The role of eukaryotic initiation factor 2 during the metabolic depression associated with estivation
1 Biochemistry and Molecular Biology, School of Biomedical and Chemical
Sciences, University of Western Australia, 35 Stirling Highway, Crawley, WA
6009, Australia
2 Department of Cellular and Molecular Physiology, Penn State College of
Medicine, 500 University Drive, Hershey, PA 17033, USA
* Author for correspondence at present address: Cellular Regulation, MRC Dunn Human Nutrition Unit, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 2XY, UK (e-mail: jlp{at}mrc-dunn.cam.ac.uk)
Accepted 31 March 2003
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Summary |
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Key words: eIF2, eukaryotic initiation factor, estivation, protein synthesis, metabolic depression, Helix aspersa, Neobatrachus sutor, phosphorylation
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Introduction |
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Given that ATP utilization decreases during metabolic depression,
energy-consuming pathways must be downregulated. If their mode of regulation
during estivation can be elucidated it may be possible to delineate the
upstream signaling mechanisms that would then point to the initial cue for
metabolic depression. Protein synthesis is a major energy-consuming pathway
and, if maintained at a similar rate during metabolic depression, would become
an impossibly costly process in terms of its contribution to total energy
consumption. Accordingly, the downregulation of protein synthesis is a
consistent phenomenon seen in metabolically depressed organisms. The extent of
this downregulation has been well characterised in a number of both in
vivo and in vitro systems and in a variety of different types of
metabolic depression, including that associated with anoxia
(Bailey and Driedzic, 1996;
Hofmann and Hand, 1994
),
developmental arrest (Podrabsky and Hand,
2000
), hibernation (Frerichs
et al., 1998
) and estivation
(Fuery et al., 1998
;
Pakay et al., 2002
).
Despite the often substantial decrease in the rate of protein synthesis
during metabolic depression, mRNA pools appear to be maintained during the
depressed state. For example, translatable mRNA can be detected in dormant
Artemia embryos, and its in vitro translation demonstrates
that there is no significant qualitative and/or quantitative differences in
mRNA between anoxic and developing embryos
(Hofmann and Hand, 1994). Also
in Artemia, the addition of exogenous mRNA does not increase the
translational capacity of lysates prepared from dormant embryos
(Hofmann and Hand, 1994
).
During anoxia in turtles, protein synthesis is undetectable in liver and white
muscle (Fraser et al., 2001
)
while translatable mRNA concentrations increase by 38% in liver and remain
constant in white muscle (Douglas et al.,
1994
). These data demonstrate that it is not message limitation
that causes a downregulation in the rate of protein synthesis. The major site
for the regulation of protein synthesis during metabolic depression must
therefore be translation.
It is now generally accepted that the rate at which mRNA is translated into
protein is limited by the rate of initiation of translation, and there are two
specific processes in the initiation pathway that have been shown to be sites
for physiological regulation (Pain,
1996). These are (1) the binding of the MettRNAi to the
43S pre-initiation complex, mediated by eIF2 (eukaryotic initiation factor 2),
and (2) the initial binding of the 43S pre-initiation complex to the 5'
end of the mRNA (mediated by eIF4E and associated factors). eIF2 is involved
in the binding of an initiation complex
(eIF2MettRNAiGTP) to the 43S ribosomal pre-initiation
complex. The formation of this ternary complex is regulated by the
phosphorylation of a conserved site on the alpha subunit of eIF2, which
results in the formation of an inactive complex comprising eIF2
and
eIF2B. This mechanism and the sequence of the phosphorylation site in
eIF2
are conserved between yeast and man. An increase in the extent of
eIF2
phosphorylation occurs concomitantly with a downregulation of the
rate of protein synthesis in normal tissues responding to a diverse array of
stresses, including heat shock (Hu et al.,
1993
), amino acid deprivation
(Kimball et al., 1991
),
reperfusion following ischemia (Martin de
la Vega et al., 2001
) and anoxia
(Tinton et al., 1997
).
Data on the role of eIF2 in the regulation of protein synthesis during
metabolic depression are limited to two systems, and, in both, the metabolic
depression is confounded by extrinsic factors; changes either in temperature
or ambient PO2. So decreased rates
of protein synthesis and the concomitant accumulation of phosphorylated
eIF2 have been observed in hibernating ground squirrels
(Frerichs et al., 1998
) but
most of the metabolic depression in this system is due to decreased
temperature. During short-term anoxia in the marine snail Littorina
littorea, there is an accumulation of phosphorylated eIF2
that
occurs concomitantly with a decrease in protein synthesis
(Larade and Storey, 2002
). But
again, protein synthesis and metabolic rate depression in this system only
occur as a result of large changes in ambient
PO2.
