Tài liệu Báo cáo Y học: Phosphorylation of initiation factor-2a is required for activation of internal translation initiation during cell differentiation ppt

Phosphorylation of initiation factor-2a is required for activation
of internal translation initiation during cell differentiation
Gabi Gerlitz
1
, Rosemary Jagus
2
and Orna Elroy-Stein
1
1
Cell Research and Immunology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Israel;
2
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, USA
The long uORF-burdened 5¢UTRs of many genes encoding
regulatory proteins involved in cell growth and differenti-
ation contain internal ribosomal entry site (IRES) elements.
In a previous study we showed that utilization of the weak
IRES of platelet-derived growth factor (PDGF2) is activated
during megakaryocytic differentiation. The establishment of
permissive conditions for IRES-mediated translation during
differentiation has been confirmed by our demonstration of
the enhanced activity of vascular endothelial growth factor,
c-Myc and encephalomyocarditis virus IRES elements
under these conditions, although their mRNAs are not
naturally expressed in differentiated K562 cells. In contrast
with the enhancement of IRES-mediated protein synthesis
during differentiation, global protein synthesis is reduced, as
judged by polysomal profiles and radiolabelled amino acid
incorporation rate. The reduction in protein synthesis rate
correlates with increased phosphorylation of the translation
initiation factor eIF2a. Furthermore, IRES use is decreased
by over-expression of the dominant-negative form of the

eIF2a kinase, PKR, the vaccinia virus K3L gene, or the
eIF2a-S51A variant which result in decreased eIF2a phos-
phorylation. These data demonstrate a connection between
eIF2a phosphorylation and activation of cellular IRES ele-
ments. It suggests that phosphorylation of eIF2a, known to
be important for cap-dependent transaltional control, serves
to fine-tune the translation efficiency of different mRNA
subsets during the course of differentiation and has the
potential to regulate expression of IRES-containing
mRNAs under a range of physiological circumstances.
Keywords: differentiation; gene expression; initiation factor
2; IRES; translation initiation.
Translation of eukaryotic gene expression is controlled both
by global mechanisms that affect the overall rate of protein
synthesis and by selective control mechanisms that affect the
translation of subsets of mRNA molecules equipped with
appropriate cis-regulatory elements. The global mechanisms
are mostly based on controlling the availability of two rate-
limiting components of the initiation step: eIF4E, the 5¢-cap
binding protein, and eIF2, a GTP-binding protein that
mediates the association of Met-tRNA
i
to the 40 S
ribosomal subunit. Control of eIF4E activity is mediated
by influencing its phosphorylation status and/or its interac-
tion with the eIF4E binding proteins, as well as by affecting
the integrity of eIF4G which serves as an adapter protein
that bridges eIF4E, the RNA helicase eIF4A, poly(A)
binding protein and eIF3 (reviewed in [1–3]). Control of
eIF2 activity is mediated through reversible phosphoryla-

tion of its a-subunit. When eIF2a is phosphorylated, the
GDP-eIF2generatedattheendofeachinitiationstep
becomes a competitive inhibitor of eIF2B, a rate-limiting
guanine nucleotide exchange factor, resulting in a reduction
of the exchange of eIF2-bound GDP for GTP. As GTP
binding to eIF2 is a prerequisite to Met-tRNA
i
binding,
phosphorylation of eIF2a effectively inhibits eIF2 recycling
and consequently inhibits additional translation initiation
steps (reviewed in [4]). The control mechanisms that govern
the rate of global protein synthesis are responsive to a
variety of conditions including nutrient deprivation, heat
shock, apoptosis and viral infection. Under conditions that
inhibit the initiation of global protein synthesis, subsets of
mRNAs remain competent to be recruited by ribosomes.
Depending on their specific cis-regulatory elements they
may gain a translational advantage over other mRNA
molecules. For instance, mRNAs encoding heat-shock
proteins are translated efficiently under conditions of
reduced eIF4E/4F activity due to their unstructured
5¢UTR (reviewed in [5]). Another example is the efficient
translation of the yeast GCN4 mRNA under conditions of
amino acid starvation due to leaky scanning of the upstream
ORFs within its 5¢UTR (reviewed in [6]).
While much data has been accumulated regarding the
control of protein synthesis in response to various stress
conditions, less is known about translational control
mechanisms that are operative during cellular differenti-
ation. Cells undergoing terminal differentiation exhibit

extensive changes in the pattern of gene expression to
acquire a specific biological function that is usually accom-
panied by cessation of proliferation. In addition to the
massive changes at the transcriptional level, mechanisms
regulating overall inhibition of protein synthesis release
most mRNAs from the polysomes and facilitate the
translation of specific mRNAs that are important for the
Correspondence to O. Elroy-Stein, Department of Cell Research &
Immunology, George S. Wise Faculty of Life Sciences,
Tel Aviv University, Tel Aviv 69978, Israel.
Fax: +972 3 642 2046, Tel.: +972 3 640 9153,
E-mail:
Abbreviations: PDGF, platelet-derived growth factor; IRES,
internal ribosomal entry site; VEGF, vascular endothelial growth
factor; CMV, cytomegalovirus; TPA, 12-O-tetradecanoylphorbol-13-
acetate; EMCV, encephalomyocarditis virus.
(Received 18 January 2002, revised 3 April 2002,
accepted 2 May 2002)
Eur. J. Biochem. 269, 2810–2819 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.02974.x
differentiation process [7]. However, there is little data
regarding the molecular mechanisms that govern such
global inhibition accompanied by activation of specific
mRNA subpopulations. The proliferation-dependent
association of polysomes with 5¢-terminal oligopyrymidine
tract-containing mRNAs [8] and the 3¢UTR-mediated
translational activation of 15-lipoxygenase mRNA during
erythrocytic differentiation [9] are the best characterized
examples.
Whereas most mRNAs are translated efficiently, a subset
of mRNAs is poorly translated due to the extraordinarily

