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INTRODUCTION |
Regulation of protein interactions and protein activity via
changes in phosphorylation status is a common theme in biology (see,
for example, Refs. 1 and 2). Eukaryotic translation is an example of a
major cellular process that may be regulated in this way at a number of
steps in the overall pathway (3). A key point of translational
regulation is at the step of ribosome binding to the 5' end of cellular
mRNA. Ribosome-mRNA binding involves interactions mediated by
the cap-binding complex
eIF4F1 (Fig. 1A).
Mammalian eIF4F comprises the eukaryotic initiation factors eIF4E,
eIF4G, and eIF4A (4). The largest eIF4F component (5), eIF4G, has
binding sites for eIF4E, eIF3, eIF4A, the poly(A)-binding protein and the mitogen-activated protein
kinase-activated protein kinase Mnk1 (5, 6-10). More recent
work indicates that yeast eIF4G also binds to the mRNA decapping
protein (Dcp1; Ref. 11). The eIF4F complex is tethered to the mRNA
cap by the 25-kDa cap-binding protein, eIF4E. eIF3 is believed to form
a bridge between the 40 S ribosomal subunit and eIF4F, whereas the
binding of eIF4G to poly(A)-binding protein may play a role in
promoting interaction between the 3' and 5' ends of mRNA. The
significance of the interaction between eIF4G and eIF4A is not clear,
but the latter factor (together with eIF4B) catalyzes
ATP-dependent RNA helicase activity that may promote
ribosomal scanning along structured mRNA (12). Mnk1 phosphorylates
eIF4E, which may in turn increase this factor's affinity for the
mRNA cap (13).
The cell regulates eIF4F activity in response to environmental changes
and various stimuli, including temperature stress and stimulation by
hormones (3, 4, 14, 15). Deviations from normal levels of eIF4F
activity can have drastic effects on cellular functions. For example,
overexpression of eIF4E leads to cell transformation and changes in
cell morphology (16, 17). The eIF4E-eIF4G interaction is of
central importance for cap-dependent initiation, and can be
blocked by small regulatory proteins that bind to eIF4E, the 4E-binding
proteins (4E-BPs; Refs. 18 and 19). Inhibition by 4E-BP1 is apparently
inversely dependent on its level of phosphorylation,
whereby the hypophosphorylated form binds most tightly to eIF4E
(19-21). Cell transformation caused by overexpression of eIF4E is
reversed by simultaneous overexpression of the 4E-BP1 gene (22).
Recent work (23-25) has shown that both the yeast and the mammalian
eIF4E proteins each interact with eIF4G via a site that maps to the
face opposite to the ventral cap-binding slot (26). The binding sites
for eIF4G of Saccharomyces cerevisiae and the yeast
eIF4E-binding protein p20 (which is also a phosphoprotein (27)) overlap
within this dorsal region on yeast eIF4E (23). There is also another
yeast 4E-binding protein, called Eap1, that is suspected to act
partially as a 4E-BP1 functional analogue (28), and possesses an
eIF4E-binding motif similar to that of p20. 4E-BP1 binds to the
equivalent region on the dorsal face of human eIF4E (24, 25), but this
regulatory protein uses a slightly different set of molecular contacts
to those involved in eIF4G binding (24, 29). The binding of protein
ligands at the dorsal site of yeast or human eIF4E is capable of
stabilizing cap binding at the ventral cap-binding slot (23, 24, 30, 31), although the physiological significance of this effect has yet to
be determined.
