Tài liệu Báo cáo khoa học: Expression of poly(A)-binding protein is upregulated during recovery from heat shock in HeLa cells doc

Expression of poly(A)-binding protein is upregulated during
recovery from heat shock in HeLa cells
Shuhua Ma*, Rumpa B. Bhattacharjee* and Jnanankur Bag
Department of Molecular and Cellular Biology, University of Guelph, Canada
In response to extracellular signals, cells regulate pro-
tein synthesis, and adapt to environmental changes
such as the level of growth factors, temperature, and
availability of nutrients [1]. The regulation of protein
synthesis is a complex process, as many factors are
required for mRNA translation. Translation initiation
is believed to be the rate-limiting step in protein syn-
thesis, and is usually regulated by several important
initiation factors, including poly(A)-binding protein
(PABP) 1 and eukaryotic initiation factors (eIFs) 4E,
4G, 2a, and 4A. eIF4E binds to the 5¢-cap of mRNA to
facilitate initiation of translation. eIF4A is a helicase
enzyme that removes the secondary structure from the
5¢-UTR of mRNA, and eIF4G acts as a bridge protein
to interact with eIF4E, PABP1, eIF4A and eukaryotic
release factor 3 [1,2]. At the beginning of the transla-
tion initiation cycle, eIF2a forms a ternary complex
with met-tRNAi and GTP [3]. Fine tuning of these
steps is achieved by regulating the interaction of these
initiation factors with each other and the mRNA [1].
PABP1 is an RNA-binding protein with a high
affinity for the poly(A) tail of mRNA [4]. It is mainly
distributed in the cytoplasm, and may shuttle between
the cytoplasm and the nucleus [5]. By interacting with
Keywords
eIF4G; HSP27; HSP70; mRNA translation;
poly(A)-binding protein

Correspondence
J. Bag, Department of Molecular & Cellular
Biology, University of Guelph, Guelph,
ON N1G2W1, Canada
Fax: 1 519 837 2075
Tel: 1 519 824 4120
E-mail:
*These authors contributed equally to this
work
(Received 5 August 2008, revised 31
October 2008, accepted 14 November
2008)
doi:10.1111/j.1742-4658.2008.06803.x
Induction of heat shock proteins (HSPs) helps cells to survive severe hyper-
thermal stress and removes toxic unfolded proteins. At the same time, the
cap-dependent translation of global cellular mRNA is inhibited, due to the
loss of function of eukaryotic initiation factor (eIF)4F complex. It has been
previously reported that, following heat shock, HSP27 binds to the insolu-
ble granules of eIF4G and impedes its association with cytoplasmic
poly(A)-binding protein (PABP) 1 and eIF4E. In the studies reported here,
in addition to heat shock, we have included results of our investigation on
the association between eIF4G, PABP1 and HSP27 during recovery from
heat shock, when cap-dependent mRNA translation resumes. We showed
here that in the heat-shocked cells, the PABP1–eIF4G complex dissociated,
and both polypeptides translocated with the HSP27 to the nucleus. During
recovery after heat shock, PABP1 and eIF4G were redistributed into the
cytoplasm and colocalized with each other. In addition, PABP1 expression
was upregulated and its translation efficiency was increased during the
recovery period, possibly to meet additional demands on the translation
machinery. HSP27 remained associated with the eIF4G–PABP1 complex

during recovery from heat shock. Therefore, our results raise the possibility
that the association of HSP27 with eIF4G may not be sufficient to suppress
cap-dependent translation during heat shock. In addition, we provide
evidence that the terminal oligopyrimidine cis-element of PABP1 mRNA
is responsible for the preferential increase of PABP1 mRNA translation
in cells undergoing recovery from heat shock.
Abbreviations
ARS, autoregulatory sequence; eIF, eukaryotic initiation factor; FITC, fluorescein isothiocyanate; b-gal, b-galactosidase; GFP, green
fluorescent protein; HSP, heat shock protein; PABP, cytoplasmic poly(A)-binding protein; TOP, terminal oligopyrimidine tract.
552 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
eIF4G and the poly(A) tail simultaneously, PABP1 is
believed to bring the 5¢-end and 3¢-end of mRNA into
close proximity and stimulate the translation of
mRNA by forming a closed-loop structure. Circular-
ization of mRNA is believed to promote the transla-
tion reinitiation by recycling 40S ribosomal subunit for
the next round of translation [1]. Interactions with
additional regulatory proteins allow PABP1 to regulate
the translation initiation step [6–11]. For example,
PAIP1 interacts with PABP1 through its ‘PABP1-inter-
acting motif 1’, containing a stretch of acidic amino
acids [8], and acts as a translational enhancer. Over-
expression of PAIP1 increases the rate of mRNA
translation in COS cells [9]. Another regulatory pro-
tein, PAIP2, disrupts the interaction of PABP1 with
PAIP1, and suppresses PABP1-dependent mRNA
translation [8,10]. Both PAIP1 and PAIP2 have similar
PABP1-binding motifs, and compete for binding with
PABP1 [10]. As such, PAIP2 works as an inhibitor of
mRNA translation by competing with PAIP1. Eukary-

otic release factor 3 also interacts with PABP1, and
facilitates termination of translation, and reinitiation
by 40S ribosomal subunits, by maintaining the quasi-
circular structure of the translating mRNA [6]. Fur-
thermore, PABP1 is believed to protect mRNAs from
degradation by binding to their poly(A) tract [12].
Studies have shown that the interaction between
PABP1 and the poly(A) tract may be regulated by
PABP1-interacting partners. For example, the AU-rich
element-binding polypeptide AUF1 may control
mRNA stability by binding to PABP1 [7].
In light of the vital roles of PABP1 in mRNA
metabolism, it is critical to control the expression of
PABP1. Expression of PABP1 is regulated primarily at
the translational level. Two cis-elements; a terminal
oligopyrimidine tract (TOP) and a unique A-rich auto-
regulatory sequence (ARS), are located in the 5¢-UTR
of PABP1 mRNA [13,14]. The ARS regulates the
expression of PABP1 by a negative feedback mecha-
nism. In the presence of excess PABP1 in mammalian
cells, PABP1 binds to the ARS element of its own
mRNA and forms a heteromeric autoregulatory ribo-
nucleoprotein complex by interacting with IMP1 and
UNR [11]. This process stalls the migration of 40S
ribosomal subunits along the 5¢-UTR of the PABP1
mRNA and limits its expression [13]. In comparison to
the role of the ARS, the precise mechanism by which
the TOP regulates PABP1 mRNA translation is not
clear. It appears that the TOP cis-element stimulates
translation of PABP1 mRNA in a developmental- and

growth-dependent manner [14]. As such, the TOP may
allow translation of PABP1 mRNA to be coordinately
regulated with a number of other TOP-containing
mRNAs encoding polypeptides involved in mRNA
translation, including elongation factors 1 and 2 and
ribosomal protein S6 kinases [15].
The PABP1 gene behaves like an early response
gene, as its expression level responds quickly to a
change in the cellular demand for protein synthesis
[16]. Under a variety of cellular stress conditions,
such as heat shock, mRNA translation undergoes
rapid changes. In response to heat shock stress, cells
induce the expression of a unique set of proteins
called heat shock proteins (HSPs) [17,18]. HSPs are
also produced at a basal level in cells under normal
conditions, and have important functions such as
facilitating appropriate protein folding as molecular
chaperones, and aiding in the assembly of protein
complexes; they also participate in translocation of
proteins across cellular membranes, as well as provid-
ing protection against cellular stresses [18]. HSPs are
also termed ‘stress proteins’, because they are overex-
pressed to improve cell survival under a variety of
additional cell-damaging conditions, such as exposure
to heavy metal ions, alcohol, and hypoxia, and glu-
cose deprivation [3]. There are several HSP families,
including HSP90, HSP70, HSP60, HSP110, and the
low molecular weight HSPs such as HSP10, HSP20,
HSP27, HSP32, and HSP40 [18]. The predominant
HSPs, HSP70 and HSP27, have been well studied.