The Australian desert frog Neobatrachus sutor and the land snail
Helix aspersa both survive extended dry periods or potential
desiccating environments by estivating. In Neobatrachus, there is a
reduction in oxygen consumption of 5070% after 812 weeks of
estivation (Withers, 1993),
and, likewise, within four weeks of the removal of food and water from
Helix aspersa there is an 84% metabolic depression
(Pedler et al., 1996
). In
contrast to the examples above, the metabolic depression in both of these
animals occurs without any changes in ambient temperature or
PO2. During metabolic depression,
protein synthesis is downregulated, by 67% in liver slices from
Neobatrachus centralis (Fuery et
al., 1998
) and by 78% and 48% in vivo in hepatopancreas
and foot muscle, respectively, from H. aspersa
(Pakay et al., 2002
). The
intrinsic nature (the absence of obvious, confounding, extrinsic effectors) of
the metabolic depression in these animals makes them ideal models in which to
study the regulation of protein synthesis and to search for intrinsic cues for
metabolic depression.
The objective of this study was to determine whether eIF2 is
involved in the downregulation of protein synthesis during metabolic
depression associated with estivation. We have measured eIF2
mRNA
levels in the snail H. aspersa and the levels and phosphorylation
status of eIF2
in both the snail and the frog N. sutor. This
comparative study highlights some fundamental differences in the expression
and phosphorylation of eIF2
in two estivating species that may have
regulatory significance and is the first study to provide evidence for a
possible mechanism by which the rate of protein synthesis is downregulated
during the metabolic depression associated with estivation.
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Materials and methods |
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Experimental animals
Helix aspersa Müller were collected, maintained and sectioned
as described in Pakay et al.
(2002). Neobatrachus
sutor Main 1957 were collected 80 km south of Newman, Western Australia.
They were not fed but either sectioned or induced to estivate within a few
days after arrival in the lab. They were kept at 20°C, individually, in
750 ml plastic containers with a sealed lid, which had a 4 mm-diameter hole
for gas exchange. They were initially in approximately 500 ml of moist soil,
which was allowed to dry out over time. All frogs cocooned as the soil dried
out. Cocooned frogs were allowed to estivate for 5 months. Frogs were double
pithed and sectioned by hand. Tissues were removed within 23 min and
were immediately frozen in liquid nitrogen.
Sample preparation
Tissues were thawed on ice and homogenised in nine volumes of assay buffer
using an Ultra-Turrax T25 homogeniser (Janke and Kunkel IKA Labortechnik) at
20 500 r.p.m. for 30 s on ice. Assay buffer consisted of 120 mmol
l-1 KCl, 2 mmol l-1 DTT, 20 mmol l-1 Hepes pH
7.2, 12 mmol l-1 MgCH3COO, 40 µmol l-1
ATP, 50 mmol l-1 NaF, 1 mmol l-1 benzamidine, 20 µg
ml-1 leupeptin, 1 mmol l-1 PMSF, 10 µg
ml-1 aprotinin and 5 µg ml-1 E64. This assay buffer
was used because it is compatible with HCR (heme controlled repressor kinase),
a specific eIF2 kinase (hence the ATP and Mg2+), and
contains a cocktail of protease inhibitors but no divalent cation chelators,
kinase inhibitors or detergent. As it was a concern that the levels of
phosphorylated eIF2
[eIF2
(P)] detected using this buffer could
be affected by the action of kinases or metalloproteases or by the fact that
not all membranes were disrupted, extraction was also performed using an
alternate lysis buffer, which, in addition to the assay buffer described
above, contained 4 mmol l-1 EDTA, 2 mmol l-1 EGTA, 0.1
mmol l-1 Na3VO4, 20 mmol l-1
ß-glycerophosphate and 1% (v/v) Triton X-100 but no MgCH3COO
and ATP. There were no differences in signal intensities on western blots
between extracts prepared in the two buffers.
Post-mitochondrial supernatant was prepared by centrifuging homogenates at 13 800 g for 20 min at 4°C. The supernatant was removed and an aliquot stored for protein determination (DC protein assay kit; Bio-Rad, Hercules, CA, USA) using BSA as a standard.