long, structured, upstream AUG-burdened 5¢UTRs that
serve as barriers for ribosomal scanning. Interestingly, such
translational inhibitors often belong to mRNAs encoding
proteins involved in cell growth and differentiation such as
growth factors, receptors, transcription factors, proto-
oncogenes, and cytokines [10]. Using the 1022 nucleotide
long 5¢UTR of platelet-derived growth factor (PDGF2) as a
model, we were able to show previously that the cumber-
some 5¢UTR is not a translational inhibitor, but rather a
translational modulator that is sensitive to changes in the
cellular milieu [11]. More specifically, the PDGF2 mRNA
leader was shown to mediate efficient translation under
conditions of megakaryocytic differentiation which provide
a permissive milieu for activation of the PDGF2 internal
ribosomal entry site (IRES) [12,13]. During the last decade,
several cellular IRES elements have been discovered
(reviewed in [14,15]), although the mechanisms of cellular
IRES-mediated ribosome recruitment remain unclear. To
further understand the mechanism of IRES activation, we
wished to assess whether the mechanisms involved in
regulation of global translation during differentiation have
a role in the translational activation of the IRES-containing
mRNA. Since internal translation is independent of the
5¢-cap structure of the mRNA, we did not focus our
attention on the changes in the activity of the 5¢-cap binding
protein, eIF4E, during differentiation. However, it
remained of interest to determine eIF2a phosphorylation
status during differentiation and to ascertain whether
phosphorylation of eIF2a is involved in IRES activation.
In this study we show that: (a) not only are differentiation

conditions permissive for the recruitment of the PDGF2
IRES, but also for the vascular endothelial growth factor
(VEGF), c-Myc and encephalomyocarditis virus (EMCV)
IRES elements, although their mRNAs are not naturally
expressed in differentiated K562 cells; (b) global protein
synthesis rate is reduced during differentiation, correlating
with increased eIF2a phosphorylation that is known to be
important for cap-dependent translational control; and (c)
inhibition of eIF2a phosphorylation during differentiation
reduces the differentiation-induced IRES activation.
MATERIALS AND METHODS
Plasmids
The pLL vector is composed of a fragment containing
Renilla luciferase from pRL-null (Promega) as the first
cistron, fused to a fragment encoding the cytosolic form of
firefly luciferase from pGL3-basic (Promega) as the second
cistron. A 22-base pair fragment containing StuIandNcoI
sites separates the stop codon of the Renilla luciferase and
the ATG initiator codon of the firefly luciferase. An NheI
(filled)–SacI fragment containing both cistrons was ligated
with a HindIII (filled)–SacIfragmentofpCL[12]to
generate pLL, which contains the dual luciferase transcrip-
tion unit downstream of the cytomegalovirus (CMV)
promoter and upstream of the SV40 intron and polyade-
nylation sites. The 5¢UTRs of VEGF and of PDGF2 were
obtained as SpeI (filled)–NcoI fragments from pSKVLUC
[16] and pCPL [12], respectively, and were ligated to the
StuI–NcoI7.5-kbfragmentofpLLtogeneratepLVLand
pLPL, respectively. The EMCV IRES fragment was
obtained as Alw26I (filled)–NcoI fragment from pTM1

[17], and was ligated to the StuI–NcoI7.5-kbfragmentof
pLL to generate pLEL. P2 c-Myc 5¢UTR (GeneBank acces-
sion # J00120) was generated by RT-PCR using total RNA
from K562 cells and the oligonucleotides 5¢-CCCCACTA
GTAATTCCAGCGAGAGGCAGA-3¢ and 5¢-AATACC
ATGGTCGCGGGAGGCTGCTG-3¢, and was ligated to
the StuI–NcoI 7.5-kb fragment of pLL to generate pLML.
pcK3L was generated by insertion of the NcoI (filled)–
BamHI 0.3-kb fragment of pTM1-K3L [18] into the HindIII
(filled)–BamHI sites of pcDNA3 (Invitrogen) under the
control of the CMV promoter. pcPKRD6 ¼ p68D6-pcD-
NAI/NEO [19] was used for expression of PKRD6, the
dominant-negative variant of PKR under the control of
the CMV promoter. pc2a-S51A (expresses eIF2a with
Ser51 fi Ala mutation) was generated by PCR of the eIF2a
cDNA from p51A and p51D [20], respectively, using
the oligonucleotides 5¢-CTGGATATCATGCCGGGTCT
AAGTTG-3¢ and 5¢-CTGCTCGAGTTAATCTTCAGCT
TTGGC-3¢, followed by ligation into the EcoRV–XhoI
sites of pcDNA3 (Invitrogen) under the control of the
CMV promoter. PEGFP-N3 plasmid (Clontech)
expressing GFP under the control of the CMV promoter
was used as control plasmid for the cotransfection
experiments.
Cells and megakaryocytic differentiation
The human chronic myelogenous leukemia cell line K562
was grown in RPMI 1640 medium (Biological Industries)
supplemented with 50 U penicillinÆmL
)1
,50lgÆmL

)1
strep-
tomycin, 0.1 mgÆmL
)1
kanamycin and 10% fetal bovine
serum. Cells at a density of 5–7 · 10
5
cellsÆmL
)1
or
1.2–1.5 · 10
6
cellsÆmL
)1
were considered as logarithmically
growing (log) or growth arrested (dense), respectively.
Megakaryocytic differentiation was induced by dilution of
cells at a density of 1.2 · 10
6
cellsÆmL
)1
, to a final concen-
tration of 5 · 10
5
cellsÆmL
)1
, with medium containing 5 n
M
12-O-tetradecanoylphorbol-13-acetate (TPA; Calbiochem)
for 48 h.

