(Received for publication, November 27, 1995; and in revised form, February 22, 1996)
From the
Translation initiation in eukaryotes is facilitated by the mRNA
5` cap structure (mGpppX, where X is any
nucleotide) that binds the multisubunit initiation factor eIF4F through
one of its subunits, eIF4E. eIF4E is a phosphoprotein whose
phosphorylation state positively correlates with cell growth. Protein
kinase C phosphorylates eIF4E in vitro, and possibly in
vivo. Using recombinant eIF4E incubated in vitro with
purified protein kinase C and analyzed by solid-phase phosphopeptide
sequencing in combination with high performance liquid chromatography
coupled to mass spectrometry, we demonstrated that the third amino acid
of the peptide SGSTTK (Ser
) is the major site of
phosphorylation. This finding is consistent with the newly assigned in vivo phosphorylation site of eIF4E (Joshi, B., Cai, A. L.,
Keiper, B. D., Minich, W. B., Mendez, R., Beach, C. M., Stepinski, J.,
Stolarski, R., Darzynkiewicz, E., and Rhoads, R. E.(1995) J. Biol.
Chem. 270, 14597-14603). A S209A mutation resulted in
dramatically reduced phosphorylation, both in vitro and in
vivo. Furthermore, the mutant protein was phosphorylated on
threonine (most probably threonine 210) in vivo. Here we show
that in the presence of the recently characterized translational
repressors 4E-BP1 or 4E-BP2, phosphorylation of eIF4E by protein kinase
C is strongly reduced. This suggests a two-step model for the
phosphorylation (and activation) of eIF4E by growth factors and
hormones: first, dissociation of eIF4E from 4E-BPs, followed by eIF4E
phosphorylation.
In eukaryotes, translation initiation is rate-limiting and is
highly regulated. The initiation factor eIF4F plays a key role in
regulating translation initiation rates. eIF4F ()is composed
of three subunits: eIF4E, the cap-binding protein; eIF4A, an RNA
helicase; and eIF4G (formerly p220), which bridges between eIF4A and
eIF4E. eIF4F binding to the cap structure (m
GpppX,
where X is any nucleotide) is mediated by the eIF4E subunit.
eIF4F, in conjunction with another initiation factor, eIF4B, is thought
to unwind the mRNA 5`-secondary structure to facilitate the binding of
ribosomes. eIF4E, and hence eIF4F, is present in limiting amounts in
cells relative to other initiation factors(1, 2) , and
plays an important role in control of cell growth and
development(3) . eIF4E overexpression in rodent cells induces
cellular transformation(4) . eIF4E also exhibits mitogenic
activity, as microinjection of eIF4E into serum-starved cells activates
DNA synthesis(5) . Conversely, expression of eIF4E antisense
RNA diminishes translation rates, reduces cellular proliferation, and
partially reverts the phenotype of transformed cells (6, 7) .
Recently, two proteins which interact with
eIF4E, 4E-BP1 and 4E-BP2 (for eIF4 E Binding Proteins
1 and 2; also known as PHAS-I for Phosphorylated Heat- and Acid-Stable protein, Insulin stimulated), were
characterized(8, 9, 10) . These proteins,
which share 56% identity, inhibit cap-dependent, but not
cap-independent, translation(9) . The two 4E-BPs inhibit eIF4E
function by competing with eIF4G for a common binding site on
eIF4E(11, 12) . 4E-BP1 and 4E-BP2 binding to eIF4E is
regulated by phosphorylation: the underphosphorylated species possess a
high affinity for eIF4E, whereas the hyperphosphorylated species do not
bind to eIF4E(9, 10) . ()In unstimulated
cells, a significant amount of 4E-BP1 is underphosphorylated and is
bound to eIF4E(9) . Following activation, either by insulin or
growth factors, 4E-BP1 becomes hyperphosphorylated, and dissociates
from eIF4E(9, 10) . Subsequently, eIF4E binds to eIF4G
to form an active eIF4F complex. Conversely, upon infection with some
picornaviruses, 4E-BP1 is rendered completely underphosphorylated
enabling 4E-BP1 to bind strongly to eIF4E, thus contributing to the
shut-off of host protein synthesis (13) . 4E-BP1 is a substrate
for MAP kinase in vitro, and data suggest that it could be
phosphorylated in vivo by the same enzyme(10) .