Preparing recombinant rat eIF2(P) for use as a positive
control and standard
A positive control for western analysis of eIF2(P) was prepared by
phosphorylating recombinant rat eIF2
FLAG (eIF2
expressed
with the FLAG fusion octapeptide) using HCR partially purified to
approximately 20% purity from rabbit reticulocytes
(Jackson and Hunt, 1985
). HCR
was lyophilised after purification and redissolved in 20 mmol l-1
Tris-HCl pH 7.5, 100 mmol l-1 KCl, 0.1 mmol l-1 EDTA, 7
mmol l-1 ß-mercaptoethanol, 30% (v/v) glycerol.
Phosphorylation involved incubating eIF2
FLAG with a sufficient
amount of the HCR preparation to ensure complete phosphorylation
(Kimball et al., 1998
). The
reaction was monitored by the addition of [
-32P]ATP, and
aliquots were removed at 0 min, 5 min, 20 min and 40 min post addition of HCR.
The aliquots were subjected to SDSPAGE followed by autoradiography and
densitometric analysis, which revealed that complete phosphorylation of
eIF2
FLAG occurred within 5 min.
Phosphorylation of endogenous eIF2 in frog and snail
extracts
It was necessary to determine the total amount of eIF2
(phosphorylated and unphosphorylated) as well as the amount of eIF2
(P)
in the post-mitochondrial supernatants. Since we were only using rabbit
anti-eIF2
(P) (an antibody specific to the phosphorylated form of the
protein only) in the western analysis, the total amount of eIF2
in
post-mitochondrial supernatant was determined by converting (by
phosphorylation) all of the endogenous eIF2
into eIF2
(P) and
then determining the total amount of eIF2
(P). Phosphorylation was
carried out using HCR. HCR (0.5 µl of the preparation described above) was
added to a duplicate sample of each post-mitochondrial supernatant, containing
100 µg of protein in a total volume of 25 µl, and incubated at 37°C.
Subsequently, the levels of total eIF2
and eIF2
(P) were
determined by quantifying the amount of eIF2
(P) in both the
phosphorylated sample (with HCR) and the unphosphorylated sample (without HCR)
from each post-mitochondrial supernatant.
To ensure that all of the endogenous eIF2 was converted to
eIF2
(P) by the HCR treatment, aliquots containing 25 µg of protein
were removed at 20 min, 40 min and 60 min after the addition of HCR and the
reaction stopped by adding SDSPAGE loading buffer. The above reaction
was also performed with 200 µmol l-1 ATP (vs 40 µmol
l-1 ATP in the normal lysis buffer) in the reaction mix to ensure
that ATP was not limiting in the reaction. In addition, to ensure that the HCR
itself was not losing activity during the time course, additional HCR (0.5
µl) was added following the 40 min time point. The time courses were done
using both awake and estivating samples.
Western analysis
The samples (equal amounts of protein) were subjected to SDSPAGE and
electrophoretically transferred using a Bio-Rad SD semi-dry trans-blot
apparatus with Towbin buffer [25 mmol l-1 Tris-HCl pH 8.3, 150 mmol
l-1 glycine, 20% (v/v) methanol] to Hybond C+ extra nitrocellulose
membranes. After air drying for 30 min, the membranes were blocked for 1 h in
5% non-fat milk in TBST [20 mmol l-1 Tris-HCl pH 7.6, 137 mmol
l-1 NaCl and 0.1% (v/v) Tween-20]. The membranes were then
incubated for 2 h with a 1:5000 dilution of primary antibody [rabbit
anti-eIF2(P)] in 1% non-fat milk in TBST.
For western analysis of H. aspersa post-mitochondrial supernatant,
the primary antibody was first pre-absorbed against H. aspersa
hemolymph in order to remove keyhole limpet hemocyanin (KLH)-related
non-specificity (Pakay et al.,
2002). The membranes were then washed in TBST and incubated for 1
h with a 1:10 000 dilution of secondary antibody in 5% non-fat milk in TBST
(sheep anti-rabbit IgG conjugated to horseradish peroxidase). All incubations
and washes were performed at room temperature. Immunoreactive bands were
detected by enhanced chemiluminescence (Supersignal West Femto Maximum
Sensitivity Substrate) followed by exposure to autoradiography film.