Plasmid transfections and luciferase assays
Twenty or forty micrograms supercoiled DNA of each of
the bi-cistronic vectors or the cotransfected plasmid,
respectively, were used per 7.5 · 10
6
K562 cells for each
electroporation sample. Electroporation was performed in
0.8 mL RPMI 1640 without serum by an electric pulse of
240 V and 1500 mF (Easy Ject1 Electroporator; Equibio).
Immediately following the electric pulse, the cells were
transferred to 10 mL RPMI 1640 medium supplemented
with 20% fetal bovine serum for 24 h. The cells were diluted
to a final concentration of 5 · 10
5
viable cellsÆmL
)1
Ó FEBS 2002 eIF2a phosphorylation and IRES activity (Eur. J. Biochem. 269) 2811
(as determined by Trypan blue staining) in RPMI 1640
supplemented with 10% fetal bovine serum, with or without
5n
M
TPA, for 48 h. Transfection efficiency was 50–60%, as
judged by the percentage of fluorescent GFP-expressing
cells. The control and differentiated transfectants were
harvested simultaneously, and assayed for Renilla and firefly
luciferase activities using the Dual-luciferase reporter assay
system (Promega) and TD-20e-Luminometer (Turner).
RNA was isolated from cells transfected with the bicistronic
constructs and analysed by Northern blotting using a LUC-
specific probe to ensure that transcripts of the correct size

were produced. Bi-cistronic transcript level from all con-
structs was approximately five times higher in differentiated
cells because of the increased activity of the CMV promoter
[12].
32
P
i
labelling, immunoprecipitation and Western analysis
of eIF2a
A total of 10
6
log, dense or megakaryocytic differentiated
K562 cells were washed twice with Hepes/saline buffer
(50 m
M
KOH/Hepes pH 7.0, 150 m
M
NaCl) and resus-
pended in 0.5 mL Dulbecco’s modified Eagle medium
lacking sodium phosphate (Sigma) supplemented with 10%
dialysed fetal bovine serum. The cells were labelled for 2 h
with 0.2 mCi
32
P
i
ÆmL
)1
(Amersham,#PBS13),followedby
twowasheswithcoldNaCl/P
i

containing 10 m
M
b-glycerophosphate and 50 m
M
NaF. Proteins were extrac-
ted from the cell pellets by using 470 lL lysis buffer
containing 25 m
M
KOH/Hepes pH 7.2, 0.5% Elugent
TM
(Calbiochem), 100 m
M
KCl, 0.05% SDS, 1 m
M
dithiothre-
itol, 2 l
M
okadaic acid, 10 m
M
b-glycerophosphate, 50 m
M
NaF and protease inhibitor cocktail (Complete
TM
, Roche).
For immunoprecipitation the sample was supplemented
with 28 lL5
M
NaCl and 0.5 lLanti-eIF2a mAb [21] and
incubated for 1 h at 4 °C. Next, rabbit anti-(mouse IgG) Ig
(Jackson Immuno Research) were added for further

incubation of 1 h, followed by addition of 10 lLpacked
protein A-Sepharose (Pharmacia Biotech) for an additional
1-h incubation. Following separation of the immunopre-
cipitate by SDS/10% PAGE, the proteins were blotted onto
a nitrocellulose membrane and quantified by phosphoi-
mager. The membrane was then used for Western analysis
using antibodies specific for Ser51-phosphorylated eIF2a
(Research Genetics, Inc.), and following stripping mAb
specific for total eIF2a were used.
Polysome fractionation
A total of 3.5 · 10
7
log, dense, or megakaryocytic differ-
entiated K562 cells were treated with 90 lgÆmL
)1
cyclohex-
imide for 10 min prior to harvest and used for fractionation
of polysomes by sedimentation through 5–47% sucrose
gradients [22].
Protein synthesis rate
One million log, dense, or megakaryocytic differentiated
K562 cells were re-suspended in 2 mL RPMI medium
containing 10% fetal bovine serum. The cells were labelled
for 20 min with 20 lCiÆmL
)1
[
35
S]
L
-methionine, [

35
S]
L
-
cysteine mix (NEN, #NEG072), followed by two washes
with cold NaCl/P
i
. Proteins were extracted from the cell
pellets using 50 lL lysis buffer containing 25 m
M
KOH/
Hepes pH 7.5, 1% Triton X-100, 100 m
M
KCl, 1 m
M
dithiothreitol, 2 l
M
okadaic acid, 10 m
M
b-glycerophos-
phate, 50 m
M
NaF and protease inhibitor cocktail (Com-
plete
TM
, Roche). Twenty micrograms total protein were
applied onto 3 m
M
filter papers (Whatman) and washed
three times for 1 min in boiling 5% (W/V) trichloroacetic

acid containing traces of cold
L
-methionine and
L
-cysteine.
Thefilterswerethenrinsedonceinethanol,driedand
counted in a scintillation counter (Beckman).
Cell cycle analysis and differentiation markers
For cell cycle analysis 5 · 10
5
cells were harvested, washed
with NaCl/P
i
and re-suspended in 0.5 mL NaCl/P
i
contain-
ing 0.1% sodium azide. Following addition of 50 lLNaCl/
P
i
containing 1% Triton X-100 and 50 lL1mgÆmL
)1
propidium iodide, the cell-cycle of the cells was analysed by
Becton Dickinson FACSort, using the Cell Quest software.
The Vav protein was used as a marker for differentiation. To
detect Vav protein level the cells were lysed using a buffer
containing 25 m
M
KOH/Hepes pH 7.5, 1% Triton X-100,
100 m
M

