The EMBO Journal Vol.19 No.3 pp.434–444, 2000
phosphorylation sites in the eukaryotic translation
initiation factor 4GI
Brian Raught, Anne-Claude Gingras,
Steven P.Gygi1, Hiroaki Imataka,
Shigenobu Morino, Alessandra Gradi,
Ruedi Aebersold1and Nahum Sonenberg2
Department of Biochemistry and McGill Cancer Centre,
McGill University, 3655 Drummond, Montre ´al, Que ´bec H3G 1Y6,
Canada and1Department of Molecular Biotechnology, University of
Washington, Seattle, WA 98195-7730, USA
B.Raught and A.-C.Gingras contributed equally to this work
The eukaryotic translation initiation factor 4G (eIF4G)
proteins play a critical role in the recruitment of the
translational machinery to mRNA. The eIF4Gs are
phosphoproteins. However, the location of the phospho-
rylation sites, how phosphorylation of these proteins is
pathways regulating eIF4G phosphorylation have not
been established. In this report, two-dimensional phos-
phopeptide mapping demonstrates that the phospho-
rylation state of specific eIF4GI residues is altered by
serum and mitogens. Phosphopeptides resolved by this
method were mapped to the C-terminal one-third of the
protein. Mass spectrometry and mutational analyses
identified the serum-stimulated phosphorylation sites
in this region as serines 1108, 1148 and 1192.
Phosphoinositide-3-kinase (PI3K) inhibitors and rapa-
mycin, an inhibitor of the kinase FRAP/mTOR
target of rapamycin), prevent the serum-induced
rylation state of N-terminally truncated eIF4GI pro-
teins acquires resistance to kinase inhibitor treatment.
These data suggest that the kinases phosphorylating
of PI3K and FRAP/mTOR, but that the accessibility
of the C-terminus to kinases is modulated by this
Keywords: eukaryotic translation initiation factor 4G/
of the translational machinery to the 5? end of mRNA.
This bridging process is accomplished via multiple pro-
tein–protein and protein–RNA interactions, coordinated
primarily by two large, modular scaffolding factors: the
eukaryotic translation initiation factor 4G (eIF4G) proteins
(for reviews, see Hentze, 1997; Morley et al., 1997;
© European Molecular Biology Organization
Gingras et al., 1999b). eIF4GI was originally identified
as a constituent of eIF4F, a tripartite complex also con-
taining an mRNA 5? cap binding protein, eIF4E, and an
RNA helicase, eIF4A (reviewed in Gingras et al., 1999b).
Viral proteases cleave the mammalian eIF4GI protein
into three fragments (N-terminal, middle and C-terminal;
Lamphear et al., 1995); biochemical and functional ana-
lyses of these fragments have clarified the role that each
plays in the formation of a functional translation initiation
complex. The N-terminal fragment interacts directly with
eIF4E (Lamphear et al., 1995; Mader et al., 1995) and
the poly(A) binding protein (PABP; Imataka et al., 1998).
The middle fragment of eIF4GI possesses binding sites
for eIF3, a multisubunit factor associated with the 40S
ribosomal subunit, and eIF4A (Lamphear et al., 1995;
Imataka and Sonenberg, 1997). The middle fragment also
possesses RNA binding activity (Pestova et al., 1996).
The C-terminal fragment of eIF4GI contains a second,
independent binding site for eIF4A (Lamphear et al.,
1995; Imataka and Sonenberg, 1997), and interacts with
Mnk1 (MAP kinase signal-integrating kinase 1; Fukunaga
and Hunter, 1997; or MAP kinase-interacting kinase 1;
Waskiewicz et al., 1997), a MAP kinase-activated serine/
threonine kinase that phosphorylates eIF4E in vitro
(Waskiewicz et al., 1997) and in vivo (Pyronnet et al.,
1999; Waskiewicz et al., 1999). A second eIF4G protein,
eIF4GII, was recently characterized (Gradi et al., 1998a),
which shares 46% identity with eIF4GI at the amino acid
level. eIF4GI and eIF4GII are functional homologs, in
that all of the features described above for eIF4GI are
conserved in eIF4GII (Gradi et al., 1998a; Imataka et al.,
1998; Pyronnet et al., 1999).
protein–RNA interactions, the eIF4G proteins perform
(i) recruitment of the 40S ribosomal subunit to the 5? end
of mRNA via interactions with eIF4E and eIF3; (ii) relief
of inhibitory secondary structure in the mRNA 5? UTR,
by delivering the eIF4A helicase to this region (Rozen
et al., 1990); (iii) circularization of mRNA through inter-
actions with both eIF4E and PABP (Wells et al., 1998);
and (iv) delivery of the kinase Mnk1 to its substrate,
eIF4E (Pyronnet et al., 1999; Waskiewicz et al., 1999).
How all of these functions are coordinated and regulated
by the eIF4G proteins is not understood.