HSP70 and HSP27 have been found both in the cyto-
plasm and in the nucleus during heat shock, although
they are constitutively localized in the cytoplasm at a
low level [18]. Expression of HSPs is induced at the
transcriptional level by heat shock factors, which are
activated during heat shock and bind to the heat
shock elements located in the promoter regions of
HSP genes [19].
During heat shock, the general translation of cap-
dependent cellular mRNAs is inhibited, whereas the
synthesis of HSPs is increased [3,20]. The mechanism
that underlies this is not well understood. It is
believed that mRNAs encoding HSPs are translated
in an internal ribosome entry site-dependent manner,
which is enhanced by heat shock [3,20,21]. It has also
been reported that heat shock enhances the transla-
tion of BiP, and viral mRNAs, which are dependent
on internal ribosome entry site-mediated translation
[20]. During heat shock, the inhibition of cap-depen-
dent translation probably takes place through inacti-
vation of the eIF4F complex and other initiation
factors, and this is supported by the following
changes observed in heat-shocked cells: decreased
phosphorylation of eIF4E and eIF4B, increased phos-
phorylation of eIF2a, and insolubilization of eIF4G
[20,22]. The phosphorylation of eIF2a is an important
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 553
mechanism for regulating global protein synthesis.
Primarily, eIF2a plays its role by forming the eIF2a–

GTP complex, which is hydrolyzed to an inactive
eIF2a–GDP complex at the end of the initiation
cycle. The GDP is then released from the eIF2a–
GDP complex, and reactivates eIF2a for the next
round of translation [3]. When eIF2a is phosphory-
lated, the GDP cannot be released from it, and the
global mRNA translation rate decreases. It has been
shown that eIF2a is increasingly phosphorylated from
mild to severe heat shock conditions as the cells are
subjected to an increasing temperature, and protein
synthesis is repressed by the presence of hyperphosph-
orylated eIF2a. However, HeLa cells subjected to a
mild heat shock (42 °C for 30 min) did not show
increased eIF2a phosphorylation, even though protein
synthesis was inhibited [3,20]. Thus, it has been sug-
gested that phosphorylation of eIF2a may not be the
dominant mechanism of inhibition of translation of
normal cellular mRNAs under heat shock, and there-
fore other regulatory mechanisms must exist.
The reduction in elF4F activity is probably also
responsible for suppression of translation of most
mRNAs during heat shock [22]. During heat shock
(44 °C, 2 h) in 293T cells, eIF4G was shown to be
trapped within the insoluble heat shock granules by
HSP27, and dissociated from PABP1. It has been sug-
gested that eIF4F-dependent mRNA translation is
inhibited by heat-induced HSP27 during heat shock
[22].
In addition to eIF4G, the PABP1 gene as an early
response gene may play a very important role in the

regulation of gene expression in response to cellular
stresses. However, in mammalian cells, there is insuf-
ficient evidence regarding the molecular behavior of
PABP1 under heat shock. Therefore, in order to
investigate how PABP1 responds to thermal stress
and whether its expression is regulated to cope with
the changing demand for mRNA translation during
and after heat shock, we examined the status of
PABP1 expression in HeLa cells subjected to heat
shock. We investigated the subcellular localization of
PABP1 and its polypeptide partner eIF4G by using
immunofluorescence confocal microscopy. The results
show that PABP1 and eIF4G become insoluble and
are translocated into the nucleus from the cytoplasm
in a time- and temperature-dependent manner. We
propose that the nuclear translocation of PABP1 and
eIF4G is associated with the induction and nuclear
translocation of HSP27 by heat shock, because both
PABP1 and eIF4G were found to be colocalized with
HSP27 around the perinuclear region before being
translocated to the nucleus. We also found that
PABP1 mRNA translation was upregulated during
recovery after heat shock, and that this was con-
trolled at the translational level by the 5¢-TOP
element located in the 5¢-UTR of PABP1 mRNA. We
suggest that increased expression of PABP1 during
the phase of recovery from heat shock may be neces-
sary to meet the cellular demand for protein synthesis
for complete recovery from stress.
Results

PABP1 expression during recovery from
heat shock
As PABP1 is important for regulation of mRNA
translation, we examined whether its cellular level is
adjusted during heat shock and subsequent recovery
periods. The results of western blot analyses (Fig. 1)
show that there was a small reduction in the cellular
abundance of PABP1 and its polypeptide partner
eIF4G immediately after the heat shock treatment.
However, during the period of recovery from heat
shock, when translation of normal cellular mRNA
resumes, there was an approximately 2.5-fold increase
in the cellular PABP1 level. In contrast, the cellular
level of eIF4G did not show a similar increase. The
increase of PABP1 abundance also correlated very well
with the increased expression of HSP27 and HSP70. In
order to assess whether the increased PABP1 level was
due to an increase in the cognate mRNA level, we
measured the PABP1 mRNA level by real time
RT-PCR. The results (Fig. 2A) show that the PABP1
mRNA levels were not altered by exposure to heat
shock or following the subsequent recovery phase. As
these results suggest translational control of PABP1
expression, we examined the distribution of PABP1
mRNA between the translationally active polysomes
and repressed subpolysomal fractions by sucrose gradi-
ent fractionation, as previously described [23]. The
results (Fig. 2B–D) show that in exponentially growing
HeLa cells, approximately 30–40% of cytoplasmic
PABP1 mRNA was present in the translated polyso-

mal fractions (Fig. 2D), and the remaining mRNA was
present in the nontranslated subpolysomal fractions. In
contrast, almost 90–100% of the b-actin mRNA was
present in the polysomal fractions. There was a signifi-
cant reduction in the translation of the b-actin mRNA
in heat-shocked cells, as the majority of this mRNA
was present in the nontranslated subpolysomal frac-
tions. However, during recovery from heat shock at
37 °C, the translation of PABP1 mRNA was
enhanced, as nearly 80–90% of PABP1 mRNA was
found in the polysomal fractions. This represents an
PABP expression during heat shock recovery S. Ma et al.
554 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
increased efficiency of PABP1 mRNA translation over
the level normally found in exponentially growing
cells. This observation suggests that the increased
abundance of PABP1 during recovery from heat shock
is controlled at the mRNA translation level. The
increase of PABP1 mRNA translation was preferential,
as the polysomal distribution profile of the b-actin
mRNA in cells during the recovery from heat shock
appeared to be similar to what was observed in the
exponentially growing cells.
Association between PABP1 and eIF4G following
heat shock and recovery
PABP1 is known to interact with eIF4G to enhance
cap- and poly(A)-dependent mRNA translation.
Therefore, we examined the association between
PABP1 and eIF4G in HeLa cells, immediately after
the heat shock treatment, and during the recovery

phase of cells, using both coimmunoprecipitation and
immunofluorescence microscopy. The results of coim-
munoprecipitation studies (Fig. 3A,C) show that
although the cellular abundance of eIF4G was not
affected significantly during heat shock and recovery,
the level of its association with PABP1 was reduced.
As compared to untreated control cells, after a 2 h
heat shock treatment, approximately 40–50% less
PABP1 coimmunoprecipitated with eIF4G. This reduc-
tion, however, was a little higher than what was
expected from the reduced abundance of both polypep-
tides in heat-shocked cells. During the recovery period
from heat shock, as compared to the non-heat-shocked
control cells, almost a three-fold increase in the associ-
ation of PABP1 with eIF4G was observed. This
increase occurred without a concomitant increase in
the cellular level of eIF4G during recovery from the
thermal stress. As the cellular abundance of PABP1 in
cells recovered from heat shock also increased approxi-
mately three-fold, our results suggest that the majority
of excess PABP1 was associated with eIF4G during
the recovery phase. We also examined the association
between PABP1 and eIF4G in RNase-treated cell
extracts, and found that the coimmunoprecipitation of
PABP1 by eIF4G antibody was independent of the
presence of intact mRNA. We used b-actin as a nega-
tive control, and did not detect this polypeptide in our
immunoprecipitated samples with the eIF4G antibody.
In addition, as a loading control, we measured the
level of b-actin in total cell extracts before they were

subjected to immunoprecipitation. As eIF4E is a
known subunit of the multimeric complex with eIF4G,
we also analyzed its coimmunoprecipitation as a posi-
tive control. The results show that there was no signifi-
cant change in the association between eIF4G and
eIF4E following heat shock and during the recovery
phase. We also used nonimmunized rabbit sera for
mock immunoprecipitation as additional negative con-
trols, and the results (Fig. 3B) show that both PABP
and eIF4G were not detectable in the immunoprecipi-
tated samples under our experimental conditions.
The average results of three independent immuno-
precipitation studies using the eIF4G antibody are
shown in Fig. 3D as the percentages of total PABP1
and eIF4G in the immunoprecipitated samples. In
A
B
Fig. 1. Changes in the cellular level of polypeptides following heat
shock and recovery. (A) Approximately 5 · 10
5
HeLa cells grown on
35 mm dishes were subjected to heat shock at 44 °C for 2 h (HS).
Cells in some dishes were allowed to recover for either 12 h (Re12h)
or 24 h (Re24h) at 37 °C. The control cells (C) were not heat shocked
and were maintained at 37 °C. Cells were directly lysed on the plate
using gel loading buffer, and lysates were analyzed by SDS ⁄ PAGE.
Individual polypeptides were detected by western blotting using
appropriate antibodies as described in Experimental procedures. The
abundance of specific polypeptides was determined by scanning the
images, and normalizing the values using the b-actin levels as load-