Densitometry was performed on scanned films using NIH Image 1.62.
Immunoreactive bands were quantified by comparing their density to the density
of known quantities of recombinant rat eIF2
(P)FLAG. In order to
verify equal loading and transfer, membranes were stained for total protein by
Ponceau S staining.
Cloning and northern analysis of H. aspersa
eIF2
Total RNA was prepared from H. aspersa hepatopancreas
using the acid guanidium thiocyanatephenolchloroform extraction
method (Chomczynski and Sacchi,
1987
). However, RNA prepared using this method contained a
contaminant that inhibited any subsequent reverse transcription reactions.
Therefore, to remove the contaminant, RNA from which the poly(A+)
RNA fraction was to be separated was first precipitated by salt, a procedure
normally used when the RNA is contaminated by glycogen
(Sambrook et al., 1989
).
Poly(A+) RNA was prepared by chromatography of total RNA on an
oligo-dT cellulose column with a single binding and elution cycle
(Sambrook et al., 1989
).
cDNA was synthesized from poly(A+) RNA using the primer
5'-GCGGCCGCTTGAATTCCCAC(T)17-3'
(Sambrook et al., 1989), and
PCR was performed using a pair of degenerate primers,
5'-GC(AGCT)GA(AG)ATGGG(AGCT)GC(AGCT)TA(TC)-3' and
5'-AT(AG)TA(AGCT)CC(TC)TT(TC)TC-(TC)TT(AG)TC-3' (Macromolecular
Resources), based on the amino acid sequences conserved between yeast,
Drosophila melanogaster and humans corresponding to residues
2733 and 7581 of human eIF2
. The PCR reaction yielded a
150 bp product that was directly cloned (pGEM®-T Easy Vector;
Promega, Annandale, NSW, Australia) and sequenced (ABI Prism BigDye Terminator
Cycle Sequencing Ready Reaction Kit; Applied Biosystems, Foster City, CA,
USA). Analysis of sequencing reactions was carried out by the Department of
Clinical Immunology at Royal Perth Hospital (Perth, Australia). A BLAST search
revealed that the 150 bp fragment corresponded to eIF2
. A primer based
on the 5' end of this sequence and an oligo-dT primer were used to
obtain a larger cDNA corresponding to eIF2
. This clone was sequenced
and used to prepare a probe in the subsequent northern analysis.
Total RNA was analysed by northern analysis by hybridisation after
formaldehyde gel electrophoresis (Sambrook
et al., 1989). The probe was prepared by 32P-labeling
of the eIF2
message fragment by random priming
(Sambrook et al., 1989
). After
transfer, hybridisation and washing, the membranes were exposed to a BAS-IIIs
phosphorimaging plate (Fuji, Stamford, CT, USA), and the plates scanned using
a BAS 1000 phosphorimager (Fuji). Densitometric analysis of the bands was
performed using the program NIH Image 1.62. Equal loading of RNA for northern
analysis was checked by densitometric analysis of the methylene-blue-stained
rRNA band (Sambrook et al.,
1989
).
Statistics
All values are quoted as means ± S.E.M. (N).
Comparisons between time points, tissues and/or treatments were made using
unpaired t-tests. Statistical significance is quoted at the 5%
level.
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Results |
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Northern analysis of total RNA from H. aspersa tissues reveals that the cDNA hybridised with two messages (estimated to be 2.6 kb and 1.4 kb in length) in hepatopancreas. Northern analysis revealed that these messages were equally expressed in awake and estivating individuals. Methylene blue staining demonstrated that there was an equal amount of RNA loaded in each lane, as judged by the intensity of the ribosomal RNA band. There were no differences in the levels of expression between the different sized transcripts within each tissue type.
Phosphorylating and detecting recombinant rat
eIF2FLAG
The eIF2FLAG(P) was detected by western analysis, which
indicated an apparent molecular mass of 41
kDa(Fig. 2A). The primary
antibody was specific for the phosphorylated form of the protein only. There
was a sigmoidal relationship between the density of the
eIF2
FLAG(P) band and the amount loaded
(Fig. 2B). Using the described
working concentration of the primary and secondary antibody, as little as 0.1
ng of eIF2
FLAG(P) could be detected.
|
Phosphorylation and detection of eIF2 in snail and frog
extracts
Endogenous eIF2 was phosphorylated by HCR in post-mitochondrial
supernatant from both H. aspersa hepatopancreas and N sutor
liver. In both tissues, the eIF2
(P) had an apparent molecular mass by
SDSPAGE of 39 kDa.