The human eIF4G family also includes two other
proteins: p97 and PAIP-1. p97 is homologous only to the
C-terminal two-thirds of the eIF4Gs, and does not possess
a region corresponding to the N-terminal one-third of the
eIF4Gs. Like the eIF4Gs, it contains binding sites for
eIF3, eIF4A and Mnk1. However, p97 does not interact
with eIF4E or PABP (Imataka et al., 1997; Levy-Strumpf
et al., 1997; Yamanaka et al., 1997), and inhibits both
cap-dependent and cap-independent translation in vivo,
eIF4GI phosphorylation sites
presumably by forming non-functional initiation com-
plexes (Imataka et al., 1997). A more distant eIF4G
homolog, PAIP-1, possesses binding sites for PABP and
eIF4A, and appears to act as a translational enhancer
in vivo (Craig et al., 1998).
eIF4G homologs have been identified in many species
(e.g. Browning et al., 1987; Goyer et al., 1993; Morley
et al., 1997; Hernandez et al., 1998). While all of the
eIF4G-like proteins identified so far possess a region
homologous to the middle fragment of the mammalian
eIF4G proteins, the N- and C-terminal regions of these
proteins diverge significantly. For example, an extended
eIF4G C-terminus is a feature present only in certain
organisms: the mammalian (Yan et al., 1992; Lamphear
et al., 1993), Drosophila (Hernandez et al., 1998) and
putative zebrafish eIF4G homologs possess an elongated
C-terminal region. However, a wheat eIF4G homolog
(Browning et al., 1987) possesses a much smaller
C-terminal region, and the Saccharomyces cerevisiae
(Goyer et al., 1993) and Schizosaccharomyces pombe
(Morley et al., 1997) eIF4G homologs do not possess
such a region at all.
These observations suggest that the middle region of
eIF4G is the ‘core’ unit required for translation. Consistent
for cap-independent translation from the encephalo-
myocarditis virus (EMCV) internal ribosome entry site
(IRES; Pestova et al., 1996). The middle one-third of the
protein plus the eIF4E binding site is the minimal fragment
required for cap-dependent translation of the β-globin
mRNA (Morino et al., 2000), and the middle region alone,
fused to iron regulatory protein-1, can direct translation of
an RNA containing an iron response element (DeGregorio
et al., 1999). Thus, the termini of the mammalian eIF4GI
protein are largely dispensable for translation. However,
recent evidence suggests that both the N- and C-termini
play important roles in enhancing and/or modulating
translation in mammalian cells. For example, the eIF4G–
PABP interaction, mediated by the eIF4G N-terminus
(Imataka et al., 1998), is posited to inhibit the synthesis
of truncated proteins from mRNAs that are not full-length
(Sachs et al., 1997). The interaction between eIF4A and
the eIF4GI C-terminus also appears to be important, as
mutation of the C-terminal eIF4A binding site decreases
the activity of eIF4GI in a reticulocyte lysate by several-
fold (Morino et al., 2000). How the activity of the eIF4G
termini may be regulated is not understood.
The eIF4Gs are phosphoproteins (Duncan et al., 1987;
Morley and Traugh, 1989, 1990; Donaldson et al., 1991;
this study). However, the intracellular signaling pathways
mediating eIF4G phosphorylation, and the location of the
phosphorylation sites within the proteins, have remained
ated residues in eIF4GI to the C-terminal one-third of the
protein. Using mass spectrometry and mutational analyses,
the serum-stimulated phosphorylation sites within this
region were identified. An intracellular signaling pathway
(Brown and Schreiber, 1996) consists of phospho-
and the FKBP12–rapamycin-associated protein/mamma-
lian target of rapamycin (FRAP/mTOR; also known as
RAFT1 or RAPT1; reviewed in Lawrence and Abraham,
1997; Thomas and Hall, 1997; Abraham, 1998; Gingras
et al., 1999b; Raught and Gingras, 1999). This pathway(s)
regulates the activity of several translation factors. We
demonstrate here that PI3K and FRAP/mTOR signaling
modulates the serum-stimulated phosphorylation of the
N-terminus appears to modulate phosphorylation of the
lacking the N-terminal region are resistant to kinase
inhibitor treatment. Unexpectedly, although the related
eIF4GII and p97 are also phosphoproteins, the correspond-
ing C-terminal fragments in these proteins are not phos-
phorylated, suggesting that this region has undergone
Phosphopeptide mapping of eIF4GI
To study the regulation of eIF4GI phosphorylation, two-
dimensional phosphopeptide mapping of
immunoprecipitated eIF4GI was conducted, essentially as
described (Gingras et al., 1998). Briefly, human embryonic
kidney 293 cells were metabolically labeled with
[32P]orthophosphate. A subset of these cells was then
stimulated with serum. The cells were lysed, and eIF4GI
was immunoprecipitated using a polyclonal antiserum
directed against the N-terminus of the protein (Craig
SDS–8% PAGE, and electrotransferred to a nitrocellulose
membrane. The membrane was autoradiographed, and the
region harboring the protein of interest excised. Membrane
pieces were subjected to tryptic digestion, and liberated
phosphopeptides visualized by two-dimensional (thin-
layer electrophoresis/thin-layer chromatography; TLE/
A typical autoradiogram of32P-labeled, immunoprecipi-
tated, gel-purified eIF4GI is shown in Figure 1A. Cerenkov
32P-labeled eIF4GI revealed no significant difference in
the quantity of
from serum-starved (–S) versus serum-stimulated (?S)
293 cells (n ? 10). However, two-dimensional tryptic
phosphopeptide mapping revealed that the relative inten-
sity of several phosphorylated peptides was significantly
altered upon serum stimulation, indicating a change in the
phosphorylation status of one or more amino acid residues.
A highly reproducible pattern of phosphorylated peptides
was observed for eIF4GI isolated from serum-starved
cells (Figure 1B). Treatment of starved cells with serum
(Figure 1C), insulin (data not shown) or phorbol ester
(data not shown) resulted in an increase in the phosphoryl-
ation state of several tryptic peptides (serum-stimulated
phosphopeptides 1–4), along with a concomitant decrease
in the phosphorylation status of several others (serum-
repressed phosphopeptides 7–9). No significant change in
the intensity of the remaining major phosphopeptides
(Figure 1C, phosphopeptides 5 and 6) was observed.