However, the mechanism of phosphorylation now appears to be more
complex, since insulin-stimulated phosphorylation is inhibited by
rapamycin, an immunosuppressant which has no effect on the MAP kinase
pathway(14, 15, 16) . In more recent
experiments, using other inhibitors, such as wortmannin and SQ20006 and
mutants in the platelet-derived growth factor receptor, it has been
demonstrated that 4E-BP1 phosphorylation is effected by the
phosphatidylinositol-3-OH (PI3) kinase wortmannin-sensitive
pathway(17) .
As eIF4E is limiting in cells(1, 2) , the regulation of 4E-BP1 binding activity by phosphorylation may explain some of the modulation of eIF4E-dependent translation. However, the activity of eIF4E itself is also regulated by phosphorylation. The phosphorylation status of eIF4E positively correlates with translation and cellular growth rates. Phosphorylation of eIF4E increases in response to treatment with phorbol esters, hormones and growth factors(3) . eIF4E also becomes phosphorylated upon maturation of T and B cells or differentiation of PC12 cells into neurons. Conversely, eIF4E is dephosphorylated in cells blocked in mitosis, or following heat-shock or infection by adenovirus, concomitant with a decrease in translation rates (for reviews, see (3) and (18) ). A biochemical function for eIF4E phosphorylation has been indicated by the reports that phosphorylation of eIF4E increases its affinity for eIF4G(19) , as well as for the mRNA cap structure (20) .
The major phosphorylated amino acid of eIF4E is
serine, but some reports also demonstrated phosphorylation, under
certain conditions, on threonine(19) . Early experiments
assigned the phosphorylation site to Ser(21) .
However, subsequent experiments showed that a mutant in which serine 53
was replaced by an alanine (S53A) was phosphorylated in vivo to the same extent as wild type eIF4E, suggesting that eIF4E was
phosphorylated on another site(22) . Recently, the in vivo eIF4E phosphorylation site has been reassigned to
Ser
(23, 24, 25) .
Protein kinase C (PKC) (26, 27, 28) and an insulin-stimulated protamine kinase (29) phosphorylate eIF4E in vitro. Activation of PKC with phorbol esters enhances the phosphorylation state of eIF4E in vivo(18, 30) . Furthermore, coinjection of PKC and eIF4E into quiescent NIH 3T3 cells leads to a synergistic effect on eIF4E mitogenic activity(31) .
In this report we identified
the PKC phosphorylation site on eIF4E and described the regulation of
eIF4E phosphorylation by 4E-BP1 in vitro. We first confirmed
that the amino acid phosphorylated on eIF4E in vivo and that
phosphorylated by PKC in vitro are the same. We assigned the
PKC phosphorylation site to Ser, and showed that
Thr
is phosphorylated to some extent. We also showed
that, when complexed to 4E-BP1 or 4E-BP2, eIF4E does not serve as
substrate for PKC. Thus, it is possible that dissociation of eIF4E from
4E-BPs is a prerequisite for eIF4E phosphorylation. A model for the
regulation of eIF4E phosphorylation is presented.
eIF4E (1 µg) was phosphorylated by 0.001 units of
protein kinase C for 15 or 30 min at 30 °C in kinase buffer (20
mM Tris-HCl, pH 7.4, 5 mM MgCl, 0.1
mM CaCl
, 10 mM dithiothreitol, 5
µM ATP, 10%, v/v, mixed micelles) in the presence of 10
µCi of [
-
P]ATP. The mixed micelles
consisted of a solution of 3.1 mg/ml L-
-phosphatidyl-L-serine, 0.6 mg/ml 1,2-diolein,
and 0.3% Triton X-100 in 20 mM Tris-HCl, pH 7.4, which was
sonicated twice for 30 s. Kinase reactions were terminated by the
addition of Laemmli sample buffer followed by analysis on SDS, 12.5%
polyacrylamide gels. Alternatively, the product of the kinase reaction
was spotted onto a phosphocellulose (P81) paper which was washed
extensively in 75 mM phosphoric acid and dried. Bound
radioactivity was measured by Cerenkov counting in a scintillation
counter.