In H. aspersa post-mitochondrial supernatant, after the addition
of HCR there was a rapid increase in the amount of eIF2(P)
(Fig. 3A). There was no
apparent difference in quantity between the 20 min and 60 min time points.
Increasing the concentration of ATP in the reaction from 40 µmol
l-1 to 200 µmol l-1 had no effect on the density of
the 39 kDa band after 60 min. Also there was no effect of adding additional
HCR after 40 min. Note that H. aspersa extracts also showed an
additional band at 42 kDa that could be detected in the absence of HCR. This
band sometimes increased in intensity after the addition of HCR, but not
consistently between samples (Fig.
3A).
|
Similarly, in N. sutor post-mitochondrial supernatant, after the
addition of HCR there was a rapid increase in the amount of eIF2(P) and
there was no difference between the 20 min and 60 min time points
(Fig. 3B). However, in N.
sutor only the band corresponding to eIF2
(P) was detected. In both
N. sutor and H. aspersa, there was no difference in the time
course of phosphorylation with HCR between the awake and estivating
states.
To calculate the ratio of eIF2(P) to total eIF2
, the amount
of eIF2
(P) before the addition of HCR was compared with that present
after phosphorylation with HCR. These levels were quantified from
eIF2
(P)FLAG standards run on the same blot. In H.
aspersa (Fig. 4A), there
was no detectable endogenous eIF2
(P) in post-mitochondrial supernatant
in either awake or estivating hepatopancreas but, since the minimum amount of
eIF2
(P)FLAG that could be detected was 0.1 ng, we can calculate
that less than 12% of the eIF2
was phosphorylated in the snail lysates.
The total amount of eIF2
was not significantly different between awake
and estivating tissues (Table
1). The total amount of eIF2
represented approximately
0.002% of the total amount of protein in post-mitochondrial supernatant.
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In N. sutor, there was more total and phosphorylated eIF2
in estivating versus awake tissues
(Fig. 4B;
Table 1). The ratio of
phosphorylated to unphosphorylated eIF2
was significantly higher in
estivating tissue compared with that in awake tissue
(Table 1; P<0.05,
N=6). The total amount of eIF2
represented approximately
0.005% and 0.008% of the total amount of protein in awake and estivating
post-mitochondrial supernatant, respectively.
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Discussion |
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There was no differential expression of the eIF2 mRNAs between the
awake and estivating states of H. aspersa. The level of eIF2
protein per mg of total protein is the same in the awake and estivating
states. The data cannot be used to draw conclusions about the rate of turnover
of either eIF2
message or protein, but if eIF2
does play a role
in regulating protein synthesis in H. aspersa during transitions
between states, neither eIF2
mRNA nor protein levels are part of the
mechanism. This leaves eIF2
phosphorylation as the only candidate
mechanism by which eIF2
could be involved.
Detection of eIF2 phosphorylation validation of the
methods
The western analysis presented us with three initial problems. First, we
needed to be able to detect eIF2 in two distantly related species. We
therefore used an antibody based on a region conserved across all eukaryotes,
the phosphorylated regulatory phosphorylation site. We were able to use this
antibody to detect eIF2
(P) in both snail and frog extracts. The
apparent size of the eIF2
(P) (39 kDa) was the same for both H.
aspersa and N. sutor and was similar to the apparent size of
mammalian eIF2
in SDSPAGE (38 kDa). The 42 kDa band detected in
H. aspersa extracts was phosphorylated by HCR in an
individual-specific manner. Since all eIF2
s described so far have
similar molecular masses (Deharo et al.,
1996
), it is unlikely that a 42 kDa protein is an eIF2
. It
is unknown if it represents a physiological substrate for HCR, but it is
likely that it shares sequence homology with the eIF2
phosphorylation
site since it is recognised by both HCR and the primary antibody.