Phosphopeptide mapping of eIF4GI from logarithmically
growing cells yielded the same phosphopeptide pattern
observed for serum-stimulated cells (data not shown).
Immunoprecipitation with antiserum directed against the
C-terminus of human eIF4GI yielded identical maps,
32P incorporated into eIF4GI isolated
B.Raught et al.
Fig. 1. Phosphorylation of specific sites in eIF4GI is modulated by serum.32P-labeled eIF4GI immunoprecipitated from 293 cells starved of serum for
36 h (–Serum or –S), or starved of serum for 36 h then stimulated with serum for 30 min (?Serum or ?S), was subjected to (A) SDS–8% PAGE, then
(B and C) to two-dimensional tryptic peptide mapping. The directions of chromatography (vertical) and electrophoresis (horizontal) as well as the loading
origin (arrow) are indicated. Major phosphopeptides are numbered.
confirming that all of the observed phosphopeptides are
derived from eIF4GI.
While the response of the phosphopeptides to serum
stimulation was very consistent (i.e. peptides 1–4 increas-
ing and peptides 7–9 decreasing in intensity), the degree
of stimulation or repression varied from experiment to
experiment. This variability appears to be due to a number
of different reasons, including the confluence level and
passage number of the cells, as well as the length of the
starvation and stimulation periods. In general (as assessed
by phosphoimaging and densitometry measurements),
phosphopeptides 2 and 4 displayed the greatest sensitivity
to serum stimulation (increasing in intensity ~3- to
10-fold), while peptides 3 (increasing ~2- to 5-fold) and 1
(increasing 1- to 3-fold; in some experiments no change
was observed for peptide 1) were less responsive (data
not shown). The intensity of peptide 7, in the serum-
repressed group, also displayed acute sensitivity (decreas-
ing by ~2- to 10-fold) to the presence of serum, whereas
peptides 8 and 9 (both decreasing by ~2- to 5-fold)
displayed a lower level of sensitivity. It is also noteworthy
that while the eIF4GI phosphopeptide mapping pattern is
quite reproducible, some variability is observed in the
minor, unnumbered peptides as well as in peptides 4 and
6. This is most likely to be due to variations in the
efficiency of tryptic digestion. Also, a streaked signal is
observed directly above the loading origin in maps of
endogenous eIF4GI (Figure 1B and C), suggesting that
other peptides that are not clearly resolved by this method
may be present. Nevertheless, the streaked signal and
unnumbered phosphopeptides account for ?10% of the
total signal, as determined by phosphoimaging (data not
Mapping of eIF4GI phosphorylation sites
To delineate the location of the eIF4GI phosphorylation
protein fragments were expressed in 293T cells. (Phospho-
peptide maps of eIF4GI from 293T cells are identical to
those from serum-stimulated 293 cells. However, since
the transfection efficiency achieved in 293T cells is much
greater than that for 293 cells, they were used in all
transfection protocols.) Logarithmically growing 293T
cells were transiently transfected (in the presence of 10%
serum), and labeled with [32P]orthophosphate. HA-tagged
proteins were then immunoprecipitated with an anti-HA
antibody, gel-purified and mapped. The constructs utilized
in this study are shown in Figure 2F. HA-tagged eIF4GI
protein fragments encompassing amino acids (aa) 614–
1560 (Figure 2B) and 1045–1560 (Figure 2C) yielded
phosphopeptide maps that were almost identical to the
endogenous protein (Figure 2A). However, further
N-terminal truncation to aa 1205 resulted in the loss of
all phosphopeptides, except peptide 5 (Figure 2D). Map-
ping of a protein encompassing aa 1045–1372 yielded all
phosphopeptides, except for peptides 5 and 6 (Figure 2E),
and mapping of a fusion protein encompassing aa 1372–
1560 yielded only peptide 5 (data not shown). Thus, all
of the phosphopeptides mapped by this procedure were
localized to the C-terminal one-third of the protein.
The inability to localize peptide 6 is likely to result
from differences in tryptic digestion efficiency between
truncation of N-terminal sequences also resulted in
a significant increase in the relative intensity of the
serum-stimulated phosphopeptides (1–4) compared with
the serum-repressed peptides (7–9; compare Figure 2A
The regions of eIF4GII and p97 corresponding to
the eIF4GI phospho-region are not phosphorylated
Alignment of the region of eIF4GI containing most of the
phosphorylation sites (aa 1045–1205; hereafter referred to
as the phospho-region) with the corresponding segments
of two other members of the human eIF4G family, eIF4GII
eIF4GI phosphorylation sites
Fig. 2. Localization of eIF4GI phosphorylation sites. (A) Tryptic map of endogenous eIF4GI. (B–E) HA-tagged eIF4GI fusion protein fragments
were expressed in 293T cells. Cells were metabolically labeled with32P, and HA-tagged proteins immunoprecipitated, gel purified, and subjected to
two-dimensional tryptic mapping. (F) Protein fragments tested in this study. The various protein binding regions of eIF4GI are indicated.
especially compared with the surrounding regions. In fact,
this region of human eIF4GI is much more closely related
to the corresponding region of eIF4G proteins in other
species than to the human eIF4GII and p97 proteins,
sharing 93% identity with the rabbit eIF4GI protein
(DDBJ/EMBL/GenBank accession No. L22090) and 47%
identity with a putative zebrafish eIF4GI protein (DDBJ/
EMBL/GenBank accession No. AI629389; data not
shown). Thus, the newly defined phospho-region appears
to have undergone divergent evolution within the human
eIF4G family members.