The mammalian 4E-BPs seem to mediate strong regulation of translation
in vivo (3, 12). This is in contrast to yeast p20 (30, 32,
33), which may possibly be involved in the fine tuning of translation
rates (23, 24). Given the key role of the 4E-BPs in the regulation of
translation, it is important to elucidate the mechanism underlying this
modulation. The binding affinities of the nonphosphorylated 4E-BPs for
human eIF4E are high (estimated KD values of
10
8-10
9 M) and comparable with
recent estimates of the binding affinity between eIF4E and the
eIF4E-binding domain of eIF4G of yeast (23, 24, 34). Thus,
hypophosphorylated 4E-BPs have the potential to compete effectively
with eIF4G for binding to eIF4E and therefore to act as potent
translational inhibitors. In the present study, we extend this
comparison by estimating the binding affinities of phosphorylated forms
of a 4E-BP.
Six sites of phosphorylation on rat 4E-BP1 (also known as PHAS-I) have
been mapped (Fig. 1B), but there is disagreement about the
roles of the respective sites in regulation, while the kinases involved
in vivo remain poorly characterized. These correspond in the
human 4E-BP1 sequence (which is almost identical to PHAS-I) to
Thr37, Thr46, Ser65,
Thr70, Ser83, and Ser112 (35, 36;
Fig. 1iB). In rat adipocytes, phosphorylation at the first four of
these sites is increased in response to insulin and partially decreased
in the presence of rapamycin (35, 37). The effect of rapamycin is
currently explained by proposing that at least two of the sites
(Thr37 and Thr46) are phosphorylated by a
FRAP/mTOR-associated pathway (38). The phosphoinositide 3-kinase and
the Akt/PKB protein kinase also seem to act via a further pathway that
does not involve FRAP/mTOR (39). However, the kinases directly
responsible for phosphorylating 4E-BP1 in vivo have not been
identified, and much uncertainty remains about the signaling pathways
leading to phosphorylation of the 4E-BPs (see Ref. 3 for review).
The other key area yet to be resolved concerns the influence of
phosphorylation on the molecular function of 4E-BP1. Some reports have
concluded that phosphorylation of Thr37 and
Thr46 (40), or of Thr46 alone (35, 41), is
capable of inhibiting the eIF4E-4E-BP1 interaction. In contrast,
another group observed no loss of binding associated with
phosphorylation at these two sites, and proposed that their primary
function is to trigger phosphorylation at the other sites on 4E-BP1
(38). This interdependence of phosphorylation events has been
questioned by others (37, 42). Heesom and Denton (42) have concluded
that the phosphorylation of Ser65 leads to dissociation of
4E-BP1-eIF4E, whereas phosphorylation at Thr37 and
Thr46 does not disrupt this complex. In this paper we focus
on the molecular consequences of phosphorylation at the sites on 4E-BP1 that are most relevant to eIF4E binding, showing how these can contribute to the regulatory function of this protein in
vivo. The result is a quantitative biophysical model of molecular
regulation that helps explain how different degrees of inhibition can
be achieved by phosphorylation at the respective phosphorylatable sites.
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MATERIALS AND METHODS |
Mutagenesis and Plasmid Construction--
Mutagenesis via a
polymerase chain reaction-based technique (43) yielded products which
could be inserted between either the NdeI and
EcoRI sites of the yeast expression vector YCpSUPEX2 (44) or
the NdeI and BamHI sites of the Escherichia
coli expression vector pCYTEXP3 (45). The genes for expression in
yeast all encode an N-terminal "Flag," MDYKDDDDKT, which appears to
enhance the stability of the protein in yeast (24, 29). The genes for
expression in E. coli all contain 12 histidine codons
between the first and second 4E-BP1 codons, thus facilitating (using
nickel ion affinity) both purification of the protein and binding to the sensor chip surface used for SPR. The nucleotide sequence of each
of the variants in both vectors was confirmed to be free of any
spurious mutations.