KCl, 1 m
M
dithiothreitol, 2 l
M
okadaic acid,
10 m
M
b-glycerophosphate, 50 m
M
NaF and protease
inhibitors cocktail (Complete
TM
, Roche). 140 lgoftotal
cell proteins were separated by SDS/10% PAGE, and
blotted onto a nitrocellulose membrane which was then used
for Western analysis using polyclonal antibodies specific for
Vav (Santa Cruz) and polyclonal antibodies specific for
CKIIa (a gift from D. Canaani, Tel Aviv University, Israel).
RESULTS
Favorable conditions for IRES-mediated translation
are established during differentiation
In previous studies we have demonstrated that in addition to
transcriptional activation of PDGF2 during megakaryocytic
differentiation, its IRES element undergoes functional
activation during the differentiation process [12,13]. Interes-
ted in elucidating the mechanism of IRES function in
general, we have used the differentiation phase to learn more
about the possible involvement of trans-acting factors.
Viewing the differentiated state as a ÔpermissiveÕ environment
for PDGF2-IRES mediated translation, we also wished to

check the effect of differentiation conditions on the beha-
viour of additional cellular and viral IRES elements.
Although normally the mRNAs of VEGF and c-Myc are
not present in differentiated megakaryocytes, it was still of
interest to check the activity of their IRES elements in the
differentiated K562 cells that are permissive for PDGF2
IRES use. The IRES elements of human VEGF, human
c-Myc and EMCV were cloned into a CMV promoter-driven
bicistronic vector, between the coding regions of Renilla and
firefly luciferases, as illustrated in Fig. 1. K562 cells were
transfected with each of the recombinant plasmids followed
by incubation under normal or differentiation conditions for
48 h prior to measurements of Renilla and firefly luciferase
enzymatic activities. As shown in Table 1, in differentiated
cells we observed elevation in the activity of both luciferases,
because of increased CMV promoter activity in this system
which results in a fivefold increase in transcript levels
(demonstrated in Fig. 3 [12]). However, upon differentiation,
2812 G. Gerlitz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
the utilization of the first cistron increased only 2.2–2.7-fold
whereas a 6–7.9-fold increase in utilization of the IRES-
mediated second cistron was observed. This was in contrast
with utilization of the second cistron from the IRES-less pLL
vector. A 2.2- to 3.1-fold increase in the firefly per Renilla
ratio was detected upon differentiation from the IRES-
containing vectors, in contrast with the 0.8-fold increase
observed for the IRES-less transcript from pLL.
Reduction of global protein synthesis
during differentiation is accompanied
by eIF2a phosphorylation

The terminal differentiation process is usually accompanied
by arrest of cellular proliferation and by decreased global
protein synthesis [7,8]. As megakaryocytic differentiated
cells cease to proliferate [23], we wished to check the status
of their global mRNA translation. The rate of radio-
labelled amino acids incorporation in logarithmically
growing cells was compared to that in density-arrested or
differentiated cells. The incorporation rate was almost two
times lower in both stationary (dense) and differentiated
cells compared with logarithmically growing cells (Fig. 2A).
The decrease in global protein synthesis was also evident
from the differences in the polysomal profiles of the above
cells. Fig. 2B demonstrates the reduced heavy polysomes
levels upon growth arrest due to high density or differen-
tiation.
As the rate of protein synthesis in higher eukaryotes is
commonly regulated at the level of eIF2a phosphorylation
[4,24], we wished to check the status of eIF2a phosphory-
lation in cells undergoing differentiation. For this purpose,
the cells were metabolically labelled with
32
P
i
followed by
immunoprecipitation using antibodies specific for eIF2a.
Fig. 3A shows the radio-labelled phosphorylated eIF2a,the
total amount of eIF2a as determined by Western analysis,
and the ratio of phosphorylated eIF2a per total eIF2a.In
dense cells, in which growth arrest was probably induced by
depletion of essential nutrients/growth factors in the

medium, a two-fold increase in eIF2a phosphorylation
was observed compared to logarithmically growing cells.
However, a more significant, 6.4-fold increase in eIF2a
phosphorylation was detected upon growth arrest induced
by the differentiation process. As the regulated phosphory-
lation of mammalian eIF2a has been shown to occur only
on Ser51 [25], we re-confirmed the
32
P
i
labelling results by
using specific antibodies against phosphorylated Ser51.
Fig. 3B shows the phosphorylated Ser51 and the total eIF2a
levels as determined by Western analysis. In agreement with
the labelling studies, the ratio of Ser51-phosphorylated
eIF2a to total eIF2a revealed an increase of 3.3-fold and
7.9-fold in eIF2a phosphorylation level in dense and
differentiated cells, respectively, compared with logarith-
mically growing K562 cells. In summary, growth arrest is
accompanied by elevation of eIF2a phosphorylation.
However, megakaryocytic differentiation involves two- to
threefold higher eIF2a phosphorylation than that resulting
from growth arrest that is induced by increased cell density.
Increased IRES-mediated translation
during differentiation requires
eIF2a phosphorylation
The phenomenon of increased IRES-mediated translation
under conditions of increased eIF2a phosphorylation,
raised the notion that eIF2a phosphorylation confers a
translational advantage on IRES-containing mRNAs. To