The use of this antibody led to the second problem, which was that this
antibody measures the levels of eIF2(P) but gives no information about
the more physiologically relevant ratio of phosphorylated to unphosphorylated
eIF2
. This is especially true when long-term changes are involved, as
changes in this ratio can occur without changes in the amount of
eIF2
(P). We overcame this problem by totally phosphorylating all of the
endogenous eIF2
in a sample, thereby enabling us to measure the total
eIF2
as well as the phosphorylated form with the same antibody. The
system we describe allows the use of SDSPAGE with a single primary
antibody, allowing simpler quantification of eIF2
and eIF2
(P)
levels. This system relies on a single antibody specific for the conserved
phosphorylation site and on phosphorylation by HCR, which has been shown to
phosphorylate eIF2
from all eukaryotes tested so far
(Mehta et al., 1986
).
Therefore, this system potentially enables the phosphorylation state of
eIF2
to be determined in any eukaryotic system.
The third problem in this study was that we needed to quantify levels of
eIF2(P) in order to accurately estimate the ratio of
eIF2
/eIF2
(P). To this end, we have:
The absolute levels of eIF2 that we report here (in terms of the
percentage of total protein present) in awake H. aspersa
hepatopancreas (0.002%) and N. sutor liver (0.005%) are of a
comparable magnitude to those reported for rat liver (0.004%;
Everson et al., 1989
) and
rabbit tissues (0.003%; Oldfield et al.,
1994
) (assuming that these mammalian tissues contain 100 mg
protein g-1 wet mass). Using the same system to determine the level
of eIF2
in neonatal rat cardiomyoctes
(Casey et al., 2002
), we have
found that approximately 0.006% of total protein is eIF2
, a value
comparable with other mammalian systems.
Changes in phosphorylation state of eIF2 with estivation
The extent of eIF2 phosphorylation required to significantly
suppress protein synthesis depends upon the eIF2/eIF2B ratio, as it is the
high-affinity binding of eIF2
(P) to eIF2B that results in the
inhibition of protein synthesis. This value is known to be somewhat variable
among different animals and tissues. For example, the ratio is 5 in calf and
rat brain (White et al.,
2000
), 2.5 in rabbit liver and 10 in rabbit brain
(Oldfield et al., 1994
). The
variation appears to predominantly depend on eIF2B levels
(Oldfield et al., 1994
). The
ratio of eIF2/eIF2B is a good predictor of the level of eIF2
phosphorylation required to completely inhibit protein synthesis
(Everson et al., 1989
). For
example, in reticulocyte lysates the ratio of eIF2/eIF2B is approximately 5,
and the level of eIF2
phosphorylation required to completely inhibit
protein synthesis is approximately 2530%
(Pain, 1996
). In Ehrlich
cells, the ratio of eIF2/eIF2B is approximately 2, and the level of
eIF2
phosphorylation required to completely inhibit protein synthesis
is approximately 5060% (Scorsone et
al., 1987
). The data for snails are inconclusive because
endogenous levels of the phosphorylated form of eIF2
are below the
limit of detection. However, the data are still useful as they define the
limits of the eIF2
/eIF2
(P) ratio, which in turn (see above) can
be used to predict the eIF2/eIF2B ratio. The eIF2/eIF2B ratio is not known for
any non-mammalian species, but our data suggest (since we can calculate that
less than 12% of the eIF2
was phosphorylated in the snail lysates) that
in H. aspersa hepatopancreas the ratio would have to exceed 10 for
phosphorylation of eIF2
to exert any control over the rate of protein
synthesis.
The data from the frog liver are easier to interpret as both endogenous
eIF2 and eIF2
(P) are detectable. The percentage of
eIF2
(P) and, in contrast to the snail, the total amount of eIF2
increases from the awake to the estivating state. The increased percentage of
phosphorylated eIF2
in the estivating state is consistent with the 67%
decrease in the rate of protein synthesis seen in N. sutor liver in
this state (Fuery et al.,
1998
). Due to the high proportion of eIF2
that is
phosphorylated, for eIF2
phosphorylation to be involved in regulating
the rate of protein synthesis in estivating N. sutor liver the ratio
of eIF2/eIF2B would only need to be approximately 2. The function of
increasing the level of eIF2
during estivation in the frog
versus the snail is unknown but perhaps the difference reflects the
degree of precision required to modulate the rate of protein synthesis
(assuming that levels of eIF2B remain constant). For example, a low eIF2/eIF2B
ratio under normal conditions would allow a high precision in controlling the
rate of protein synthesis, as large changes in the percentage phosphorylation
would result in relatively small changes in the rate of protein synthesis.