Immunoprecipitation of eIF4GII and p97 from meta-
bolically labeled 293 cells revealed that they are both
phosphoproteins (data not shown). To determine whether
eIF4GII and p97 are phosphorylated in the same region
as eIF4GI, glutathione S-transferase (GST) fusion proteins
encompassing fragments corresponding to the eIF4GI
phospho-region (aa 1035–1206 for eIF4GI, aa 1057–1225
for eIF4GII, aa 395–547 for p97) were expressed in
293T cells, which were then metabolically labeled with
[32P]orthophosphate. The transfected 293T cells were
lysed, and GST fusion proteins isolated via incubation with
glutathione-coupled Sepharose beads. Western blotting,
using anti-GST antiserum (Morino et al., 2000), confirmed
that all three GST fusion proteins were expressed
(Figure 3B, upper panel), albeit at different levels. The
same blot was then autoradiographed directly (Figure 3B,
lower panel). Interestingly, only the GST–eIF4GI fusion
protein incorporated significant amounts of32P. (A weak
background signal at approximately the same location as
the GST–eIF4GI and GST–eIF4GII proteins is detected
in all lanes; however, the intensity of this signal did not
correlate with the amount of GST fusion protein expressed,
and the signal did not co-migrate with GST–p97.) To
ensure that all of the cells were efficiently metabolically
labeled, endogenous eIF4GI was immunoprecipitated from
the same lysates and found to incorporate similar amounts
both eIF4GII and p97 are phosphoproteins, the regions
corresponding to the eIF4GI C-terminal phospho-region
do not appear to be phosphorylated significantly in these
members of the eIF4G family.
32P in all samples (data not shown). Thus, while
Identification of the serum-inducible
phosphorylation sites in eIF4GI
To identify the C-terminal eIF4GI residues phosphorylated
in response to serum treatment, mass spectrometric and
mutational analyses were performed. Briefly, phospho-
peptide mapping was conducted as above, except that
eIF4GI immunoprecipitated from 2 ? 108serum-stimu-
lated 293 cells (1/20 of which were metabolically labeled
with [32P]orthophosphate) was loaded onto two TLC
plates. The cellulose harboring peptides 1–4 was scraped
from the plastic backing of the plates, and the phospho-
peptides eluted and identified by capillary liquid chromato-
graphy–electrospray ionization tandem mass spectrometry
(LC–MS/MS). The tandem mass spectrum for phospho-
peptide 1 corresponds to EAALPPVSPLK, with the
phosphorylation site being Ser1192 (Figure 4A). The
tandem mass spectrum for phosphopeptide 3 corresponds
to SFSKEVEER, with the phosphorylation site being
Ser1148 (Figure 4B). The tandem mass spectrum for
phosphopeptide 4 corresponds to SSLSRER, with the
phosphorylation site being Ser1108 (data not shown).
B.Raught et al.
Fig. 3. The phospho-region of eIF4GI is poorly conserved within the human eIF4G family. (A) An alignment through the newly defined eIF4GI
phospho-region of the members of the human eIF4G family. Locations of defined protein binding sites in eIF4G proteins are indicated, as well as
percentage identity to the corresponding region in eIF4GI. Below the diagram is an alignment of the amino acid sequences of eIF4GI, eIF4GII and
p97 in this region. Identical residues are boxed in black, similar residues are boxed in gray. (B) Fragments encompassing the phosphorylated region
of eIF4GI (aa 1035–1206) and the corresponding regions of eIF4GII (aa 1057–1225) and p97 (aa 395–547) were expressed in 293T cells (5, 10 and
20 µg of DNA transfected) as GST fusion proteins. Cells were metabolically labeled with32P. Proteins isolated with glutathione-coupled Sepharose
beads were washed, gel-purifed, and transferred to nitrocellulose. Upper panel, Western blot using anti-GST antiserum. Lower panel, direct
autoradiogram of the same nitrocellulose membrane.
Mass measurements for phosphopeptide 2 predicted the
peptide SSLSR (data not shown), indicating that peptide 2
is an alternative cleavage product of peptide 4. The identity
of the phosphorylated residue could not be determined for
To confirm the mass spectrometric results, site-directed
mutagenesis of serines 1108, 1148 and 1192, along with
several other phosphorylation site candidates, was per-
formed. To simplify the cloning and mapping procedures
these mutants were constructed in the background of the
GST fusion protein containing only the eIF4GI phospho-
region (aa 1035–1206). A typical two-dimensional phos-
phopeptide map of the GST–eIF4GI phospho-region
protein is shown in Figure 5A. As observed for the
HA-1045–1372 fragment (Figure 2E), phosphorylation of
the GST fusion protein is almost exclusively limited to
peptides 1–4, while serum-repressed peptides 7 and 8 are
almost undetectable. A phosphopeptide that migrates in
the same region as peptide 9 remains visible on most
maps. A novel phosphopeptide (denoted by an asterisk,
Figure 5A–D) that does not co-migrate with any endogen-
ous eIF4GI peptides is observed in maps of these proteins,
and is most likely to be derived from the GST moiety.