For capillary HPLC, a
320-µm inside diameter 15-cm long C
reversed
phase HPLC column (Michro-Tech Scientific, Saratoga, CA) was connected
to the electrospray ionization interface of the MS with a 50-cm-long
50-µm inside diameter
150 outside diameter, fused
silica capillary. The solvent gradient was delivered by a Michrom
Ultrafast Microprotein analyzer. The chromatography mobile phases were
0.05% trifluoroacetic acid, 2% acetonitrile in water (phase A) and
0.045% trifluoroacetic acid, 80% acetonitrile in water (phase B).
Samples (10 µl) were introduced onto the column isocratically for 5
min with phase A followed by a gradient from 0% phase B to 50% phase B
over another 35 min at a flow rate of 5 µl/min. A pre-column flow
split was installed to reduce the flow rate from 100 µl/min being
delivered by the pumps in the HPLC.
The mass spectrometer was an API-III triple quadrupole instrument equipped with an electrospray ionization interface (PE/Sciex, Thornhill, Ontario, Canada). The nebulization sheath gas was supplied via 1-mm inside diameter Teflon tubing at a rate of 2 liters/min while a drying nitrogen curtain gas at ambient temperature was supplied at a flow rate of 1.8 liters/min to the region between the ionspray ionization source and the MS orifice. Tuning and calibration of the MS was performed either with polypropylene glycol or myoglobin. Typically, the quadrupole was scanned from m/z 300 to 2000 at 2.9 s/scan unless otherwise stated. Background was subtracted from all mass spectra data.
We first performed a time course of PKC kinase assay to determine the extent of incorporation of phosphate into eIF4E (Fig. 1A). At maximal incorporation, up to 0.32 pmol of phosphate were incorporated into 1 pmol of eIF4E. However, most of the experiments described were performed in the linear portion of this curve (at 15 min), where 0.15 pmol of phosphate are incorporated into eIF4E. This value is higher than that reported earlier with native eIF4E from rabbit reticulocytes(28) .
Figure 1: PKC phosphorylates eIF4E at the site that is phosphorylated in vivo. A, time course of incorporation of phosphate into eIF4E was performed as described under ``Materials and Methods'' using 10 pmol of eIF4E bacterially expressed and 1 nmol of phosphate (ratio hot/cold phosphate is 1/2000) and radioactivity bound to the phosphocellulose paper was quantified by scintillation counting (the incorporation in PKC alone was substracted from the total counts before the conversion into picomoles of phosphate); results presented here are the mean of two experiments with different purifications of eIF4E. B, immunoprecipitated eIF4E from orthophosphate labeled NIH 3T3 cells and murine recombinant eIF4E phosphorylated by PKC for 15 min were analyzed by SDS-PAGE and autoradiography as described under ``Materials and Methods.'' C, phosphoamino acid analysis was performed on the samples shown in B, as described under ``Materials and Methods.'' Positions of cold amino acid standards are indicated. D, samples eluted from the gel in B were digested with trypsin and analyzed on two-dimensional phosphopeptide maps as described under ``Materials and Methods.'' Arrows indicate the origin of application. E, Samples eluted from the gel in A were digested with V-8 protease and separated by HPLC, as described under ``Materials and Methods.''
We reproduced earlier results (26) showing that the eIF4E peptide that is phosphorylated in vivo and that phosphorylated in vitro by PKC
comigrate on two-dimensional tryptic maps. For in vivo labeling, NIH 3T3 cells were incubated with
[P]orthophosphate, treated with okadaic acid,
and eIF4E was immunoprecipitated. For in vitro labeling,
bacterially expressed eIF4E was phosphorylated by PKC in vitro in the presence of [
P]ATP (Fig. 1B). Phosphoserine was the only detectable
species in both the in vivo and in vitro samples (Fig. 1C). A single major spot was detected for the
tryptic maps of both samples (Fig. 1D). When the two
samples were mixed together prior to the analysis, a single
phosphorylated species was observed (Fig. 1D). To
further establish that the two peptides are identical rather than
having fortuitously the same migration, reverse phase HPLC was
performed. Peptides from both in vivo and in vitro samples were eluted from SDS-polyacrylamide gels, digested with V8
protease, and analyzed by HPLC. The radioactive material eluted at the
same time (50-53 min) for both in vivo and in vitro labeled samples (Fig. 1E).
Figure 2: Phosphopeptide sequencing of tryptic peptides. Peptides eluted from the TLC plates of Fig. 1D were subjected to phosphopeptide sequencing. Radioactivity released in each cycle was quantified as described under ``Materials and Methods.'' A, in vivo sample; B, in vitro sample.