However, with a low ratio it would be more difficult to completely
downregulate protein synthesis, as a greater percentage of the eIF2
would need to be phosphorylated. Therefore, by upregulating the amount of
eIF2
it would be easier to obtain the percentage of eIF2
(P) that
is required to downregulate protein synthesis during estivation. Possibly, the
snail needs less precision in its control over protein synthesis during normal
conditions and therefore maintains a high eIF2/eIF2B ratio, which allows it to
completely downregulate the rate of protein synthesis during estivation
without needing to synthesise more eIF2
. This idea would seem
consistent with the greater metabolic depression during estivation in the
snail compared with the frog.
An alternative explanation is that the differences are not functional but
obligatory. Possibly, the phosphorylated form of eIF2 is less
susceptible to proteolytic breakdown; thus, upregulating the phosphorylated
species [eIF2
(P)] is necessarily associated with an increase in the
total level of eIF2
. There is evidence for a similar mechanism in
apoptotic HeLa cells, where caspase cleaves eIF2
but shows a preference
for the unphosphorylated form and does not cleave eIF2
(P) complexed
with eIF2B (Marissen et al.,
2000
). If this is a similar case, then the increased level of
eIF2
expression seen in the estivating frog is not the result of an
active upregulation of eIF2
levels but is due to a higher proportion of
the more stable species [eIF2
(P)]. The data are consistent with this
hypothesis, as the increase in the amount of eIF2
in N. sutor
during estivation is equal to the increase in the amount of
eIF2
(P).
Potential mechanisms for changes in eIF2
phosphorylation status in N. sutor
There are several possible mechanisms by which the percentage of
eIF2(P) could increase in the liver of estivating N. sutor.
The downregulation of protein synthesis in other systems has been attributed
to an increase in eIF2
kinase activity. Specific eIF2
kinases
that are upregulated in activity in response to diverse stimuli, including
heme deprivation, dsRNA, amino acid deprivation and endoplasmic reticulum
stress, have been identified.
Alternatively, an increase in eIF2(P) can occur due to a decrease in
eIF2
phosphatase activity. For example, during hibernation in ground
squirrels, the accumulation of eIF2
(P) has been attributed to a
decrease in phosphatase (PP1) activity modulated by the stress-inducible
growth arrest and DNA damage protein GADD34
(Connor et al., 2001
). A
similar mechanism may regulate protein synthesis in turtles that depress both
metabolic rate and protein synthesis during anoxia, as during anoxia there is
a decrease in PP1 activity in liver and brain
(Mehrani and Storey,
1995
).
Aside from modulation of kinase and/or phosphatase activity, the
accumulation of eIF2(P) in N. sutor could also be due to a
change in accessibility of eIF2
to kinase or phosphatase activity. For
example, changes in the affinity of p67 (a cellular glycoprotein found in
reticulocyte lysate, which binds the
-subunit of eIF2 and masks the
regulatory phosphorylation site of eIF2
) could regulate changes in
eIF2
phosphorylation state (Datta
and Datta, 1999
).
Summary
This is the first study to investigate the regulation of the initiation of
translation by eIF2 in estivating organisms and provides evidence for a
possible mechanism by which the rate of protein synthesis is downregulated
during the metabolic depression associated with estivation. By finding a
change in the phosphorylation status of a key translation regulatory protein
(eIF2
) in N. sutor during estivation, we have provided the
potential starting point for a link between the downregulation of an
energy-consuming pathway and the initial intrinsic cue for metabolic
depression associated with estivation. Future work will need to establish the
ratio of eIF2
/eIF2B and also estimate changes in eIF2B activity in
these tissues to determine if the changes in the percentage of eIF2
phosphorylation in N. sutor could account for the observed changes in
the rate of protein synthesis. If the increase in phosphorylated eIF2
does lead to a decrease in guanine nucleotide exchange on eIF2, then
identifying whether the increased phosphorylation is due to increased kinase
or decreased phosphatase activity will be the next necessary step in
delineating the mechanism of signal transduction between the intrinsic cue for
estivation and the downregulation of protein synthesis. In the case of H.
aspersa, future investigation is needed to determine whether there is a
role for eIF2
phosphorylation in the downregulation of protein
synthesis, possibly by directly assaying guanine nucleotide exchange on eIF2
in the awake and estivating states. Virtually nothing is known about the
potential regulatory mechanisms in any estivating animal, but the data are
accumulating and suggest that research in this direction will be an effective
strategy to delineate the mechanisms of this common, but inscrutable, form of
metabolic depression.
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