test this hypothesis, we looked at the effect of expression of
eIF2a phosphorylation inhibitors on IRES use. We used
either the vaccinia virus K3L gene that encodes an eIF2a
homologue and pseudo-substrate inhibitor of eIF2a protein
Fig. 1. The bicistronic transcription units used. The bicistronic tran-
scription unit expressing Renilla and Firefly luciferase reporter genes as
the first and second cistrons, respectively, under the control of CMV
promoter. The 5¢UTRs of the human PDGF2, VEGF, c-Myc or
EMCV were placed in the intercistronic space as indicated to create
plasmids pLPL, pLVL, pLML and pLEL, respectively. The bicistronic
IRES-less vector pLL served as a control plasmid.
Table 1. Effect of differentiation on IRES activity. Each of the bicistronic plasmids harbouring the IRES elements indicated in Fig. 1 was
transfected into K562 cells followed by further incubation under nondifferentiation or differentiation conditions and subsequent analysis of Renilla
(R) and Firefly (F) luciferase activity. Each value represents the mean ± SE of three independent experiments. The fold induction values represent
the F/R ratio in differentiated cells relative to the F/R ratio in nondifferentiated cells.
Non-differentiated cells Differentiated cells
Fold F/R
induction
Renilla
(U per 10
6
cells)
Firefly
(U per 10
6
cells) F/R
Renilla
(U per 10
6
cells)

Firefly
(U per 10
6
cells) F/R
pLPL 59 ± 3.5 7.5 ± 0.6 0.13 ± 0.01 131 ± 18 53 ± 6 0.4 ± 0.05 3.1 ± 0.2
pLML 94 ± 10 6.4 ± 0.07 0.068 ± 0.01 253 ± 29 38 ± 5 0.15 ± 0.02 2.2 ± 0.3
pLVL 89 ± 10 42 ± 3.3 0.47 ± 0.06 240 ± 33 312 ± 30 1.3 ± 0.2 2.8 ± 0.1
pLEL 78 ± 9 3.4 ± 0.4 0.04 ± 0.006 205 ± 25 27 ± 3.5 0.13 ± 0.02 3.1 ± 0.4
pLL 52 ± 7 0.6 ± 0.08 0.01 ± 0.002 125 ± 15 1.0 ± 0.16 0.008 ± 0.001 0.8 ± 0.2
Ó FEBS 2002 eIF2a phosphorylation and IRES activity (Eur. J. Biochem. 269) 2813
kinases [18,26], or PKRD6, a dominant-negative variant of
the dsRNA activated eIF2a-kinase, PKR [19]. To confirm
the connection between eIF2a phosphorylation during
differentiation and IRES activation, we also used a plasmid
encoding a variant form of eIF2a in which Ser51 is replaced
by an alanine residue (eIF2a-S51A). As this variant protein
cannot undergo phosphorylation, it serves as a competitor
that reduces the translational inhibitory effect of phosphor-
ylated endogenous wild-type eIF2a [20]. Plasmids expres-
sing K3L, PKRD6, eIF2a-S51A variant or GFP as control,
were cotransfected along with the different IRES-containing
bi-cistronic vectors into K562 cells followed by their
incubation under normal or differentiation conditions for
48 h prior to measurements of Renilla and firefly luciferase
enzymatic activities. Tables 2 and 3 show the effects of the
transfected gene products on the absolute levels of the
translation products of both cistrons. Overexpression of
K3L, PKRD6oreIF2a-S51A led to enhanced translation of
both cistrons in nondifferentiated cells, whereas in differen-
tiated cells it led to decreased IRES-mediated translation of

the second cistron. Fig. 4A summarizes the sensitivity of the
differentiation-induced IRES activation to the various eIF2a
phosphorylation inhibitors. Expression of eIF2a-S51A,
K3L, or PKRD6 in differentiated cells reduced the level of
eIF2a-P to 80%, 70% or 40% compared with GFP-
transfected control, respectively (Fig. 4B). The effect of the
various transfections on eIF2a phosphorylation is underes-
timated as not all the cells were successfully transfected. The
reduction in IRES use in differentiated cells by expression of
K3L, eIF2a-S51A and PKRD6 was shown to be correlated
with a reduction in the level of eIF2a-P. These data suggest
that eIF2a phosphorylation is required for more efficient
IRES use during the differentiation process.
Fig. 3. Phosphorylation of eIF2a in logarithmically growing, dense and
differentiated K562 cells. (A) A total of 10
6
logarithmically growing
(Log), density-induced growth arrested (Dense) or differentiated
(Dif f ) K562 cells were metabolically labelled with
32
Pi, followed by
immunoprecipitation using an antibody specific for eIF2a.The
immunoprecipitated phospholabeled proteins were separated by SDS/
10% PAGE and blotted onto a nitrocellulose membrane. Phosphor-
ylated eIF2a was observed following exposure of the membrane to an
X-ray film and the intensities of the bands were determined using a
phosphoimager. The same membrane was analysed for total eIF2a
level by Western analysis and the intensities of the bands were deter-
mined by densitometry. The [
32