ing controls. The values below each lane represent the relative abun-
dance of each polypeptide using an arbitrary scale where the level in
control cells was considered to be 1.00. (B) The experiment
described in (A) was repeated three times, and the averages are
shown here as mean ± standard error (SE). The linear response of
the western blotting experiments is shown in Fig. S1.
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 555
non-heat-shocked control cells, approximately 50% of
total cellular PABP1 was associated with eIF4G. In
heat-shocked cells, the level of eIF4G-associated
PABP1 was reduced to approximately 25% of total
cellular PABP1. However, following 12 or 24 h of
recovery, the level was increased to approximately
75% of total cellular PABP1. In addition, as expected,
all samples showed 90–100% of total eIF4G in the
immunoprecipitates.
PABP1 is a phosphoprotein, and its interaction with
poly(A) and eIF4G is enhanced by phosphorylation
[24]. Therefore, we investigated PABP1 phosphoryla-
tion by two-dimensional gel electrophoresis, followed
by western blotting as previously described [25]. In
exponentially growing cells (Fig. 3B), PABP1 was pres-
ent in multiple phosphorylated states, as shown by its
different isoelectric points. Upon inhibition of phos-
phorylation by U0126, an inhibitor of the MKK2
kinase pathway [25,26], PABP1 migrated as a more
basic protein with a higher isoelectric point, and
appeared as a single spot in two-dimensional gels. Fol-
lowing 24 h of recovery from heat shock, the abun-

dance of PABP1 increased, and nearly all of this
polypeptide was found in hyperphosphorylated forms
migrating with lower isoelectric points than what was
observed for the nonphosphorylated polypeptide.
Thus, the increased PABP1 phosphorylation might
explain the observed enhanced association of PABP1
with eIF4G in our coimmunoprecipitation studies
during recovery from heat shock.
In order to observe the association of PABP1 with
eIF4G in individual cells, confocal immunofluorescence
microscopy was performed after heat shock and recov-
ery of HeLa cells. The results (Fig. 4) show that in expo-
nentially growing cells at 37 °C, PABP1 was colocalized
with eIF4G. However, a significant amount of PABP1
also appeared within distinct cytoplasmic locations that
were not colocalized with eIF4G. Both eIF4G and
PABP1 showed diffuse distribution within the cyto-
plasm. Following heat shock, both PABP1 and eIF4G
B
40s
60s
80s
Polysomes
1
3 5 7 9 11
Fraction Number
A254nm
0.4
0.2
D

β-actin
80
90
100
PABP
30
40
50
60
70
0
10
20
% in polysome
Fraction Number (6-11)
-HS
+HS
Re24
1.0
1.2
1.4
con HS
Re12 h Re24 h
0.2
0.4
0.6
0.8
Relative mRNA level
A
0.0

PABP
β-actin
C
-HS (control)
HS
β-actin
-HS (control)
Re24 h
Re24 h
PABP
HS
Fractions: 1 2 3 4 5 6 7 8 9 10 11
Fig. 2. The abundance and polysomal distribution profiles of mRNA following heat shock and recovery. Cells were subjected to heat shock
and recovery as described in the legend to Fig. 1. Either the total cellular RNA was isolated and analyzed by real time RT-PCR (A) or the
cytoplasmic extract was subjected to sucrose density gradient centrifugation as described in Experimental procedures (B). The gradient was
fractionated, and RNA was isolated from individual fractions using Triazole (Roche). The abundance of PABP1 and b-actin mRNAs in each
fraction was measured by RT-PCR as described in Experimental procedures. For each sample, a different number of amplifications cycles
was used to determine the linear range. Twenty-two and 25 cycles were found to be optimum for b-actin and PABP1 mRNA, respectively.
Each RNA fraction was also subjected to the PCR step without the prior reverse transcription step to determine DNA contamination in our
samples, and none was found. The amplicons were analyzed by 1% agarose gel electrophoresis, and quantified by scanning the digital
image. Representative results of three separate experiments are shown in (C). The distribution of mRNA in the subpolysomal region (fraction
numbers 1–5) and the polysomal region (fraction numbers 6–11) of the gradient was determined by quantifying the mRNA level in each frac-
tion using an arbitrary scale. The value of mean ± standard error was derived from three separate experiments and is presented in (D).
PABP expression during heat shock recovery S. Ma et al.
556 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
were translocated to the nucleus in a time- and tempera-
ture-dependent fashion. After 1.5 h of heat shock at
44 °C, nearly all of the eIF4G and PABP1 was present
at the perinuclear site and remained colocalized,
although some granular structures were also visible. The

cytoplasmic b-actin also showed some aggregated gran-
ular structures and perinuclear localization in approxi-
mately 30–40% of cells. In contrast, the perinuclear
granular localization of eIF4G and PABP1 was more
pronounced and noticeable in a much larger percentage
(> 85%) of cells. Following 2 h of heat shock at 44 °C,
almost complete translocation of both eIF4G and
PABP1 within the cell nucleus as granules was observed.
During the same treatment, b-actin remained mostly
cytoplasmic, with some granular aggregates. Similar
nuclear translocation was also observed within a shorter
time period of 1–1.5 h when the cells were heat shocked
at 46 °C (results not shown). Following nuclear translo-
cation, we observed detectable dissociation of some
eIF4G granules from PABP1 (Fig. 4, enlarged inset).
Furthermore, neither polypeptide was present within the
nucleolus. Interestingly, during the recovery period,
PABP (-RNase)
eIF4G
PABP (+ RNase)
eIF4E
β-actin (Co-IP)
PABP
eIF4G
3
3.5
4
4.5
con
HS

1
1.5
2
2.5
Re12 h
Re24 h
con HS
0
0.5
eIF4G PABP
60
80
100
Re12 h
Re24 h
0
20
40
60
% of input
PABP eIF4G
Relative amount in
co-immunoprecipitates
β-actin
total cell extract
1.0 0.5 3.0 4.0
Con
AC
D
-+

Control
Re24 h
+
20um U0126
PABP
Re24 h
β-actin
Control
pH 116
E
B
Re12 h Re24 h
1.0 0.5 3.4 4.0
1.0 1.0 1.1 1.0
1.1 0.9 0.9 1.0
1.0 0.9 1.0 0.9
HS
Fig. 3. Coimmunoprecipitation of polypeptides with antibody to eIF4G and analyses of PABP1 phosphorylation. (A) Approximately
5 · 10
5
HeLa cells following heat shock and recovery were lysed in a lysis buffer and reacted with the eIF4G antibody as described in Exper-
imental procedures. The antigen–antibody complex was captured with protein A–Sepharose beads and eluted with SDS containing gel load-
ing buffer. The eluted samples were subjected to SDS ⁄ PAGE and western blotting using antibodies against PABP1, eIF4G, eIF4E and
b-actin (Santa Cruz) to detect coimmunoprecipitated polypeptides, according to the method described in Experimental procedures. Some
samples were treated with 1 lgÆlL
)1
RNase A and T1 for 10 min at 20 °C to degrade RNA before addition of the antibody. The effective-
ness of this treatment was determined by examining the absence of intact rRNA by gel electrophoresis of RNA samples prepared from the
treated cell extracts. (B) Mock immunoprecipitation was performed using 1.5 lg of preimmunized rabbit serum, and the eluted fractions
from protein A–Sepharose beads were examined for the presence of PABP1 and eIF4G as described above. (C) Western blots of three sepa-

rate experiments were scanned and quantified to determine the value of mean ± standard error (SE). (D) Equivalent cellular levels of total
cell lysate and immunoprecipitated fractions eluted from the protein A–Sepharose beads were examined for the presence of eIF4G and
PABP1 by western blotting. Total cell lysate and eluted fractions were analyzed together in the same blot, and quantified as described
above. The averages of three independent experiments are shown. (E) Cells subjected to different treatments were lysed and subjected to
two-dimensional gel electrophoresis as described in Experimental procedures. The separated polypeptides were transferred to a nitrocellu-
lose membrane for immunoblotting with either a PABP1 or b-actin antibody. Control, cells before heat shock; Re24h, cells were heat
shocked and allowed to recover for 24 h as previously described; U0126, cells were treated with the inhibitor of MKK1 ⁄ 2 U0126 (20 l
M) for
the last 12 h of the 24 h recovery period of heat-shocked cells.
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 557
PABP1 and eIF4G were gradually redistributed to the
cytoplasmic compartment. In addition, the colocaliza-
tion of PABP1 and eIF4G was re-established. After
12 h of recovery at 37 °C, both eIF4G and PABP1
appeared in the perinuclear cytoplasmic space, with
some granules. Both polypeptides showed a predomi-
nantly diffuse distribution almost throughout the cyto-
plasm after 24 h of recovery. This cytoplasmic
redistribution occurred in the absence of new protein
synthesis. In cycloheximide-treated cells during a 20 h
Fig. 4. Colocalization of eIF4G and PABP1. HeLa cells, grown on coverslips placed in 35-mm tissue culture dishes, were subjected to heat
shock and recovery or maintained at 37 °C as described in the legend to Fig. 1. Cells were fixed in methanol, and treated with the appropri-
ate primary antibody followed by a secondary antibody: either FITC-labeled anti-goat (for eIF4G) IgG or Texas Red-labeled anti-mouse IgG (for
PABP1). The b-actin signal was detected by using an FITC-labeled mouse secondary antibody. Cells were examined by confocal microscopy
using FITC- and Texas Red-specific filters. Approximately 200 cells were examined in each of three separate experiments, and the results
given here represent more than 80% of cellular images. The inset shows the enlarged view of one nucleus marked with an arrowhead.
PABP expression during heat shock recovery S. Ma et al.
558 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
recovery period in the absence of protein synthesis, both