Mutation of Ser1108 to alanine resulted in the loss of
both peptides 2 and 4 (Figure 5B). Thus, tryptic cleavage
after arginine 1109 appears to be relatively inefficient
(most likely to be due to the presence of a phosphate
group near the cleavage site), resulting in the formation
of two tryptic peptides (SSLSR and SSLSRER) containing
the same phosphoserine. Elimination of phosphopeptides
2 and 4 was specific for the Ser1108Ala mutant, as maps
of Ser1105Ala and Ser1106Ala mutants (other serines
present in the same tryptic peptide) retained peptides 1–4
(data not shown). Mutation of Ser1148 to alanine specific-
ally abolished phosphopeptide 3 (Figure 5C), and mutation
of Ser1192 to alanine eliminated phosphopeptide 1
(Figure 5D). Mutation to alanine of serines 1085, 1095,
1125, 1155, 1160 and 1170, or of threonines 1093, 1096
and 1140 had no effect on the phosphopeptide pattern
(see Figure 5E for a summary of the mutational analysis).
Thus, using a combination of mass spectrometric and
mutational analyses, the serum-stimulated phosphorylation
sites were identified as Ser1108, Ser1148 and Ser1192.
PI3K and FRAP/mTOR signaling modulates eIF4GI
Several translation regulatory factors have been identified
as targets of the PI3K–FRAP/mTOR signaling pathway(s)
(Thomas and Hall, 1997; Abraham, 1998; Gingras et al.,
1999b). It was thus pertinent to test whether signaling to
eIF4GI is also mediated by this pathway. Specific kinase
inhibitors may be used to determine whether phosphoryl-
ation of a given protein is modulated by these kinases.
Wortmannin inhibits PI3K by covalently binding to its
catalytic subunit (reviewed in Ui et al., 1995), and
LY294002 is a structurally unrelated, reversible PI3K
inhibitor (Vlahos et al., 1994). Rapamycin is a fungal
macrolide that, in a complex with the immunophilin
FKBP12, binds to FRAP/mTOR to inhibit its kinase
activity (Abraham, 1998).
Serum-starved 293 cells metabolically labeled with
[32P]orthophosphate (Figure 6A) were pre-treated (for
20 min) with kinase inhibitors, then serum was added to
the culture media to a final concentration of 10%. After
eIF4GI phosphorylation sites
Fig. 4. Identification of serum-stimulated phosphorylation sites by mass spectrometry. (A) Tandem mass spectrum resulting from the analysis of
excised phosphopeptide 1 from the map in Figure 1C. This spectrum contained sequence information for a single phosphopeptide. Two separate ion
series were recorded simultaneously; b- and y-ion series represent sequencing inward from the N- and C-termini, respectively. The computer program
Sequest (Eng et al., 1994) was utilized to match this tandem mass spectrum to the sequence shown, with the serine residue being phosphorylated.
(B) Tandem mass spectrum resulting from the analysis of excised phosphopeptide 3. The position of the phosphorylated serine residue is indicated.
30 min in the presence of serum (Figure 6B), or serum
plus inhibitor (Figure 6C and D), cells were lysed and
eIF4GI was subjected to phosphopeptide mapping.
LY294002 (5 µM; Figure 6C), wortmannin (100 nM; data
not shown) and rapamycin (25 nM; Figure 6D) abrogated
the serum-induced hyperphosphorylation of peptides 1–4,
and prevented the hypophosphorylation of peptides 7–9.
(In this experiment, peptide 3 responded only weakly to
rapamycin addition.) Addition of solvent [dimethylsulf-
oxide (DMSO) for wortmannin and LY294002, ethanol
for rapamycin] alone had no effect on the phosphopeptide
pattern (data not shown). Thus, serum-induced modulation
of eIF4GI phosphorylation appears to be mediated by
PI3K and FRAP/mTOR signaling.
The N-terminus confers kinase inhibitor sensitivity
to the phospho-region
Two models may be proposed to explain the effects of
kinase inhibitors on the phosphorylation state of eIF4GI:
(i) the kinases phosphorylating this region are rapamycin-
and LY294002-sensitive; or (ii) the kinases themselves
are not sensitive to these inhibitors, but a change in the
conformation of eIF4GI (due to alterations in intramolecu-
lar interactions, or in the interaction of eIF4GI with
B.Raught et al.
Fig. 5. Identification of serum-inducible phosphorylation sites by mutational analysis. Mutations of serine residues identified by mass spectrometry as
phosphorylation sites were introduced into the eIF4GI phospho-region (aa 1035–1206)–GST fusion proteins. Proteins were expressed in 293T cells,
32P-labeled, isolated and mapped. Tryptic maps of the GST–eIF4GI (aa 1035–1206) wild-type protein (A), and point mutants Ser1108Ala (B),
Ser1148Ala (C) and Ser1192Ala (D). A novel phosphopeptide that does not co-migrate with any endogenous eIF4GI peptide is indicated with an
asterisk. (E) Complete mutational analysis summary. An alignment of the eIF4GI phospho-region with the corresponding regions of eIF4GII and
p97. Identical residues are boxed in black, similar residues are boxed in gray. eIF4GI residues mutated to alanine are indicated by an ‘x’ or arrow.
Locations of the serum-stimulated, phosphorylated residues, as well as the phosphopeptides identified in this analysis, are also indicated.
Fig. 6. Serum-induced phosphorylation of eIF4GI is sensitive to inhibitors of PI3K and FRAP/mTOR. Two-dimensional tryptic peptide mapping of
endogenous eIF4GI isolated from 293 cells (A) starved of serum, (B) then treated with serum for 30 min, (C) pre-treated with LY294002 for 20 min,
then treated with serum for 30 min, or (D) pre-treated with rapamycin for 20 min, then treated with serum for 30 min. The map in (A) is the same
as that shown in Figure 1B.
other binding partners) modulates the accessibility of
the C-terminal phosphorylation sites. In this regard, the
N-terminal truncation mutants used in this study were no
longer sensitive to kinase inhibitor treatment. Instead,
phosphorylation of the truncation mutants was constitu-
tively in the ‘serum-stimulated’ state (data not shown).