Next,
bacterially expressed eIF4E was extensively phosphorylated in vitro by PKC and digested with trypsin. One major phosphopeptide (1) and a minor phosphorylated form (2) were detected
by TLC tryptic phosphopeptide analysis (Fig. 3A). In
contrast to the samples analyzed in Fig. 1, this tryptic digest
contained a significant amount of phosphorylated threonine, as
determined by two-dimensional phosphoamino acid analysis (Fig. 3B). In earlier reports, it was noted that PKC
phosphorylation of eIF4E yielded two phosphorylated peptides and that
radioactive phosphate was incorporated into threonine in addition to
serine(40) . In our hands, this occurred when the reaction was
carried out for an extended period of time or with excess kinase
(conditions used for Fig. 3A). The tryptic digest was
also analyzed by liquid chromatography coupled with mass spectrometry
(LC-MS). The LC-MS chromatogram is shown in Fig. 3C.
Two peaks (a and b) accounted for all the
radioactivity in the sample that was injected onto the HPLC column. The
mass spectra of peaks a and b are shown in Fig. 3, D and E, respectively. The mass
spectra of peak a shows an intense peak at an m/z ratio of 659 (Fig. 3D), which corresponds to the m/z ratio of the molecular ion of the tryptic peptide SGSTTK
of eIF4E, bearing one phosphate
group. The spectrum also contains smaller peaks at m/z values
corresponding to molecular ions resulting from partial fragmentation of
the parent phosphopeptide
SGSTTK
. From this
fragmentation pattern we have narrowed down the site of phosphorylation
to either Ser
, Thr
, or Thr
,
since the first serine and the glycine can be cleaved from the parent
phosphopeptide without the loss of the phosphate group. The mass
spectra of the component in peak b in Fig. 3C corresponds to that of the same tryptic peptide
SGSTTK
, but bearing two phosphate groups.
The radioactivity in this fraction was, however, much lower than that
of the material in peak a (data not shown), suggesting that
phosphorylation occurs predominantly at only one amino acid residue. By
combining the data obtained from the solid phase phosphopeptide
sequencing with that from the mass spectra analysis, we conclude that
Ser
is the major PKC phosphorylation site on eIF4E, while
a minor site of phosphorylation may occur on Thr
.
Figure 3: Identification of the PKC phosphorylated peptide on eIF4E by LC-MS. eIF4E was phosphorylated in vitro by PKC for 30 min and resolved by SDS-PAGE. eIF4E was eluted from the gel and digested with trypsin. A, a tryptic phosphopeptide map was performed as in Fig. 1. B, two-dimensional phosphoamino acid analysis was performed as described under ``Materials and Methods.'' C, tryptic digests, prepared as for A, were fractionated on an HPLC column. Peaks containing radioactivity are indicated by arrows a and b. D and E, phosphorylated peptides from peaks a and b, respectively, were analyzed directly by mass spectrometry as described under ``Materials and Methods.'' The position of two peaks resulting from partial fragmentation of the parent phosphopeptide SGSTTK is indicated by letters. S indicates the peak resulting from the cleavage of the first serine of the peptide, and G indicates that from the cleavage of the glycine.
The
assignment of Ser as the phosphorylation site in eIF4E
was further substantiated with mutants of Ser
in in
vivo experiments. HA-epitope tagged eIF4E wild type and S209A were
transiently transfected into 293 cells followed by labeling with
[
P]orthophosphate. Immunoprecipitation with
anti-HA antibody showed a 30% decrease in the
P
incorporation in the mutant relative to the wild type protein (Fig. 4A). The phosphorylated peptide migrated at a
similar position for both the wild type and the mutant S209A in
two-dimensional TLC (data not shown). However, whereas serine was the
predominant form in the wild type eIF4E (Fig. 4B), in
the mutant S209A only threonine was detected by phosphoamino acid
analysis (Fig. 4C). When Ser
is mutated
to alanine, a threonine residing on the same peptide as
Ser
, which is most probably Thr
(because
radioactivity was detected in cycle 4, but not in 5, by phosphopeptide
sequencing; Fig. 2A), became phosphorylated.