eIF4G and PABP1 were able to relocate to the cyto-
plasm and were mostly colocalized. This suggests that
the pre-existing nuclear PABP1 and eIF4G in heat-
shocked cells could travel to the cytoplasm and reassoci-
ate during the recovery phase. However, the cellular
abundance of both polypeptides was reduced as a result
of inhibition of new protein synthesis. As mRNA trans-
lation was inhibited by cycloheximide treatment, we
could not determine whether the relocated PABP1 and
eIF4G were capable of participating in mRNA transla-
tion. eIF4G and PABP1 appeared to be completely
colocalized in cells that had recovered for 12 and 24 h.
As all of the PABP1 in exponentially growing cells was
not colocalized with eIF4G, these observations support
the coimmunoprecipitation results, which suggest an
increased association between the two polypeptides dur-
ing the recovery period. It is possible that post-transla-
tional modifications of polypeptides during the recovery
period might allow PABP1 to interact with eIF4G at a
stoichiometry of more than 1 : 1. It should be noted
here that, in addition to the C-terminal domain, PABP1
has another eIF4G-binding domain within its N-termi-
nal RNA-binding domain 1 [27]. As a negative control,
we also examined the colocalization of b-actin with
eIF4G in exponentially growing cells. As is evident from
the confocal images, b-actin was not colocalized with
eIF4G in exponentially growing cells. Furthermore, fol-
lowing recovery from heat shock, the cytoplasmic distri-
bution of b-actin returned to what was observed in
exponentially growing cells.

It has been previously reported that after heat
shock, eIF4G is present in detergent-insoluble gran-
ules [22]. Therefore, we examined whether PABP1 is
also present in similar granules, and whether transi-
tion to the normal soluble form takes place for both
PABP1 and eIF4G during recovery from heat shock.
Analyses of nonionic detergent-treated cells by immu-
nofluorescence confocal microscopy (Fig. 5) show that
detergent treatment of normal cells removed almost
all of their PABP1 and eIF4G content. In heat-
shocked cells, prior to the recovery period the major-
ity of nuclear-translocated PABP1 and eIF4G was
present as detergent-insoluble granules. During the
recovery period, as shown in Fig. 4, both PABP1 and
eIF4G were redistributed to the cytoplasm, and fol-
lowing the nonionic detergent treatment, both poly-
peptides were extracted from the cells. We also
examined the detergent solubility of another nuclear
protein, PABPN1. Cells were transfected with a PAB-
PN1-A10–green fluorescent protein (GFP) fusion pro-
tein expression vector [28] before being subjected to
the detergent treatment. We show here that most of
the PABPN1–GFP was extracted from the control
cells. In untreated controls, approximately 80–90% of
cells showed diffuse GFP signal throughout the
nucleoplasm. In detergent-treated samples, the diffuse
GFP signal was lost from most of the cells. Approxi-
mately 10–20% of cells showed the presence of some
detergent-insoluble nuclear aggregates. This was
expected, as PABPN1 is known to form nuclear

aggregates, especially at a very high expression level
[28]. Together, our results suggest that, following heat
shock treatment, both eIF4G and PABP1 translocate
to the cell nucleus as detergent-insoluble granular
aggregates.
Cis-acting translational control element involved
in regulating PABP1 mRNA translation during
recovery from heat shock
The mRNA encoding PABP1 consists of two well-
known translational control elements, the TOP and the
ARS in the 5¢-UTR [13]. To examine which of the two
cis-elements are involved in upregulating translation of
the PABP1 mRNA during recovery from heat shock,
we introduced both cis-elements either individually or
in combination at the 5¢-UTR of the reporter b-galac-
tosidase (b-gal) mRNA. Following transfection with
different constructs, cells were subjected to 2 h of heat
shock and recovery for 12 and 24 h, and the level of
b-gal polypeptide was measured by western blotting.
The results (Fig. 6) show that the presence of the ARS
had no stimulatory effect on the b-gal expression level
during recovery from heat shock. In contrast, the pres-
ence of the TOP element in the 5¢-UTR of the reporter
construct resulted in an increase in b-gal abundance
during recovery from heat shock. Furthermore, the
presence of the ARS with the TOP did not prevent the
increase in b-gal level from occurring during recovery
from heat shock. However, as the ARS has an inhibi-
tory effect on mRNA translation [23], its presence
together with the TOP element in the reporter con-

struct reduced the overall expression level of b-gal
under all conditions examined. Nevertheless, our
results suggest that the TOP can exhibit its stimulatory
function on mRNA translation in the presence of the
ARS. To further elucidate whether the effect on the
b-gal level was due to changes in the abundance of the
cognate mRNA, we measured its levels by real-time
RT-PCR. The reporter mRNA abundance was found
to be similar in cells transfected with different con-
structs (Fig. 6D) and under different experimental con-
ditions of cellular stress and recovery. Thus, these
results suggest that the presence of the PABP1 mRNA
TOP or ARS sequences in the reporter construct did
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 559
not affect the transcription and ⁄ or the stability of the
reporter mRNA. Although we did not directly measure
the translation of the reporter mRNA, on the basis of
the enhancement of PABP1 mRNA translation during
recovery from heat shock (Fig. 2), it can be deduced
that the increase in b-gal expression in cells recovering
A
B
Fig. 5. Solubility of eIF4G and PABP1 in nonionic detergent. (A) HeLa cells grown on coverslips were subjected to heat shock and recovery
or maintained at 37 °C as described previously. The cells were treated with 0.5% Triton X-100 containing buffer for 5 min on ice before fixa-
tion in methanol. The detergent-treated specimens were immunostained with eIF4G and PABP1 antibodies, and viewed by confocal micros-
copy as described in the legend to Fig. 4. The localization pattern of eIF4G and PABP1 representing more than 90% of the cells are
presented here. These results were reproducible in three separate experiments. (B) Cells were transfected with 2 lg of PABPN1–GFP
expression vector [28] as described in Experimental procedures. At 36 h following transfection, the cells were either fixed directly or pre-
treated with 0.5% Triton X-100 for 5 min and viewed by confocal microscopy. Representative images of more than 90% of the cells are

shown.
PABP expression during heat shock recovery S. Ma et al.
560 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
from heat shock was due to enhancement of TOP-con-
taining reporter mRNA translation.
Association of HSPs with eIF4G and PABP1
The association of HSP27 with eIF4G following heat
shock has been previously described as a possible
mechanism for repression of translation of normal cel-
lular mRNA [22]. Therefore, to extend our knowledge
further, we expanded our investigation to examine the
association of both eIF4G and PABP1 with the two
predominant HSPs, such as HSP27 and HSP70,
following heat shock, as well as during the recovery
period. We used both coimmunoprecipitation and
immunofluorescence confocal microscopy to study the
interaction between the polypeptides. The results of
coimmunoprecipitation studies (Fig. 7A,B) show that
the antibodies to both eIF4G and PABP1 immunopre-
cipitated HSP27 from extracts of heat-shocked and
recovered cells. A small amount of HSP27 was also
detected in the immunoprecipitated samples of expo-
nentially growing cells. By analyzing the percentage of
total cellular HSP27 found in the coimmunoprecipitat-
ed samples, we found that approximately 25–30% of
cellular HSP27 was associated with eIF4G in both
control and heat-shocked cells. However, owing to the
induction of HSP expression following heat shock, the
total amount of eIF4G-bound HSP27 was increased
almost two-fold (Fig. 7A). As there was very little

change in the cellular eIF4G level in heat-shocked
cells, our results suggest that, due to the increased
cellular abundance of HSP27, there was an overall
CMV β-gal
β-gal
β-gal
β-gal
(1) β-gal
A
C

D
B
(2) ARS-β-gal
CMV
AR S
5’-aaaaaatccaaaaaaaatctaaaaaaatcttttaaaaaaccccaaaaaaatttacaaaaaa-3’
(3) TOP-β-gal
CMV
(4) TOP-ARS-β-gal
5’-ccttctccccggcggttagtgctgagagtgc-3’
CMV
Transcription start site
Transcription start site
Transcription start site
Transcription start site
ARS TOP
TOP
β-gal β-actin
CMV-β-gal

Transfection
ARS-β-gal
TOP-β-gal
Top- ARS-β-
gal
1.00 1.00 0.95 1.00 1.02
1.00
1.00 1.21 1.10 1.15
1.
00
1.
0
1 1.
0
1 1.
0
7
1.07 1.03 1.02 1.00
1.00 0.74
0
.
68

1.
98
2.
0
4
2.25 2.10
1.