These data argue for the second model, and suggest that
the N-terminal region of eIF4GI modulates the phospho-
rylation state of the C-terminal phospho-region.
It was of particular interest to determine whether this
N-terminal regulatory region could be the eIF4E binding
site, since binding of eIF4E to eIF4G is also rapamycin-
and LY294002-sensitive, and because eIF4E binding
induces a conformational change in the eIF4GI protein
(Haghighat et al., 1996; Ohlmann et al., 1997; Hershey
et al., 1999) that could theoretically alter the accessibility
of the C-terminus to kinases. To address this question,
HA-tagged eIF4GI fragments aa 550–1560 (consisting of
the eIF4E binding site ? middle ? C-terminus) and aa
614–1560 (consisting only of the middle ? C-terminus)
were expressed in 293T cells. Cells were metabolically
labeled, treated with kinase inhibitors, and both the HA-
tagged eIF4GI fragments and endogenous eIF4GI were
immunoprecipitated from the cell lysates and mapped
ive to rapamycin- (compare Figure 7B and D with A and
C) and LY294002-treatment (data not shown); serines
1108, 1148 and 1192 remained phosphorylated, while
eIF4GI phosphorylation sites
Fig. 7. N-terminal sequences confer kinase inhibitor sensitivity to the
C-terminal phospho-region. HA-tagged eIF4GI fragments were
expressed in 293T cells. Cells were metabolically labeled and
incubated in 10% serum (A, C and E) or 10% serum ? 25 ng/ml
rapamycin (B, D and F) for 1 h. Following lysis, immunoprecipitation
was conducted sequentially with anti-HA and anti-eIF4GI antisera.
Isolated proteins were gel-purified and mapped. The HA-eIF4GI 550–
1560 fragment interacts with eIF4E, while HA-eIF4GI 614–1560 does
phosphopeptide 7 was almost absent, even in the presence
of rapamycin. Endogenous eIF4GI immunoprecipitated
from the same lysates retained rapamycin- (Figure 7E and
F) and LY294002-sensitivity (data not shown). (In this
experiment, phosphopeptides 2, 3, 4 and 7 displayed acute
sensitivity to rapamycin treatment, whereas peptide 1,
which is the least responsive to serum, did not.) These
data indicate that eIF4E binding per se is not responsible
for conferring LY294002- and rapamycin-sensitivity
The protease sensitivity of a 98 aa N-terminal fragment
of the yeast eIF4GI homolog (TIF4631) is reduced upon
binding to yeast eIF4E, as it progresses from a less
structured to a more structured state (Hershey et al., 1999).
Thus, it remained possible that eIF4E binding to human
eIF4GI could also cause a conformational change invol-
ving a much larger region of the N-terminus than is
present in the aa 550–1560 fragment. To address this
possibility, and to localize further the eIF4GI N-terminal
region conferring rapamycin sensitivity to the C-terminal
phospho-region, a much longer truncation mutant (aa 157–
1560, the original eIF4GI clone; Yan et al., 1992) was
phosphorylation of this fragment displayed rapamycin
resistance (data not shown). Endogenous eIF4GI immuno-
precipitated from the same cell lysates retained rapamycin
sensitivity (data not shown). Thus, intramolecular inter-
actions between the extreme N-terminus (aa 1–156) and
the C-terminal phospho-region, or between a second
protein that interacts with the eIF4GI N-terminus (and
appear to confer rapamycin- and LY294002-sensitivity.
Finally, these data also indicate that eIF4GI C-terminal
phospho-region kinases are not rapamycin sensitive them-
selves, but that, instead, a conformational change that
alters the accessibility of several key residues is effected
by serum, and is inhibited by rapamycin, wortmannin and
LY294002. A similar phenomenon has been reported for
p70 S6 kinase 1 (Dennis et al., 1996; Mahalingam and
Here, we have identified a set of serum-stimulated
phosphorylation sites (Ser1108, Ser1148 and Ser1192)
within the highly phosphorylated C-terminal one-third of
the eIF4GI protein. Phosphorylation of these sites is
demonstrated to be responsive to various extracellular
stimuli, and is shown to be sensitive to specific inhibitors
of the PI3K–FRAP/mTOR signaling pathway(s). Interes-
tingly, the extreme N-terminus of eIF4GI appears to
modulate the phosphorylation state of the C-terminal
phospho-region. Taken together, our data suggest a two-
step model for the phosphorylation of eIF4GI. The eIF4GI
protein in serum-starved (or kinase inhibitor-treated) cells
lar interactions between the N- and C-termini, or due to
interactions with unknown eIF4G binding partners. In step
one, the repressed eIF4GI molecule is derepressed by the
PI3K–FRAP/mTOR pathway(s), either by direct phospho-
rylation of the N-terminus of the protein, or via modulation
of an eIF4GI binding partner. This event renders the
C-terminal serine residues accessible to other kinases.
Phosphorylation of these residues results in a ‘fully active’
eIF4GI. However, how phosphorylation may affect the
activity of eIF4GI in translation initiation is unknown.
