2-month-old (Y) and ﬁve 20-month-old (O) B6D2F1
speciﬁc pathogen free mice were subjected to immunoblot analysis using monoclonal antibodies speciﬁc
for murine PKR (panel 1), P58IPK (panel 2, or eIF-2a (panel 3). Fig. 1 is representative of the results
obtained from each of the ﬁve mouse pairs (summarized in Table 1). Each lane contains 50 mg of
protein. For PKR, P58IPK, and eIF-2a, the level of each band was quantitated by imaging densitometry,
normalized as a percentage of the corresponding level of the actin control (% actin), and used to
calculate data for Table 1.

Additional evidence that PKR is part of a cellular stress response pathway was
recently reported by Melville et al. (1997a) who identiﬁed a molecular chaperone
known as hsp40 which maintains P58IPK, the inhibitor of PKR, in an inactive state.
It may be that some viruses, such as inﬂuenza virus, are able to block the interferon
antiviral effect mediated by PKR by recruiting the hsp40–P58IPK –PKR cellular
stress response pathway (Melville et al., 1997b).
P58IPK is the best described of several cellular regulators of PKR (Tang et al.,
1996). P58IPK has been identiﬁed and cloned (Korth et al., 1996) and found to be
a member of the tetratricopeptide repeat family of proteins (Lee et al., 1994). In
addition, the C-terminal portion of P58IPK shares homology with the J region of the
DNAJ heat shock protein of E coli placing it within the heat shock family of
proteins (Melville et al., 1997a). P58IPK appears to be present in all tissues examined
so far in both mice and humans, with especially high levels in the liver and pancreas
(Korth et al., 1996). We had similar ﬁndings, with very strong expression in the
pancreas of both young and older mice. In general, P58IPK was expressed in tissues
from both young and old mice but at a more intense level in the older mice. Our
explanation for the correlating levels of expression of PKR and P58IPK in tissues
from young and older mice is the need for additional P58IPK to continually guard
against overproduction of PKR. It may be that PKR, at this age in the older
B6D2F1 mice, is still able to strongly compensate for oxidative ‘hits’, or other types
of stress exposure, and still requires P58IPK to prevent overcompensation and

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129

complete shutdown of protein synthesis. Evaluation of even older mice might
possibly show exhausted levels of PKR and P58IPK as suggested by Miller (1994)
who saw a decline in both serine/threonine and tyrosine speciﬁc protein kinase
signals after activation of T lymphocytes in aging mice.
Expression of eIF-2a was detected in all tissues, as expected, similar to expression
of PKR and P58IPK. In contrast to the increased expression of PKR and P58IPK in
tissues of older mice compared with younger mice, eIF-2a levels were generally
decreased in tissues, except for liver and lung, of older mice relative to tissues of
younger mice, similar to ﬁndings in tissues from aging rats (Kimball et al., 1992).
However, isoelectric focusing showed that eIF-2a in its hyperphosphorylated form
was actually increased in selected tissues (kidney and liver) from the older mice
compared with tissues in younger mice (Fig. 2) indicating a correlation between
PKR expression and kinase function. This would suggest that translational activity
is slowing down during the aging process in mice possibly as the result of increased
levels of PKR, and could be a factor in the reduction of the rate of protein
synthesis during senescence seen in other organisms (Sohal et al., 1993). For
example, it has recently been reported that the rate of synthesis of total tissue
protein is slower in skeletal muscle of healthy older humans compared with skeletal
muscle of young adults (Welle et al., 1997). There was no difference in total mRNA
between young and old muscle suggesting that the slower protein synthesis might be
the result of decreased translation activity by eIF-2a.
The concept that down-regulation of protein translation is mediated by phosphorylation of eIF-2a in aging mouse tissues ﬁts best with our data for kidney, colon,
brain, and testis. Several tissues showed inconsistencies. Lung and liver from old
mice expressed higher levels of non-phosphorylated eIF-2a than respective tissues
from young mice. It may be that liver and lung have different environmental
demands reﬂective of increased need for high levels of eIF-2a associated with aging
in these tissues. There was deﬁnitely evidence of kinase activity in liver tissue from
old mice as indicated by the presence of phosphorylated eIF-2a. Lung tissue was

Fig. 2. Isoelectric focusing of eIF-2a in tissue homogenates from kidney and liver (arbitrarily selected as
representative tissues) from two 2-month-old (Y) and two 20-month-old (O) B6D2F1 speciﬁc pathogen
free male mice. Lane 1 is a positive control prepared from a rabbit reticulocyte lysate. Lanes 2 – 9 each
represent kidney or liver tissue from a single young or old mouse. The more acidic hyperphosphorylated
eIF-2a (eIF-2a-P) can be seen as the upper row of bands, while the more basic hypophosphorylated
eIF-2a can be seen as the lower row of bands. Each lane contains 20 mg protein.

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not tested for kinase activity but it would be interesting to determine if levels were
similar to liver. Our data suggest some unique aspect of protein translation is
associated with aging in the liver and lung of mice. In addition, pancreas showed a
strong age-associated increase of PKR, a high level of P58IPK that did not change
with age, and an abundant level of eIF-2a that showed minimal change. Heart
tissue also showed an age-associated up-regulation of PKR and P58IPK, but a
dramatic loss of eIF-2a. These are likely characteristics unique to speciﬁc cellular
functions of these tissues, and further experiments are necessary to determine
relevant translation activity.
Our explanation for the difference in expression of PKR in tissues of aging versus
young mice has focused on the cellular stress pathway, speciﬁcally oxidative stress,
leading to an inhibition, or at least slowing, of mRNA translation and protein
synthesis. However, it has recently been shown that PKR is an activator of
apoptosis (Williams, 1997), and that PKR-dependent apoptosis requires eIF-2a
phosphorylation (Srivistava et al., 1996). The process of programmed cell death, or
apoptosis, is activated as a defensive mechanism for eliminating damaged or
harmful cells within a multicellular organism. It may be that aging increases the
requirement for PKR in mediating different forms of stress-related apoptosis.
Another possible mechanism that may be a functional part of PKR activity during
aging is the ability to phosphorylate the inhibitor of nuclear factor-kB (IkB) (Der
et al., 1997). IkB, when phosphorylated, is prevented from inhibiting NF-kB, thus
allowing the activation of this ubiquitous transcription factor (Baeuerle and Baltimore, 1988). NF-kB stimulates the expression of inﬂammatory cytokines, immunoregulatory receptors, and acute phase proteins. It has been suggested that
NF-kB is primarily an oxidative stress-responsive transcription factor since its
activity can be inhibited by antioxidant compounds such as N-acetylcysteine, lipoic
acid, vitamin E derivitives, and metal chelators (Schreck et al., 1992). It was not
apparent that this was an active event in our aging mice showing expression of
PKR since we did not observe any pathology to suggest inﬂammatory conditions,
although we did not examine NF-kB activity directly. Helenius et al. (1996) have
recently shown that the NF-kB transcription factor pathway is activated during
aging in cardiac tissue of aging mice, but provided little insight into the activation
mechanisms. We have recently generated transgenic mice over-expressing PKR in
an effort to investigate this point more thoroughly.

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