00

1.06
0.97 1.02 1.06
1.00 0.98
3
2
Con
HS
Re12 h
Re24 h
Relative abundace of β-gal
0.5
1.5
1
0
CMV-β-gal ARS-β-gal TOP-β-gal Top-ARS-β-gal
2.5
Relative β-gal mRNA
level
1.4
1.2
1
0.8
0.6
0.4
0.2
0
con HS re12h re24 h
CMV-β-gal ARS-β-gal TOP-β-gal Top-ARS-β-gal

Fig. 6. Characterization of the translational control element of PABP1 mRNA responsible for upregulation of its translation during recovery
from heat shock. (A) Reporter b-gal constructs with different PABP1 mRNA cis-elements were prepared as described in Experimental proce-
dures. The cytomegalovirus (CMV) promoter was used to drive b-gal expression; TOP, ARS and TOP + ARS sequences were placed within
the 5¢-UTR of the b-gal gene, and the parental construct pCMV-SPORT–b-gal (Invitrogen) was used as a control. The nucleotide sequences
of the TOP and ARS cis control elements of the PABP1 mRNA are shown. (B) Approximately 2 · 10
5
HeLa cells were transfected with dif-
ferent CMV– b-gal expression vectors, and 48 h after transfection, cells were heat shocked at 44 °C for 2 h and allowed to recover at 37 °C
for 0, 12 and 24 h as described in Experimental procedures. The control cells were not heat shocked, and were harvested after 48 h of
transfection. For mock transfection, cells were treated with the transfection reagent without any plasmid DNA. The cellular level of b-gal
was measured by western blotting and quantified by scanning images. The abundance of b-actin was measured as a loading control. The
numbers at the bottom of each lane represent cellular abundance relative to the transfected control cells maintained at 37 °C, after adjusting
for the loading control. (C) The values of mean ± standard error (SE) of three independent experiments are shown here. (D) Transfected cells
were treated as described in (B), total cellular RNA was isolated, and the abundance of different mRNAs was measured by real-time RT-PCR
as outlined in Experimental procedures. The average of three independent experiments is shown.
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 561
increase in the association of HSP27 with eIF4G in
these cells. During recovery from heat shock, the
percentage of input HSP27 present in the immuno-
precipitated samples remained almost unchanged
(Fig. 7D). The association of PABP1 with HSP27
appeared to change more significantly in heat-shocked
and recovered cells than what was observed for eIF4G.
Approximately 7% of cellular HSP27 was coimmuno-
precipitated by PABP1 antibody from extracts of heat-
shocked cells. When cells were allowed to recover for 12
and 24 h, approximately 25–30% of cellular HSP27 was
found in the immunoprecipitated samples. Therefore, it
appears that the association of PABP1 with HSP27

depends on whether or not PABP1 is bound to eIF4G.
In contrast to HSP27, only a small but reproducible
level of HSP70 was detected in all samples tested in these
studies (Fig. 7A,B). As judged by analyzing the percent-
age of input HSP70 found in the samples immunopre-
cipitated with both eIF4G and PABP1 antibody, the
majority of HSP70 (90–95%) was not associated with
either eIF4G or PABP1 (Fig. 7D,E). As a negative
control, we also examined the association of b-actin
with eIF4G and PABP1 by using the respective anti-
body; as shown in Fig. 3, immunoprecipitation of
b-actin by either antibody was not detected in our
experiments. To further examine the specificity of the
antibodies, we used nonimmunized rabbit serum in
mock immunoprecipitation experiments (Fig. 7C).
Neither HSP27 nor HSP70 was immunoprecipitated
under our experimental conditions.
To further confirm the association between HSP27
and eIF4G and PABP1 in individual cells, we used
in situ double immunofluorescence confocal micros-
copy. As very little HSP70 was immunoprecipitated by
either the eIF4G or the PABP1 antibody, we did not
further study the colocalization of HSP70 with eIF4G
or PABP1 by this approach. As expected, the results
(Fig. 8) show induction and nuclear translocation of
HSP27 following heat shock. In heat-shocked cells,
fractions of both eIF4G and PABP1 were colocalized
with HSP27 in the nucleus. However, as compared to
eIF4G, a much lower level of colocalization between
PABP1 and HSP27 was visible in heat-shocked cells.

As a fraction of the nuclear PABP1 was still associated
with eIF4G (Fig. 3), this observation can be explained
if PABP1 does not bind HSP27 directly but associates
with it through eIF4G. During recovery from heat
shock, within 12 h, eIF4G, PABP1 and most of the
HSP27 were found in the cytoplasm. In addition, both
eIF4G and PABP1 remained colocalized with HSP27.
-HS HS Re12h Re24h
HSP 27
10 17 25 20
HSP70
1.0 1. 2.5 2.0
1.0 1.7 0.7
eIF4G
HSP 27
1.0 0.9 0.9 0.8
HSP70
1.0 1.3
(0.6) (2.2) (2.5)
PABP
1.0 0.7 1.9 1.8
HSP 27
HSP70
50
AD
EB
C
con HS Re12 h Re24 h
20
30

40
0
10
HSP27 HSP70
50
con HS Re12
h
Re24
h
20
30
40
0
10
HSP27
% of input % of input
HSP70
Fig. 7. Coimmunoprecipitation of HSP27and HSP70 with PABP1 and eIF4G. Cells were subjected to heat shock and recovery as described
in the legend to Fig. 1, and the cell lysates were subjected to immunoprecipitation with either the eIF4G antibody (A) or the PABP1 antibody
(B). The presence of HSP27, HSP70 and eIF4G or PABP in the eluted fractions from the protein A–Sepharose beads were analyzed by wes-
tern blotting as described in Experimental procedures. The individual bands were quantified using an arbitrary scale as described in the leg-
end to Fig. 1. (C) Mock immunoprecipitation was performed using 1.5 lg of preimmunized rabbit serum, and the eluted fractions from
protein A–Sepharose beads were examined for the presence of HSP27 and HSP70 by western blotting as described previously. (D, E) Equiv-
alent cellular levels of total cell lysate and eluted fractions from the protein A–Sepharose beads following immunoprecipitation with either
eIF4G (D) or PABP1 (E) were examined for the presence of HSP27 and HSP70 by western blotting. Total cell lysate and eluted fractions
were analyzed together in the same blot, and quantified as described above. The averages of three independent experiments are shown.
PABP expression during heat shock recovery S. Ma et al.
562 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
Similar results were observed after 24 h of recovery.
Although most of the eIF4G and PABP1 was detected

with the HSP27, a significant fraction of the HSP27
was also localized independently. This was expected, as
HSP27 also functions as a chaperone to properly fold
cellular polypeptides [18].
A
B
Fig. 8. Cellular colocalization of HSP27 with eIF4G and PABP1 following heat shock and recovery. Cells were immunostained with appropri-
ate antibodies and viewed by laser scanning confocal microscopy as described in the legend to Fig. 4. HSP27 was labeled using Texas Red-
conjugated secondary antibody, and eIF4G and PABP1 were labeled with an appropriate FITC-labeled secondary antibody.
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 563
Our results suggest that eIF4G could associate with
HSP27 in the cell nucleus and translocate to the cyto-
plasm as a complex with HSP27 during recovery from
heat shock. This process perhaps facilitates refolding
of eIF4G into its native form during recovery from
heat shock. We propose that PABP1 associates with
HSP27 by virtue of its interaction with eIF4G. As
capped mRNAs are translated during recovery from
heat shock, it is likely that the association of eIF4G
with HSP27 may not be the sole basis for the reduced
translation of capped mRNA in heat-shocked HeLa
cells, and therefore these results differ from what was
reported previously for H293T cells [22].
Discussion
Translational control of PABP1 expression during
recovery from heat shock
PABP1 is considered to be a genuine translation initia-
tion factor [29], and its activity and expression level
are controlled by a multitude of regulatory networks