Figure 4:
Mutant S209A is phosphorylated on
threonine in vivo. HA-tagged eIF4E wild type and S209A mutant
were expressed in 293 cells. Following labeling of the cells with
[P]orthophosphate, HA-tagged eIF4E was
immunoprecipitated using an anti-HA antibody as described under
``Materials and Methods.'' A, immunoprecipitated
proteins were separated on an SDS-polyacrylamide gel and
autoradiographied. B and C, two-dimensional
phosphoamino acid analysis was performed on proteins eluted from the
polyacrylamide gel, for the wild type and the S209A mutant,
respectively.
Similar studies were done in vitro. Wild type eIF4E or S53A and S209A mutants were expressed in bacteria and purified on a cap column(33) . The S53A mutant protein was phosphorylated by PKC to the same extent as the wild type (Fig. 5). In contrast, eIF4E S209A mutant was phosphorylated to a much lesser extent than wild type eIF4E (Fig. 5), further indicating that serine 209 is the main site phosphorylated by PKC.
Figure 5: In vitro phosphorylation of eIF4E wild type, S53A, and S209A by PKC. Recombinant eIF4E was expressed in Escherichia coli and purified by cap affinity chromatography as described under ``Materials and Methods.'' Proteins were phosphorylated in vitro by PKC and separated on an SDS-polyacrylamide gel followed by autoradiography as described under ``Materials and Methods.''
Figure 6: 4E-BPs prevents in vitro phosphorylation of eIF4E. A, 4E-BP1 (0.5 µg) or 4E-BP2 (0.5 µg) were mixed with eIF4E (1 µg) and incubated in the kinase buffer (without lipids) on ice for 30 min prior to the kinase reaction. Control reactions consisting of either rat 4E-BP1 (PHAS-I), 4E-BP2, myelin basic protein (MBP), 4E-BP1 and MBP, or 4E-BP2 and MBP were processed in parallel. The in vitro kinase assay was performed for 15 min as described under ``Materials and Methods.'' Phosphorylated proteins were separated by SDS-PAGE and autoradiographied.
eIF4E modulates cell growth in response to a wide array of stimuli. eIF4E is regulated at three different levels: (a) it is the most limiting factor in amount in cells as compared to other initiation factors and ribosomes. This limitation is thought to play an important role in regulation of translation rates and growth control (for a review, see (3) ). Consistent with this idea, eIF4E overexpression transforms rodent cells, and microinjection of eIF4E into quiescent cells activates DNA synthesis(4, 5) ; (b) the translational repressors, 4E-BP1 and 4E-BP2, inhibit the association of eIF4E with eIF4G and thus the formation of the eIF4F complex(9, 11, 12) ; and (c) phosphorylation modulates eIF4E activity, as the phosphorylated form of eIF4E binds more tightly to the cap structure and has an enhanced affinity for eIF4G(19, 20, 41) .
Several
reports demonstrated the phosphorylation of eIF4E by PKC in
vitro(26, 27, 28) . The peptide
phosphorylated in vitro co-chromatographed with the in
vivo phosphorylated peptide(26) . However, the PKC
phosphorylation site has not been identified directly. Smith et al.(31) have shown that a bacterially expressed S53A mutant
could not be phosphorylated in vitro by PKC. However, here we
found no differences in PKC phosphorylation between S53A and wild type
eIF4E. It is possible that there is a variability in the renaturation
of S53A mutant and, as a result, a change of the mutant protein to
serve as substrate for PKC. We determined directly by LC-MS analysis
that the main phosphorylation site of PKC in vitro is
Ser. This agrees well with the recent findings that
Ser
is the major site of phosphorylation of eIF4E in
vivo(23) . Also, in agreement with this assignment is our
finding that bacterially expressed mutant S209A was not phosphorylated
by PKC to a significant extent. In addition, Ser
conforms
very well with the PKC consensus phosphorylation site(42) .
When the kinase reaction was performed for an extended period of time,
an additional amino acid residing on the same peptide, most probably
Thr
, became phosphorylated. When expressed in mammalian
cells, the mutant S209A was not phosphorylated on serine, but rather on
threonine, probably Thr
. Taken together, these data
provide direct and unambiguous evidence that the phosphorylation site
of PKC on eIF4E is Ser
.