This work also outlines a technique to evaluate how
any extracellular stimulus or cellular stress modulates
eIF4GI phosphorylation, and provides the basis for further
studies to assess how phosphorylation may modulate
eIF4GI activity. In addition, this study points out previ-
ously unsuspected differences between members of the
human eIF4G family, in that the corresponding region of
two closely related proteins, eIF4GII and p97, is not
phosphorylated. This observation is intriguing, in that it
suggests that the various members of the eIF4G family
may be differentially regulated by intracellular signaling
Contrary to previous reports suggesting that treatment
of cells with mitogenic stimuli leads to a net increase in
eIF4G phosphorylation (Morley and Traugh, 1989, 1990;
Donaldson et al., 1991), we did not observe a change in
the quantity of32P incorporated into eIF4GI after starva-
tion, serum stimulation (Figure 1A) or kinase inhibitor
treatment(B.Raught andA.-C.Gingras,unpublished obser-
vation). In these earlier reports, however, eIF4G (most
likely to be a mixture of eIF4GI and eIF4GII) was
isolated via m7GTP affinity purification as an eIF4E–
eIF4G complex. Subsequent to these reports, the eIF4E-
binding proteins (4E-BPs) were discovered, and found to
compete with the eIF4Gs for an overlapping binding site
on eIF4E (Lin et al., 1994; Pause et al., 1994; Haghighat
B.Raught et al.
et al., 1995; Mader et al., 1995). In quiescent or starved
293 cells, a relatively high proportion of eIF4E is bound
to 4E-BPs, and a low amount of eIF4G protein is recovered
after cap column purification of eIF4E (A.-C.Gingras and
B.Raught, unpublished observation). Mitogenic stimula-
tion leads to an increase in phosphorylation of the 4E-BPs,
and a consequent decrease in their affinity for eIF4E. The
liberated eIF4E is then free to interact with eIF4Gs,
resulting in a significant increase in the amount of eIF4G
recovered by cap column purification. Thus, the earlier
observed increase in eIF4G phosphorylation in response
to mitogen treatment probably reflected an augmentation
in the quantity of eIF4G bound to eIF4E. We cannot,
however, exclude the possibility that following other types
of treatments, or in other cell types, the quantity of
phosphate incorporated into eIF4GI changes.
It is clear that while both the human eIF4GI and eIF4GII
proteins can function in translation initiation (Gradi et al.,
1998a), these molecules differ in several respects. For
instance, the eIF4GII protein is present at ~1/4 of the
level of eIF4GI in HeLa cells (Svitkin et al., 1999), and
the cleavage kinetics of eIF4GII in virus-infected cells
differs markedly from that of eIF4GI (Gradi et al., 1998b;
defined eIF4GI phospho-region is not phosphorylated in
eIF4GII. Thus, it appears that these proteins have under-
gone some degree of divergent evolution, and that the
activity of the eIF4G proteins may be regulated quite
differently. These observations may have important
implications regarding the regulation of translation initi-
ation in response to different types of intracellular signals,
or in the regulation of different mRNA populations.
We previously reported that Mnk1 can phosphorylate
eIF4GI in vitro (Pyronnet et al., 1999). After further study,
it does not appear that this observation is relevant in vivo.
Mnk1 is activated via the MAPK pathway, which is
stimulated in response to serum treatment in 293 cells
(von Manteuffel et al., 1996). However, truncation of the
Mnk1 binding site in eIF4GI does not negatively impact
upon the phosphorylation state of the serum-stimulated
phosphopeptides (e.g. Figure 2E), and mapping of a
recombinant eIF4GI fragment phosphorylated in vitro by
Mnk1 revealed that it is phosphorylated only on serum-
repressed phosphopeptides 7 and 8 (B.Raught and
A.-C.Gingras, unpublished observation). Thus, it appears
unlikely that this kinase plays a role in mediating eIF4GI
A signaling pathway that may be dedicated to transla-
tional control (Brown and Schreiber, 1996) consists of
PI3K, Akt/PKB and FRAP/mTOR. These kinases signal
to several factors involved in the regulation of translation,
including the p70 S6 kinases (Jefferies and Thomas,
1996; Gout et al., 1998; Shima et al., 1998), eukaryotic
elongation factor 2 (Redpath et al., 1996) and the 4E-BPs
(Gingras et al., 1999b). eIF4GI may now be added to this
list. However, even though the serum-stimulated eIF4GI
phosphorylation sites identified here are rapamycin sensit-
ive, they are not directly phosphorylated by FRAP/mTOR
or p70 S6 kinases 1 and 2 in an in vitro kinase assay
(A.-C.Gingras and B.Raught, unpublished data). In vivo
evidence also argues against a role for rapamycin-sensitive
kinases such as the p70 S6 kinases and FRAP/mTOR in
the phosphorylation of the serum-stimulated eIF4GI sites:
the phosphorylation state of N-terminal truncation mutants
is rapamycin- and LY294002-insensitive. Thus, an
N-terminal region of eIF4GI appears both to repress
phosphorylation of peptides 1–4 in the serum-starved state,
and confer LY294002- and rapamycin-sensitivity to the
How might phosphorylation modulate eIF4GI activity?