Yeast Growth Assay for 4E-BP Function--
4E-BP1 function was
assayed in yeast strain Jo56 (cdc33::LEU2
his3 ura3 ade3 (YCpTrp-hu4E TRP1), Ref. 29) after
transformation to uracil prototrophy in glucose-containing selective
medium with the YCpSUPEX2 plasmid derivatives. The cdc33
disruption in Jo56 was complemented by the human eIF4E gene in the
plasmid YCpTrp-hu4E. 4E-BP1 expression was induced by transferring the
cells to galactose-containing selective medium. Expression of each of
the 4E-BP1 derivatives was also tested in a strain with intact
CDC33 as control. The phenotypes of two independently
isolated derivatives of each 4E-BP1 allele were tested, and each allele
was re-tested after re-cloning into a fresh YCpSUPEX2 vector background.
Synthesis and Purification of Recombinant Proteins in E. coli--
Expression of the 4E-BP1 genes contained in pCYTEXP3 was
achieved by a temperature shift from 30 to 42 °C in the
protease-deficient E. coli strain CAG629 (46). The proteins
were then purified denatured on nickel-nitrilotriacetic acid-agarose (Qiagen).
Phosphorylation of 4E-BP1 in Vitro--
For analysis of the
sites of phosphorylation, 2 µg of 4E-BP1 were incubated in 10 mM HEPES, pH 7.4, 1 mM dithiothreitol, 2 mM magnesium chloride, 1% (v/v) glycerol, and 5 mM ATP with 5 µCi of [
-32P]ATP and 0.45 units of activated recombinant ERK2 in a total volume of 20 µl for
2 h at 23 °C. The reference activity of ERK2 was estimated
using myelin basic protein as substrate. ERK2 was generously given by
Dr. C. Armstrong, Division of Signal Transduction Therapy,
University of Dundee. For SPR analysis, 20 µg of recombinant 4E-BP1
was phosphorylated with 2.25 units of ERK2 under the same conditions,
but in a total reaction volume of 100 µl and excluding radiolabeled ATP.
Phosphopeptide Mapping--
The phosphorylation reaction was
stopped by adding SDS and
-mercaptoethanol, each to 1%, and heating
at 95 °C for 5 min. The proteins were separated by
SDS-polyacrylamide gel electrophoresis and radiolabeled 4E-BP1 was
eluted from the gel by shaking overnight in 50 mM Tris-HCl,
pH 7.5, 0.1% SDS, and 5%
-mercaptoethanol. Gel pieces were removed
with Spin-X centrifuge tube filters (Costar) and the labeled protein
was precipitated in 10% trichloroacetic acid. After removal of the
trichloroacetic acid by extensive washing, the protein was digested
overnight at 30 °C with trypsin (sequencing grade, Roche Molecular
Biochemicals) in 20 mM ammonium bicarbonate, 0.1%
Zwittergens, or with trypsin and chymotrypsin (Roche Molecular Biochemicals) in 20 mM ammonium bicarbonate and 0.2%
n-octylglucopyranoside (Calbiochem). The peptide mixtures
were acidified by adding 3 volumes of 0.1% (v/v) trifluoroacetic acid
in water and analyzed by high pressure liquid chromatography (HPLC)
(Gilson) using a Vydac C18 (250 mm long × 4.6-mm
internal diameter) reversed-phase column developed with 0.1%
trifluoroacetic acid in water/acetonitrile. The main radiolabeled
fractions were analyzed by matrix-assisted laser desorption ionization
time-of-flight mass spectrometry (47). Two-dimensional mapping was
performed as previously described (27, 48). Tryptic peptide mixtures
were dried, then redissolved in buffer for the first dimension
separation. Electrophoresis in the first dimension was performed at pH
1.9, and chromatography in the second dimension with buffer containing
isobutyric acid.