P]eIF2a/eIF2a ratio in logarithmically
growing cells was set as 1. (B) Fifty lg total protein extract from Log,
Dense or Diff K562 cells were separated by 10% SDS/PAGE and
blotted onto a nitrocellulose membrane. Phosphorylated eIF2a was
detected using antibodies specific for phosphorylated Ser51. The same
membrane was stripped and used for Western analysis using antibodies
specific for total eIF2a.TheeIF2a-P/eIF2a ratio in logarithmically
growing cells was set as 1.
Fig. 2. The effect of differentiation on the overall protein synthesis level.
(A) Logarithmically growing (Log), density-induced growth arrested
(Dense) or differentiated (Diff) K562 cells were metabolically labelled
with [
35
S]
L
-methionine and [
35
S]
L
-cysteine followed by determination
of their incorporation level by trichloro-acetic acid precipitation, as
described in Materials and methods. The incorporation level (cpmÆlg
)1
protein) in log cells was termed 100%. The values are means ± SE of
three independent experiments. (B) A total of 3.5 · 10
7
log, dense or
differentiated K562 cells were harvested and their cytoplasmic com-
partments were subjected to fractionation on linear 5–47% sucrose
gradients. Relative absorbance at 260 nm was monitored continuously

as the gradient was collected. The vertical bars on the abscissa indicate
the boundaries of the polysomal (P) and subpolysomal (SP) fractions.
Peaks at the top of the gradient containing the 40 S, 60 S and 80 S
ribosomal subunits are indicated.
2814 G. Gerlitz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
Reduced phosphorylation of eIF2a has no significant
effect on megakaryocytic differentiation
The differentiation process is a cascade of events leading to
major changes in gene expression. Reduced global protein
synthesis is a consequence of upstream events, as inhibition
of protein synthesis per se does not lead to differentiation.
However, it seemed important to ascertain the effect of
reduced eIF2a phosphorylation level on the differentiation
process. As megakaryocytic differentiation involves growth
arrest and polyploidy [27], DNA content evaluation by flow
cytometry was chosen as a tool to detect the reduced
number of cells in S-phase and enhanced number of cells
harbouring two- to fourfold higher DNA content [23,28].
K562 cells were transfected with plasmids expressing K3L,
PKRD6, eIF2a-S51A, or GFP under similar conditions to
those used to assess the effect of inhibition of eIF2a
phosphorylation on IRES activity. The transfection effi-
ciency in these experiments was 50–60%, as judged by the
percentage of fluorescent GFP-expressing cells. As shown in
Fig. 5A, the differentiation process was not significantly
affected by any of the transfected plasmids, as judged by the
decreased number of cells in S-phase and increased number
of polyploid cells. For additional confirmation we checked
the level of the Vav proto-oncogene, which is known to
increase early during megakaryocytic differentiation [27,29].

CKIIa protein level was used as a control. As shown in
Fig. 5B, the level of Vav protein was increased due to
differentiation, regardless of the transfected plasmid. These
results suggest that interference with eIF2a phosphorylation
does not prevent the early differentiation steps, e.g. the
global changes in gene expression upstream of mRNA
translation. Instead, it interferes with the ability to fine-tune
the translation efficiency of specific mRNA groups.
DISCUSSION
Cells undergoing terminal differentiation exhibit extensive
changes in the pattern of gene expression. Much data has
been accumulated regarding transcriptional regulation, but
less is known about the mechanisms that inhibit the
translation of most transcripts while activating the transla-
tion of specific mRNAs during the course of differentiation.
During the early developmental stages of Xenopus, Caenor-
habditis elegans and Drosophila, the translation of subclasses
of mRNAs is regulated. However, during differentiation of
mammalian cells, only a few individual mRNAs are known
to be subjected to translational regulation due to their cis-
regulatory elements (reviewed in [30]). Initial attempts to
identify groups of translationally regulated genes during
HL60 cell differentiation towards monocytes/macrophages
has revealed that while most mRNAs are released from
polysomes early in the differentiation process, a subset of
transcripts is retained or even mobilized onto polysomes [7].
The data presented in this study suggest that mRNAs
harbouring an IRES within long, structured, uORF-
burdened 5¢UTRs, comprise a subgroup which is specific-
ally translationally activated during differentiation (Fig. 1

Table 3. Effect of eIF2a-S51A expression on IRES activity. Absolute values of Renilla and Firefly activities from experiments described in Fig. 4A.
Non-differentiated cells Differentiated cells
Renilla
(U per 10
6
cells)
Firefly
(U per 10
6
cells) F/R
Renilla
(U per 10
6
cells)
Firefly
(U per 10
6
cells) F/R
Fold F/R
induction
pLPL + GFP 87 ± 6 7.8 ± 1.4 0.09 ± 0.02 184 ± 20 59 ± 5 0.32 ± 0.08 3.5 ± 0.3
+ eIF2a-S51A 145 ± 11 14 ± 2 0.1 ± 0.02 200 ± 18 42 ± 3 0.2 ± 0.02 2.1 ± 0.2
pLVL + GFP 105 ± 11 42 ± 5 0.4 ± 0.06 273 ± 33 382 ± 37 1.4 ± 0.2 3.5 ± 0.2
+ eIF2a-S51A 180 ± 20 72 ± 8 0.4 ± 0.1 304 ± 31 273 ± 28 0.9 ± 0.1 2.2 ± 0.2
Table 2. Effect of K3L and PKRD6 expression on IRES activity. Absolute values of Renilla and Firefly activities from experiments described in
Fig. 4A.
Non-differentiated cells Differentiated cells
Fold F/R
induction
Renilla