Kinase inhibitor treatment does not appear to affect
the binding of several known eIF4GI binding partners,
including eIF3, eIF4A (B.Raught, A.-C.Gingras, S.Morino
and H.Imataka, unpublished observations) and MnkI
(S.Pyronnet, personal communication). These data are
consistent with the fact that the phospho-region does not
overlap with the binding site of any known eIF4GI binding
protein. Secondary structure predictions suggest that
this region is relatively unstructured (B.Raught and
A.-C.Gingras, unpublished data). The phospho-region also
containsa caspase cleavage
S.J.Morley, personal communication), suggesting that it
is solvent exposed. We therefore posit that this region acts
as a flexible ‘hinge’ between the middle and C-terminal
domains of eIF4GI. In the absence of any evidence for
quantitative changes in protein–protein interactions, we
suggest that phosphorylation alters intramolecular inter-
actions to cause short- or long-range changes in eIF4GI
structure. How these changes modulate the ability of
eIF4GI to initiate translation remains to be determined.
site (M.Bushell and
Materials and methods
Cell culture and [32P]orthophosphate metabolic labeling were carried
out as described (Gingras et al., 1998). Wortmannin and LY294002 were
purchased from Calbiochem and diluted in DMSO at 1000?. Rapamycin
was purchased from the same source, dissolved in EtOH at 25 µM, and
used at a final concentration of 25 nM. 293T cells were transfected
using a modified calcium phosphate procedure (Chen and Okayama,
1988). Bovineinsulinwas purchased
12-O-tetradecanoylphorbol 13-acetate (TPA) from Sigma.
HA-tagged eIF4GI constructs were described previously (Imataka and
Sonenberg, 1997). Fragments encompassing aa 1035–1206 of eIF4GI,
aa 1057–1225 of eIF4GII and aa 395–547 of p97 were amplified by
PCR, and cloned in-frame into EcoRI–XhoI sites of pcDNA3–GST
(Imataka et al., 1998). Point mutations of eIF4GI were generated using
pcDNA3–GST–eIF4G(1035–1206) as a template, and mutants were
cloned into EcoRI–XhoI sites of pcDNA3–GST. All inserts were
Antiserum raised against the eIF4GI N-terminus was characterized
previously (Craig et al., 1998). Antiserum prepared against the eIF4GI
C-terminus was derived from a New Zealand White rabbit injected with
the peptide CRSRERPSQPEGLRKAASLTEDRDRGR, corresponding
to aa 1154–1179 (Sheldon Biotechnologies, McGill University), conjug-
ated to keyhole limpet hemocyanin. This antiserum does not cross-react
with eIF4GII (A.Gradi and B.Raught, unpublished data).
Tryptic phosphopeptide mapping
To isolate labeled eIF4GI protein, cell culture plates were washed twice
with phosphate-buffered saline, and lysed in 1 ml of lysis buffer (Gingras
et al., 1998) containing a 1:500 dilution of the Sigma protease inhibitor
cocktail, 10 mM NaF, 0.25 mM sodium orthovanadate and 10 nM
calyculin. Cells were lysed for 15 min at 4°C, and all subsequent steps
were carried out at 4°C. Lysate was scraped from the plates and
centrifuged for 15 min at 14 000 g. Supernatant was pre-cleared by
adding 50 µl of protein A–Sepharose, and incubated with end-over-end
mixing for 60 min. eIF4GI was immunoprecipitated with 25 µl of protein
A–Sepharose beads coupled to 5 µl of antiserum for 3.5 h. Beads were
eIF4GI phosphorylation sites
collected by centrifugation at 3000 g, washed twice in lysis buffer and
twice in RIPA (50 mM Tris–HCl pH 7.5, 150 mM NaCl, 1% NP-40,
0.5% sodium deoxycholate, 0.1% SDS). Laemmli loading buffer was
added to the washed beads, and samples were boiled and subjected to
SDS–8% PAGE. Separated proteins were transferred to a 0.2 µm
nitrocellulose membrane (Schleicher & Schuell), which was exposed to
film, and the region of the membrane harboring labeled eIF4GI was
excised and Cerenkov counted. HA-tagged eIF4GI proteins were
immunoprecipitated with anti-HA11 (BabCO) coupled to protein G–
Sepharose beads, and subjected to SDS–12% PAGE. Tryptic digests and
TLE/TLC mapping were carried out as described (Gingras et al., 1998)
using Kodak (Figures 1, 2 and 6) or Merck (Figures 5 and 7) TLC
plates. Phosphorylation intensity was quantified using the BAS 2000
Large-scale, keratin-free TLE/TLC mapping was conducted to obtain
samples of sufficient mass to subject to mass spectrometry, as described
(Gingras et al., 1999a). Cellulose from regions harboring radioactivity
was scraped from the plate backing and prepared for analysis by LC–
MS/MS, as described (Watts et al., 1994). The system used was as
described (Gingras et al., 1998; Gygi et al., 1999).
The authors thank Colin Lister for exceptional technical assistance. We
also thank Drs S.Pyronnet, S.J.Morley, M.Bushell, L.Beilsolell and
S.K.Burley for sharing unpublished data, and Drs G.Thomas and
S.Kozma for p70 S6 kinases 1 and 2. B.R. is the recipient of a Medical
Research Council of Canada (MRC) post-doctoral fellowship. A.-C.G.
is the recipient of an MRC doctoral award. S.P.G. is supported by
National Institutes of Health grant T32HG00035 and a grant from Oxford
Glycosciences. Work in R.A.’s laboratory was supported by the National
Science Foundation Science and Technology Center for Molecular
Biology, and by NIH grant R01 AI41109-03. Work in N.S.’s laboratory
was supported by grants from the National Cancer Institute of Canada,
the Medical Research Council of Canada and the Howard Hughes
Medical Institute. N.S. is a distinguished MRC of Canada scientist and
a Howard Hughes Medical Institute International Scholar.
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Received August 25, 1999; revised and accepted November 29, 1999