[13,14,23,25]. As such, the PABP1 gene behaves like an
early response gene. Its expression level is modulated
by growth and developmental changes [14,30]. In our
studies, we showed that PABP1 abundance is upregu-
lated during recovery of HeLa cells from heat shock.
This upregulation occurs without a concomitant
change in the cellular level of its polypeptide partner
eIF4G. Previous studies have shown that the expres-
sion level and activity of factors involved in mRNA
translation, including initiation factors, elongation fac-
tors, and ribosomal proteins, increase whenever global
mRNA is reactivated for translation [1,2,31]. We
believe that the change in PABP1 abundance during
recovery from heat shock is related to the renewal of
translation of normal cellular mRNAs during the
recovery phase from a thermal stress. In a previously
published study [22], it was shown that the expression
of eIF4G remained unchanged in heat-shocked cells.
However, the solubility of eIF4G and its association
with PABP1 was reduced in heat-shocked cells. In our
studies, eIF4G behaved similarly in heat-shocked cells
but, as we extended our experiments to examine
changes during the recovery phase, we detected a spe-
cific change in the abundance of PABP1. This change
in PABP1 abundance was mediated at the level of
mRNA translation, as no increase in PABP1 mRNA
level was observed during recovery. We also showed
here that in exponentially growing cells, only 30–40%
of cellular PABP1 mRNA was translated, as judged by
its distribution with the polysomal fraction. As such,

the PABP1 mRNA is translated less efficiently than
the control b-actin mRNA, which was present predom-
inantly (90%) in the polysomal fraction. However,
during recovery from heat shock, the efficiency of
PABP1 mRNA translation reached the same level as
that of the b-actin mRNA. Similar preferential
enhancement of PABP1 mRNA translation was also
observed during liver regeneration and growth stimula-
tion of cells following serum starvation [14,30]. Thus,
it appears that a preferential increase of PABP1
mRNA translation occurs whenever there is a demand
for an increase in global mRNA translation. The
increase in the cellular PABP1 level may act as a cue
for cells to stimulate global mRNA translation. The
PABP1 mRNA is generally inefficiently translated
under normal growth conditions through feedback
inhibition mediated by binding of PABP1 to its own
ARS cis-element [23]. It is not known how the TOP
overrides the inhibition by the ARS during recovery
from heat shock. However, a small reduction of
PABP1 abundance after heat shock could conceivably
relieve the feedback control, and enhance PABP1
mRNA translation through its TOP cis-element.
Association between PABP1 and eIF4G during
heat shock and recovery
The function of PABP1 in mRNA translation depends
on its interaction with eIF4G. Earlier, it was suggested
that this interaction is inhibited by sequestration of
eIF4G into insoluble granules in a complex with
HSP27; as a result, cap-dependent mRNA translation

of normal cellular mRNA is suppressed in heat-
shocked cells [22]. We have shown here that as the
cap-dependent translation resumes after cells are trans-
ferred to the physiological temperature, PABP1 and
eIF4G interaction is re-established. In situ localization
studies of these two polypeptides also show a dramatic
change in the subcellular location of both polypeptides
after heat shock. Both eIF4G and PABP1 were found
predominantly in the nucleus of heat-shocked cells as
nonionic detergent-insoluble granules, and significant
amounts of these polypeptides were not colocalized in
the nucleus. However, both coimmunoprecipitation
and immunofluorescence studies showed detectable
association of PABP1 and eIF4G in the nucleus of
heat-shocked cells. This observation can be explained
if these two polypeptides enter the nucleus as a com-
plex that later dissociates. The presence of colocalized
PABP1 and eIF4G in the perinuclear space after a
shorter heat shock period also supports this possibility.
After recovery, PABP1 and eIF4G were not detectable
in the cell nucleus, and were colocalized in the cyto-
plasm. These changes took place while a large amount
PABP expression during heat shock recovery S. Ma et al.
564 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
of HSP27 was present in cells and remained colocal-
ized with eIF4G during the recovery period. Therefore,
our studies suggest that the presence of HSP27 and its
association with eIF4G is not responsible for altering
the solubility and subcellular location of eIF4G. Thus,
our results presented here are different from those of a

previous study, where eIF4G was not found in the
nucleus of H293T cells following heat shock at 44 °C
for 2 h. However, even in this previous report, most of
the eIF4G accumulated as granules near the perinucle-
ar site, which we also observed during a somewhat
shorter heat shock period. The observed difference in
the nuclear localization of eIF4G distribution between
the two studies could be simply due to the use of dif-
ferent cell lines. Cultured immortal cell lines may differ
with respect to the time and temperature for the opti-
mum stress response. In our studies, we found that the
nuclear localization of eIF4G and PABP1 was depen-
dent on the length of exposure of cells as well as the
temperature. Therefore, under more stringent condi-
tions, similar nuclear localization of those two poly-
peptides might also occur in H293T cells. Under
normal conditions, both PABP1 and eIF4G are
predominantly localized in the cell cytoplasm; never-
theless, both polypeptides contain weak nuclear locali-
zation signals to facilitate nuclear entry under some
circumstances [32,33]. A small fraction of eIF4G has
been previously shown to be present in the nuclear
fraction, and to bind the cap-binding complex involved
in splicing [34]. It is believed that the nuclear eIF4G
may associate with pre-mRNA through its interaction
with the polypeptide partners of the cap-binding com-
plex, such as CBP20 and CBP80, and accompany the
mRNA to the cytoplasm, where partner switching
occurs to form the eIF4F complex. This process could
conceivably couple mRNA translation with mRNA

export [34]. The possible mechanism of nuclear trans-
location of eIF4G in heat-shocked cells is uncertain,
but it is possible that inhibition of cap-dependent
translation after heat shock uncouples mRNA export
and translation, and prevents the export of eIF4G-
associated mRNA from the nucleus, causing a nuclear
build-up of this polypeptide. A number of studies sug-
gest that PABP1 also enters the nucleus and binds to
the 3¢-poly(A) tract, and accompanies the mRNA to
the cytoplasm [5,32]. Thus, the nuclear accumulation
of PABP1 in heat-shocked cells may result, at least in
part, from uncoupling of translation and the export
process after heat shock. Interestingly, as significant
amounts of nuclear PABP1 and eIF4G were not colo-
calized in heat-shocked cells in our experiments, it is
likely that interaction between PABP1 and eIF4G
occurs only in the cytoplasm. Another related issue is
how the nuclear PABP1 and eIF4G exit the nucleus.
Whether or not a nuclear exit signal is present in
PABP1 and eIF4G is not known. Both polypeptides
might help the mRNA to exit the nucleus, as the pro-
cessing, transport and translation of normal cellular
capped transcripts is resumed during recovery from
heat shock. Our studies have shown that the nuclear
exit of both eIF4G and PABP1 occurs in the absence
of new protein synthesis, and thus suggest that the
pre-existing molecules can be exported to the cyto-
plasm. Although the precise mechanism of this process
is unknown, it is tempting to speculate that both poly-
peptides piggy-back the HSP27 as it translocates to

the cytoplasm during the recovery period. During the
recovery period, we have also shown an increase in
the association of PABP1 with eIF4G, and a concom-
itant increase in its phosphorylation during the recov-
ery period. Thus, it appears that the excess PABP1
produced during recovery is mostly utilized for mRNA
translation.
Translational control elements of PABP1 mRNA
The main function of the ARS element of PABP1
mRNA is to regulate the cellular PABP1 level under
all circumstances [13]. The TOP cis-element is involved
in regulating PABP1 mRNA translation during growth
stimulation [14]. A number of mRNAs coding for fac-
tors involved in mRNA translation, such as ribosomal
proteins and elongation factor eEF1a, contain a simi-
lar TOP element [14,15]. It is believed that the presence
of this common cis-element allows coordinated regula-
tion of translation of mRNAs that participate in the
same biochemical step, such as mRNA translation. We
have shown here that the TOP cis-element of PABP1
mRNA is involved in the upregulation of PABP1
mRNA translation during recovery from heat shock.
Previous studies have shown that translation of TOP-
containing mRNAs is stimulated during growth by
changing the size of the polysomes [14]. This could
occur either by moving the 40S ribosomal subunit at a
faster rate along the 5¢-UTR, or by increasing the rate
of elongation. However, these two mechanisms are not
necessarily mutually exclusive. We have previously
shown that the presence of the ARS in the 5¢-UTR of