(U per 10
6
cells)
Firefly
(U per 10
6
cells) F/R
Renilla
(U per 10
6
cells)
Firefly
(U per 10
6
cells) F/R
pLPL + GFP 53 ± 3 6.4 ± 0.6 0.12 ± 0.02 125 ± 17 50 ± 6 0.4 ± 0.05 3.2 ± 0.2
+ K3L 184 ± 13 20 ± 2 0.1 ± 0 249 ± 27 48 ± 7 0.2 ± 0.03 2.0 ± 0.2
+ PKRD6 132 ± 12 17 ± 2 0.13 ± 0.02 213 ± 16 37 ± 4 0.17 ± 0.03 1.2 ± 0.15
pLML + GFP 101 ± 12 7.3 ± 1.1 0.07 ± 0 235 ± 23 38 ± 14 0.16 ± 0.02 2.3 ± 0.3
+ K3L 303 ± 27 20 ± 3.5 0.06 ± 0.01 318 ± 27 34 ± 3.6 0.1 ± 0.02 1.6 ± 0.2
+ PKRD6 222 ± 25 22 ± 3 0.1 ± 0.03 286 ± 28 22 ± 3 0.08 ± 0.01 0.8 ± 0.1
pLVL + GFP 100 ± 11 52 ± 7 0.5 ± 0.1 253 ± 32 368 ± 40 1.43 ± 0.2 2.9 ± 0.2
+ K3L 260 ± 21 130 ± 10 0.5 ± 0 390 ± 38 286 ± 20 0.73 ± 0.08 1.5 ± 0.15
+ PKRD6 186 ± 20 120 ± 15 0.6 ± 0.1 300 ± 30 191 ± 5 0.6 ± 0.1 1.0 ± 0.1
pLEL + GFP 51 ± 6 2.0 ± 0.9 0.04 ± 0.01 226 ± 25 31 ± 5 0.13 ± 0.01 3.2 ± 0.4
+ K3L 228 ± 24 8.5 ± 0.3 0.04 ± 0.01 330 ± 5 33 ± 4 0.11 ± 0.03 2.7 ± 0.25
+ PKRD6 98 ± 10 5.7 ± 0.5 0.06 ± 0.01 192 ± 20 17 ± 3.3 0.09 ± 0.01 1.5 ± 0.2
Ó FEBS 2002 eIF2a phosphorylation and IRES activity (Eur. J. Biochem. 269) 2815
and Table 1) under conditions of eIF2a phosphorylation
and substantial inhibition of protein synthesis (Figs. 2 and

3). Other recent studies demonstrate a correlation between
differentiation with reduction of global protein synthesis
and enhanced eIF2a phosphorylation [31–34].
The list of recently identified IRES elements within
cumbersome 5¢UTRs of growth factors, cytokines, tran-
scription factors and oncogenes is constantly growing
(reviewed in [14,15]). Cellular IRES elements have been
implied to confer a translational advantage under reduced
levels of active 5¢-cap binding complex. However, the
current study shows that under certain physiological
conditions, for instance during differentiation, translation
mediated by cellular IRES elements benefits from phos-
phorylation of eIF2a. Supplementary mechanisms for
inhibition of global protein synthesis, such as reduced
availability of the 5¢-cap binding complex, may also take
place during differentiation and contribute to the observed
enhancement of 5¢-cap independent translation. However,
such mechanisms were beyond the focus of this study.
Phosphorylation of eIF2a which leads to decreased binding
of initiator tRNA to the small ribosomal subunit, has been
mostly studied in relation to growth inhibition induced in
response to starvation for growth factors/nutrients, heat
shock, and virus infection (reviewed in [35]). PKR, the
interferon-induced (double-stranded) RNA-activated eIF2a
kinase, has been implicated in cellular growth control, as
well as in differentiation and apoptosis (reviewed in [36–39]).
It seems likely that the regulatory function of eIF2 depends
on the delicate balance of phosphorylated eIF2a with other
cellular components, and on the physiological status of the
cell. The data presented in this study support this idea.

Inhibition of PKR activity by over-expression of its
dominant-negative variant PKRD6, or reduction of eIF2a
phosphorylation level by over-expression of its variant
form, eIF2a-S51A, or the pseudosubstrate K3L, resulted in
reduced IRES activity in differentiated cells. However, it did
not have any inhibitory effect on IRES activity in nondif-
ferentiated cells (Fig. 4). The fact that expression of PKRD6
had a greater impact on the levels of eIF2a phosphorylation
and IRES-mediated translation compared with the efficient
general eIF2a kinase inhibitor K3L suggests that PKR is the
primary activated kinase during differentiation. Interest-
ingly, the over-expression of the eIF2a phosphorylation
Fig.4.EffectofeIF2a phosphorylation inhibitors on IRES activity. (A)
Each of the bicistronic vectors pLPL, pLVL, pLML, pLEL (described
in Fig. 1A) harbouring the IRES elements of PDGF2, VEGF, c-Myc
or EMCV, respectively, was cotransfected into K562 cells together
with a plasmid expressing the PKRD6, K3L, eIF2a Ser51 fi Ala
mutant ( pc2a-S51A), or GFP coding region from the CMV promoter.
The cells were further incubated under normal or differentiation con-
ditions for 48 h and subsequently analysed for Renilla (R) and firefly
(F) luciferase activity. The absolute values are presented in Tables 2
and 3. Each value represents the mean ± SE of three independent
experiments. The fold induction values represent the F/R ratio in
differentiated cells relative to the F/R ratio in nondifferentiated cells.
The graph demonstrates the effect of K3L (stippled bars), PKRD6
(dark bars), or eIF2a-S51A (hatched bars) on the differentiation-
induced IRES activation relative to the fold induction value with GFP
that was set as 100% (light bars). (B) Fifty lg of total protein extract
from differentiated cells transfected with plasmids expressing GFP,
K3L, PKRD6oreIF2a-S51A were separated by 10% SDS/PAGE and