There are many lines of
evidence that support a role for PKC in phosphorylation of eIF4E in
cells. Treatment of cells with phorbol esters that activate PKC induces
eIF4E phosphorylation(30, 32, 43) . In many
cell types, down-regulation of PKC by phorbol esters prevents
stimulation of eIF4E phosphorylation by phorbol 12-myristate 13-acetate
or insulin (reviewed in (3) ). Furthermore, PKC coinjection
with eIF4E into NIH 3T3 cells has a synergistic effect on eIF4E
mitogenic activity(31) . However, in PC12 cells,
down-regulation of PKC with phorbol esters has no effect on the nerve
growth factor-induced phosphorylation of eIF4E(43) . There are
two possible explanations for this finding: (a) there exists
an alternative pathway leading to phosphorylation of eIF4E and (b) phosphorylation of eIF4E could be mediated at least partly
through a phorbol ester-insensitive isoform of PKC, such as
PKC(44) .
The results in this report indicate that
phosphorylation of eIF4E can be regulated by 4E-BPs in vitro.
We demonstrated here that the phosphorylation of eIF4E by PKC is
prevented when 4E-BP1 is bound to eIF4E. Thus, phosphorylation of
4E-BP1 or of 4E-BP2 might be an obligatory step for eIF4E
phosphorylation. Consistent with this finding, the phosphorylation of
eIF4E and that of 4E-BP1 follow similar kinetics in response to serum
stimulation. ()Phosphorylation of eIF4E is thus likely to
depend on the activation of two kinases: one, unknown as yet, that
phosphorylates 4E-BPs and releases eIF4E, and the second, PKC, which
phosphorylates eIF4E.
Fig. 7shows a model for the
phosphorylation of eIF4E. Insulin treatment activates one or more
pathway(s) that leads to the phosphorylation of 4E-BP1. Earlier results
suggested that the phosphorylation of 4E-BP1 is carried out by the MAP
kinases ERK1 and ERK2(45) . Recent evidence (reviewed in 3)
indicate that this pathway is incorrect and that the major 4E-BP1
phosphorylation pathway is the PI3K/p70 rapamycin-sensitive pathway, rather than the MAP kinase pathway.
However, it is not known what rapamycin-sensitive kinase phosphorylates
the 4E-BPs in vivo, since 4E-BP1 is not a substrate for the
rapamycin-sensitive kinase p70
(45) . (
)Also, phosphorylation of 4E-BP1 by ERKI and ERKII is
prevented in vitro when eIF4E is bound to 4E-BP1(15) .
This result is also compatible with the model presented in Fig. 7, since the kinase that phosphorylates 4E-BP1 in the
rapamycin-sensitive pathway might still be able to phosphorylate 4E-BP1
when bound to eIF4E. The model in Fig. 7posits that, following
phosphorylation of 4E-BP1 and disruption of the complex between eIF4E
and 4E-BP1 (step 1), eIF4E associates with eIF4A and eIF4G to
form the eIF4F complex (step 2). Two subunits of eIF4F, eIF4E
and eIF4G, are phosphorylated by PKC (step 3). Phosphorylation
of eIF4E by PKC might occur on the eIF4F complex, since eIF4E as a part
of eIF4F was reported to be a better substrate for protein kinase C
than free eIF4E(28) . Phosphorylation of eIF4E (or of eIF4E and
eIF4G) by PKC enhances binding to the cap structure of the mRNA and
stabilizes the interaction between eIF4G and
eIF4E(19, 20, 39) , leading to an increase in
the translation (step 4).
Figure 7: Proposed model for the events leading to eIF4E phosphorylation. In resting cells, a significant portion of eIF4E is bound to 4E-BP1. Insulin first causes the dissociation of the 4E-BP1/eIF4E complex by phosphorylating 4E-BP1. Consequently eIF4E becomes free to associate with eIF4G and eIF4A and can be phosphorylated by PKC.
Future studies will be necessary
to define the biological significance of Ser phosphorylation. Joshi et al.(23) reported that
a mutant S209A retained the ability to associate with 48 S
preinitiation complex, in contrast to a S53A mutant of eIF4E. It would
be important to test the activity of the double mutant S209A/T210A,
since, as shown here, Thr
is phosphorylated in the S209A
mutant of eIF4E in vivo. It would also be critical to examine
the transforming activities of the various eIF4E mutants, including
S209E and S209D, in order to ascertain the importance of eIF4E
phosphorylation in control of cell growth and transformation.