an mRNA stalls the 40S ribosomal subunit before the
ARS. Therefore, the ARS-containing mRNA is
arrested at the translation initiation step [13]. As the
TOP may increase the overall initiation rate during
growth [14] and recovery from heat shock, more ribo-
somal subunits may be present at the 5¢-UTR at any
given time; as a result of shear numbers, some of these
ribosomes could escape the steric hindrance by the
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 565
heterotrimeric complex of PABP1, IMP1 and UNR at
the ARS [11]. This will result in a shift of mRNA from
the repressed nonpolysomal fraction to the translated
polysomal fraction. The precise mechanism by which
the TOP promotes translation during growth stimula-
tion is not clear. Nevertheless, the P13 kinase and ⁄ or
mTOR kinase pathways appear to be important for
TOP function [31,35]. In addition, whether or not one
of the heat shock polypeptides, particularly the HSP70
or HSP27, which have been previously shown to bind
mRNA [36,37], can bind to the TOP control element
of the PABP1 mRNA and stimulate its translation
remains to be investigated. The TOP-mediated regula-
tion of PABP1 mRNA translation is distinct from that
mediated by the ARS. The TOP appears to upregulate
mRNA translation when the demand on the cellular
translation machinery increases. On the other hand,
the ARS monitors and controls the overall cellular
level of PABP1 and suppresses PABP1 mRNA transla-
tion through a feedback mechanism [13,23]. In our

studies, the stimulatory effect of the TOP on reporter
mRNA translation was maintained even when the
ARS was also present. However, the overall cellular
abundance of the reporter b-gal was less in cells trans-
fected with both ARS- and TOP-containing construct
than what was observed with the construct containing
TOP alone. This is in good agreement with previous
observations that the ARS slows the rate of translation
initiation of the endogenous PABP1 mRNA in the
presence of the TOP control element [23].
Association of HSPs with PABP1 and eIF4G
during heat shock and recovery
The HSPs function as molecular chaperones, and inter-
act with many cellular proteins. Previous studies have
linked HSP27 with the eIF4G subunit of the eIF4F
complex [22]. Here we showed colocalization of HSP27
with both eIF4G and PABP1 in heat-shocked cells. Fol-
lowing 24 h of recovery from heat shock, most of the
HSP27 accumulated in the cytoplasm with eIF4G and
PABP1, and possibly remained colocalized within a
complex. However, it is not known whether or not the
HSP27-associated eIF4G participates in translation.
Thus, the previous observation regarding the sequestra-
tion of eIF4G by HSP27 may still explain, at least in
part, the reduced cap-dependent translation of mRNA
in heat-shocked cells [22]. In our studies, we have shown
that PABP1 also associates with HSP27, albeit at a
much reduced level in heat-shocked cells than what was
observed with eIF4G. Perhaps HSP27 does not directly
interact with PABP1 but was coimmunoprecipitated

with the PABP1 antibody as a complex with eIF4G.
Heat shock and translational control
Cells respond to thermal stress by producing large
amount of specific polypeptides (HSPs) designed to
protect cellular proteins from stress-induced denatur-
ation [17,18]. Prior to the induction of HSPs, a mul-
titude of changes take place that prepares the cell to
synthesize HSPs while shutting down expression of
normal cellular proteins. This is achieved at least in
part by preventing translation of normal cellular
capped mRNAs [3]. We have shown here that disso-
ciation of PABP1 from the eIF4F complex and
nuclear translocation of both PABP1 and eIF4G
occur following heat shock, and suggest that seques-
tration of both PABP1 and eIF4G within the cell
nucleus may be an important contributing factor in
suppressing translation of normal cellular mRNA.
Our studies also show that during recovery, PABP1
and eIF4G functions are restored. In addition, we
propose that the observed increase in both PABP1
abundance and phosphorylation may be crucial in
meeting the cellular demand to restore the normal
level of translation during recovery from heat shock.
We have shown that the increase in PABP1 level
was due to a preferential increase in TOP-mediated
translation of PABP1 mRNA. Collectively, our
results show that a cascade of changes in the cellular
translation machinery is involved in the suppression
of translation in heat-shocked cells, and subsequent
resumption of normal mRNA translation during

recovery is accompanied by a preferential upregula-
tion of PABP1 expression.
Experimental procedures
Cell culture and heat shock treatment
HeLa cells were grown at 37 °C in the DMEM supple-
mented with 10% fetal bovine serum for 2–3 days, until
cells were fully spread and the desired degree of confluence
was obtained. Cells were subjected to heat shock at 44 °C
for 2 h, and incubated at 37 °C for 0, 12 and 24 h to allow
them to recover. The cells were washed and fixed for
immunofluorescence analysis, or harvested for biochemical
studies.
Transfection of cells
Approximately 2–5 · 10
5
subconfluent HeLa cells grown on
35 mm dishes were transfected with 2 lg of plasmid DNA
using 10 lg of lipofectamine (Invitrogen, Burlington,
Canada), according to protocols supplied by the manufac-
turer. In short, the DNA was mixed with lipofectamine and
PABP expression during heat shock recovery S. Ma et al.
566 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
100 lL of Opti-MEM (Invitrogen) for 30 min before being
added to the culture dish and incubated for 6 h with an
additional 0.9 mL of Opti-MEM. After 6 h, 1 mL of
growth medium containing 20% fetal bovine serum was
added. Following a total of 12 h of incubation, the medium
was replaced with fresh DMEM containing 10% fetal
bovine serum. Cells were usually harvested after an addi-
tional 24–36 h of incubation at 37 °C.

Western blotting
To prepare cell lysate, approximately 2 · 10
5
cells were har-
vested into 150 lLof2· Laemmli sample buffer (4% SDS,
20% glycerol, 120 mm Tris ⁄ HCl, pH 6.8, 200 mm dith-
iothreitol, and 0.1% bromophenol blue) and boiled for
5 min at 95 °C. The level of expression of eIF4E, eIF4G,
PABP1, HSP27 and HSP70 polypeptides was measured by
immunoblot analysis as previously described [25]. Cell
lysates containing equal amounts of protein were separated
by SDS ⁄ PAGE [38]. The polypeptides were transferred to a
nitrocellulose membrane and incubated with a primary anti-
body; this was followed by incubation with an appropriate
horseradish peroxidase-conjugated secondary antibody. The
expression level of polypeptides was visualized by chemilu-
minescence using a Western lighting kit (Perkin Elmer, Bos-
ton, MA, USA), and quantified by scanning. Antibodies
for eIF4G, eIF4E, PABP1, HSP27, HSP70 and b-actin
were obtained from Santa Cruz Biochemical (Santa Cruz,
CA, USA).
Coimmunoprecipitation
Cells (1 · 10
6
) were washed with NaCl ⁄ P
i
three times, and
harvested into 450 lL of chilled lysis buffer [50 mm
Hepes ⁄ KOH, pH 7.4, 250 mm NaCl, 1% (v ⁄ v) NP-40,
5mm EDTA, 200 UÆmL

)1
aprotinin, 0.1 mm phen-
ylmethanesulfonyl fluoride, and 10 lgÆmL
)1
leupeptin]. The
cells were lysed by passing them through a 27G syringe.
The cell lysate was centrifuged at 5000 g for 5 min, and the
supernatant was used for analysis. Immunoprecipitation
was carried out by adding 1.5 l g of goat monoclonal anti-
eIF4G or rabbit polyclonal PABP1 antibody (Santa Cruz)
to the cell extract. After overnight incubation at 4 °Cona
rotary mixer, 50 lL of protein A–Sepharose bead slurry
(Amersham Biosciences, Piscataway, NJ, USA) was added,
and the incubation was continued for 3 h at 4 °C. After
washing six times with 1 mL of lysis buffer, resin-bound
proteins were eluted from the beads by adding 30 lLof2·
Laemmli sample buffer (4% SDS, 20% glycerol, 120 mm
Tris ⁄ HCl, pH 6.8, 200 mm dithiothreitol, and 0.1% brom-
ophenol blue) and heating the mixture for 5 min at 95 °C.
The proteins were then resolved by 12% SDS ⁄ PAGE, and
the presence of coimmunoprecipitated polypeptides was
examined in the eluted samples by western blotting using
the appropriate antibody.
Immunofluorescence confocal microscopy
HeLa cells plated on glass coverslips were used for exam-
ining the cellular localization of polypeptides by using
immunofluorescence confocal microscopy. Cells were
washed with NaCl ⁄ P
i
and fixed with methanol at )20 °C