blotted onto a nitrocellulose membrane. Phosphorylated eIF2a was
detected using antibodies specific for phosphorylated Ser51. The same
membrane was stripped and used for Western analysis using antibodies
specific for total eIF2a.TheeIF2a-P/eIF2a ratio in GFP-transfected
cellswassetas1.
Fig. 5. The effect of eIF2a phosphorylation inhibitors on the differenti-
ation process. (A) A total of 5 · 10
5
K562 cells were transfected by
electroporation with a plasmid expressing GFP, eIF2a-S51A, K3L or
PKRD6, as indicated. The transfected cells were incubated under
normal (–TPA) or differentiation (+ TPA) conditions for 48 h, and
subjected to DNA content analysis by flow cytometry. (B) One-hun-
dred and forty lg of total protein extracted from the transfected cells as
detailedin(A)wereseparatedbySDS/10%PAGE,blottedontoa
nitrocellulose membrane, and subjected to Western analysis using
antibodies specific for Vav and for CKIIa.
2816 G. Gerlitz et al. (Eur. J. Biochem. 269) Ó FEBS 2002
inhibitors did not interfere with the global process of
megakaryocytic differentiation as judged by their morphol-
ogy (not shown), cell-cycle, and enhanced Vav protein
expression (Fig. 5). The latter result suggests that both
eIF2a phosphorylation and IRES activation are late events
during the differentiation process. The peak of PDGF2
IRES activation at 48 h after induction of differentiation
[11,12] is in agreement with this notion. It is therefore
conceivable that eIF2a phosphorylation serves to fine-tune
the translation efficiency of specific mRNA groups.
Increased translation of certain IRES-containing
mRNAs has also been implicated in apoptosis [40–43], a

cellular process that includes activation of eIF2a phos-
phorylation [44–47]. Furthermore, the recently discovered
cell cycle-dependent IRES elements are activated specifically
at the G
2
/M boundary [48–50], when increased phosphory-
lation of eIF2a is found in correlation with decreased
overall rate of protein synthesis [51]. Moreover, the IRES
elements of the amino acid transporter protein cat-1 and
c-Myc mRNAs have recently been shown to function
efficiently where there is an increase in eIF2a phosphory-
lation, under conditions of amino acid starvation and
genotoxic stress, respectively [52,53].
What is the mechanism underlying IRES-mediated
translation under conditions of enhanced eIF2a phosphory-
lation? In nondifferentiated cells, in which global translation
is active, the IRES-containing mRNAs compete with the
cap-dependent mRNAs for the translation machinery. The
decrease in global protein synthesis and reduced competi-
tion might be beneficial for IRES-mediated translation
during differentiation. An interesting possibility may be the
ability of IRES elements to direct efficient translation
initiation in the absence of eIF2 and Met-tRNA
i
. Recently,
internal initiation without Met-tRNA
i
has been demonstra-
ted in two picorna-like insect viruses, Plautia stali intestinal
virus and Cricket paralysis virus [54–56]. It is an open

question whether cellular IRES elements known to contain
conserved secondary and tertiary structural motifs can also
direct internal translation from noncognate initiation
codons in the absence of Met-tRNA
i
. Direct binding to
the 40 S ribosomal subunit followed by joining of the 60 S
subunit may provide a significant advantage to IRES
elements that confer efficient translation under conditions of
global translation inhibition mediated by eIF2a phosphory-
lation. Another possibility could be that a mechanism exists
that is similar to the translational regulation of GCN4 in
yeast. In this case, the induction of GCN4 translation in
response to eIF2a phosphorylation is modulated by four
short uORFs in the 5¢UTR. Reduced rates of ternary
complex formation leads to bypass of the uORFs and
initiation at the downstream GCN4 major ORF [6]. A
comparable mechanism of translational regulation was
recently demonstrated for the stress-induced transcription
factor ATF4, in mammalian cells under conditions of
enhanced eIF2a phosphorylation due to stress [57]. Simi-
larly, the short ORFs that furnish many of the cellular IRES
elements may have a role in translational regulation, as
seems to be the case for the activation of the cat-1 mRNA
[52]. Internal ribosomal binding upstream of the translation
initiator codon may be followed by subsequent scanning to
the initiation codon. For instance, in the case of the PDGF2
5¢UTR, which contains three uORFs, the IRES has been
mapped to the central part of the 5¢UTR at the vicinity of
the first uORF [13]. The need to scan through the second

and third uORFs towards the major coding region cannot
be ruled out at this point. Another possibility may be that
eIF2a phosphorylation induces the synthesis of a protein
that interacts with the IRES. This can be achieved by direct
translational regulation of its mRNA akin to GCN4/ATF4
mRNAs, or by regulating the translation of a GCN4/
ATF4-like transcription factor that activates the transcrip-
tion of the potential IRES activator. Current experiments
are designed to elucidate the mechanism(s) by which eIF2a
phosphorylation serves to enhance IRES-mediated transla-
tion.
ACKNOWLEDGEMENTS
This work was supported by the Israel Science Foundation adminis-
tered by the Academy of Sciences and Humanities – the Charles H.
Revson Foundation to O. E. S., by a grant from the Israeli Chief
Scientist’s Office of the Ministry of Health to O. E. S. and NSF 9808401
to R. J. We thank B. White and G. Krause for the antibody against
phosphorylated eIF2a, D. Canaani for antibody against CKIIa,
N. Sonenberg for PKRD6 construct, R. J. Kaufman for eIF2a-S51A
construct, and B Z. Levi for the pSKVLUC construct. We are grateful
to T. Dever for comments on the manuscript.
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