for 10 min as described previously [28]. The fixed cells were
incubated with an appropriate antibody, and subsequently
the specimens were treated with an appropriate secondary
antibody conjugated with either fluorescein isothiocyanate
(FITC) or Texas Red (Santa Cruz). Colocalization of two
proteins was examined by simultaneously incubating the
sample with primary antibodies against two different pro-
teins, and then with two different FITC-labeled secondary
antibodies at the same time. The specimens were mounted
in an NaCl⁄ P
i
buffer containing 70% glycerol (pH 7.5) for
microscopy. The localization of proteins was visualized and
imaged with a Leica Microsystems Confocal Laser S Micro-
scope (CLSM, Microsystems, Inc., Heidelberg, Germany)
equipped with a Plan-Achromat 63 ·⁄NA1.4 objective. In
some experiments, cells were treated with a 0.5% Triton X-
100 containing buffer (0.5% Triton X-100, 10 mm
Pipes,10 mm NaCl, 2.5 mm magnesium acetate, and 0.3 m
sucrose) at 22 ° C for 5 min before fixation to remove solu-
ble proteins [31]. Optical sections of representative cells were
obtained by laser confocal fluorescence microscopy. Two
hundred cells in different fields of view were visualized, and
representative images of the majority of cells were used for
presentation. Controls were performed by using nonimmu-
nized serum. FITC signals were obtained through a filter set
with excitation at 488 nm (450–490 nm), a beam splitter of
510 nm, and an emission filter of > 520 nm (525–555 nm).
Texas Red signals were detected through a filter set with
excitation at 543 nm and emission at > 600 nm, and 63·

objectives was used for all cells. Images were recorded with
a resolution of 512 · 512 pixels. A series of optical slices
that spanned the depth of the cell was obtained (normally
eight slices, 0.5 lm apart). Other parameters were used with
default settings. In order to avoid cross-talk between FITC
and Texas Red channels, the signals were scanned individu-
ally with one channel on and the other off. The images for
the same cells from two channels were merged with the
overlap function. To differentiate the autofluorescence, con-
trol cells without immunostaining were examined to set up
the settings for the stained cells.
Measurement of mRNA levels
Total cellular RNA was isolated using the High Pure
RNA Isolation Kit (Roche Biochemical, Indianapolis, IN,
USA). The quality and quantity of the RNA were deter-
mined by 1.5% agarose gel electrophoresis and spectropho-
tometry, respectively. The levels of specific mRNAs,
including PABP1, b-actin and b-gal, in the samples were
determined either by quantitative real-time RT-PCR using
S. Ma et al. PABP expression during heat shock recovery
FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS 567
a Rotor-gene 3000 (Corbett Research, NSW, Australia) or
by RT-PCR. An aliquot of total RNA (1 lg) was reverse
transcribed at 42 °C for 1 h in a total reaction volume of
25 lL using SuperScript II reverse transcriptase (Invitrogen,
Burlington, Canada) and 150 ng of random primers. After
the reaction, 5 lL of the cDNA sample was amplified by
PCR in a total reaction volume of 25 lL, using the plati-
num SYBR green qPCR supermix-UDG kit (Invitrogen,
Burlington, Canada) and 100 ng of primers specific for

PABP1, b-actin and b-gal (Table 1). The amplification was
performed using an initial denaturation step at 95 °C for
4 min, followed by 40 cycles of denaturation at 95 °C for
20 s, annealing at 60 °C for 20 s and extension at 72 °C
for 20 s. Specificity of the PCR product was examined after
the final cycle by generating a melting curve with a heating
rate of 1 °CÆs
)1
between 72 and 99 °C. The data were ana-
lyzed using Rotor-gene 3000 software and the 2
)DDCt
method [39]. The relative expression values of all mRNAs
were normalized by the b-actin mRNA level. Reaction con-
ditions for the RT-PCR were similar to those used for the
real-time RT-PCR, except that each sample was analyzed
at a different cycle number to remain within the linear
range of amplification. In all samples, this was found to be
20–25 cycles, depending on the cellular level of the individ-
ual mRNA.
Subcellular fractionation of polysomes and RNA
isolation
Samples corresponding to equal numbers of cells were used
for analysis. The cells were lysed in 200 lL of polysomal
buffer (10 mm Mops, pH 7.2, 250 mm NaCl, 2.5 mm
MgOAc, 0.5% Nonidet P-40, 0.1 mm phenylmethane-
sulfonyl fluoride, 200 lgÆmL
)1
heparin, and 50 lgÆmL
)1
cycloheximide) [23]. After removal of the nuclei and cell

debris by centrifugation at 12 000 g for 10 min, the super-
natant fraction was centrifuged in a 12 mL 10–50% sucrose
gradient, containing 25 mm Hepes (pH 7.0), 50 mm KCl,
2mm MgOAc, 50 lgÆmL
)1
cycloheximide and 15 mm
2-mercaptoethanol at 100 000 g in a Beckman SW 41Ti
rotor for 3 h as previously described [23]. Gradient frac-
tions of approximately 1 mL each were collected using an
Auto Densi-Flow IIC apparatus (Buchler Instruments, Fort
Lee, NJ, USA). Total RNA from each fraction was isolated
using the Triazole RNA isolation kit as described by the
manufacturer (Roche), and precipitated with ethanol using
5 lg of yeast tRNA as a carrier.
Plasmid construction
A reporter b-gal construct containing the ARS region
(nucleotides 71–131) of human PABP1 (GeneBank ID:
Y00345) was generated by ligating double-stranded oligode-
oxynucleotides with BamHI and NheI sticky ends into the
site between BamHI and NheI of the pCMV-Sport–b-gal
plasmid vector. To generate the TOP–b-gal construct, the
TOP region (nucleotides 1–31) of human PABP1 was
inserted into the transcription start site of pCMV-Sport–
b-gal by using a megaprimer-based method [40]. A mega-
primer was produced by PCR with primer 1 containing an
EcoRI site and primer 2 (primer sequences are listed in
Table 2). The PCR product was used as the megaprimer,
and treated with exonuclease I to digest the remaining sin-
gle-stranded primers. The megaprimer was used as the
upstream primer to amplify another region of the vector by

PCR using primer 3 carrying an NcoI site. The final ampli-
fied fragment was gel purified and cloned into the site
between EcoRI and NcoI in the pCMV-Sport–b-gal vector.
To construct the TOP–ARS–b-gal expression vector, sense
ARS and antisense ARS oligomers with extra unrelated
sequences were annealed to produce a double-stranded
ARS oligodeoxynucleotide, and digested with PstI and SalI
to produce sticky ends. This double-stranded ARS was
inserted at the PstI and SalI sites of our TOP–b-gal con-
struct. The restriction enzymes used in cloning were pur-
chased from MBI Fermentas (Amherst, NY, USA), and the
parent pCMV-Sport–b-gal was obtained from Invitrogen.
Two-dimensional gel electrophoresis
Cellular proteins were analyzed by two-dimensional gel
electrophoresis using immobilized pH 6–11 IEF dry strips
as previously described [25]. Sample preparation and elec-
trophoresis conditions were according to the procedure pro-
vided by the supplier of the immobilized strips (Amersham
Biosciences).
Table 1. Primers used for the real-time RT-PCR. S, sense oligonucleotide; AS, antisense oligonucleotide.
mRNA Nucleotide sequence (5¢-to3¢) GenBank ID
Human
b-actin (S)
CTCTTCCAGCCTTCCTTCCT (780–799) BC013835
Human
b-actin (AS)
CACCTTCACCGTTCCAGTTT (963–982)
PABP1 (S) GCACAGAAAGCTGTGGATG NM_002568
PABP1 (AS) TTTGCGCTTAAGTTCCGTC (1341–1323)
b-gal (S) GCTGGATAACGACATTG (2435–2453) U02451

b-gal (AS) CAGCACCGCATCAGCAAG (2560–2577)
PABP expression during heat shock recovery S. Ma et al.
568 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS
Acknowledgements
This work was supported by funds from a discovery
grant from the Natural Science and engineering
Research council (NSERC) of Canada.
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Primer name Primer sequence (5¢-to3¢)
Primer 1 with EcoRI site GACCCGGGAATTCCGGACCGG (nucleotides 4222–4242 of PCMV-Sport–b-gal plasmid)
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Antisense ARS with PstI and SalI gcattGTCGACttttttgtaaatttttttggggttttttaaaagatttttttagattttttttg
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TOP Ccttctccccggcggttagtgctgagagtgc
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Supporting information
The following supplementary material is available:
Fig. S1. Dose response of western blotting. HeLa cells
were lysed in gel sample buffer as described in Experi-
mental procedures and the indicated volume of cell
lysate was used for western blotting using PABP,
HSP70 and beta-actin antibodies to examine the linear
response of the detection technique.
This supplementary material can be found in the
online version of this article.
Please note: Wiley-Blackwell is not responsible for
the content or functionality of any supplementary
materials supplied by the authors. Any queries (other
than missing material) should be directed to the corre-
sponding author for the article.
PABP expression during heat shock recovery S. Ma et al.
570 FEBS Journal 276 (2009) 552–570 ª 2008 The Authors Journal compilation ª 2008 FEBS