Reticulocyte Lysate Translation Assays--
Capped and
polyadenylated luciferase mRNA was synthesized in vitro
from a luciferase gene inserted into plasmid pHST7 (11). 16.5 µl of a
rabbit reticulocyte lysate (micrococcal nuclease-treated, Promega) was
mixed with 125 ng of 4E-BP1 in 0.5 µl of buffer A (20 mM
HEPES, pH 7.4, 100 mM KCl, 2 mM
MgCl2), plus RNasin (final concentration 1 unit/µl) and
amino acids (final concentration of each, 40 µM) in a
total volume of 24 µl. The mixture was preincubated at 30 °C for
10 min, programmed by the addition of 1 µl containing 0.1 µg of
mRNA and incubated a further 90 min. Luciferase activity was then
measured using a luminometer.
m7GTP-Sepharose Chromatography--
2 µg of 4E-BP1
were labeled with 32P, as described above, and separated
from [
-32P]ATP by gel-filtration through Sephadex G-25
(Amersham Pharmacia Biotech). The sample was mixed with a 5-fold molar
excess of eIF4E, to give a total volume of 370 µl (in buffer A, see
above), then incubated at 15 °C for 15 min before mixing with 30 µl of m7GTP-Sepharose resin (Amersham Pharmacia Biotech)
and incubating for a further 90 min with gentle agitation. The resin
was subsequently washed three times with 400 µl of the same buffer,
and bound proteins were eluted with 100 µl of 0.1 mM
m7GTP in the same buffer. In controls, further elution
steps were performed to demonstrate that all of the eIF4E (and, if
present, associated proteins) had been eluted. The protein fractions
were precipitated with ice-cold acetone and resolved by denaturing 15%
polyacrylamide gel electrophoresis. The quantities of radiolabeled proteins in the gel were determined using a Molecular Dynamics Typhoon
8600 image analyzer (Amersham Pharmacia Biotech) with Image Quant software.
Purification of Phosphorylated 4E-BP1--
Phosphorylated 4E-BP1
was separated from nonphosphorylated 4E-BP1 by native gel
electrophoresis after loading the phosphorylation reaction mixture
directly onto a 12.5% polyacrylamide gel (49), with an aliquot of
32P-labeled 4E-BP1 run in parallel as a marker.
Phosphoprotein was eluted from macerated gel slices directly into
buffer B (10 mM HEPES pH 7.4, 150 mM NaCl,
0.005% surfactant P20 (Biacore), and 50 µM EDTA). Gel
pieces were removed using a Spin-X centrifuge tube filter (Coster) and
the solution was concentrated using a Vivaspin polyethersulfone
membrane with a 5-kDa exclusion limit (Vivascience).
Surface Plasmon Resonance Analysis--
All SPR assays were
performed using a Biacore 3000 instrument essentially as described
previously (24). The 4E-BP1 proteins in Buffer B (described above) were
immobilized on the sensorchip of type NTA (Biacore). For estimation of
the dissociation constant (KD), six different eIF4E
concentrations, ranging from 10 nM to 1 µM,
were allowed to interact with the immobilized 4E-BP1 protein. The
resulting sensorgrams were evaluated using the BIA Evaluation software
package. The curves were analyzed using local fittings for Langmuir
binding and averaged for each protein.
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RESULTS |
Modification of 4E-BP1 Phosphorylation Sites to Glu and
Asp--
We examined to what extent modification at each of the
threonine/serine-proline ((T/S)P) phosphorylation sites affect the translation regulatory function of 4E-BP1 by substituting acidic amino
acids, either glutamic acid (Glu) or aspartic acid (Asp), for each of
the threonines (Thr) or serines (Ser) at positions 37, 46, 65, 70, and
83 individually (Fig. 1B and
the substitutions listed in Fig. 3). Ser112 was not
included in this study because its phosphorylation is neither
rapamycin-sensitive nor is it thought to affect 4E-BP1 binding to eIF4E
(36).

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Fig. 1.
A, schematic representation of how a
4E-BP is thought to block eIF4F formation and association with the cap
structure at the 5' end. The 4E-BP competes with eIF4G for binding to
the dorsal site on eIF4E, forming a stable heterodimeric complex that,
although still able to bind to the 5' cap (not shown here; see Ref.
24), is no longer able to interact with eIF4G. Phosphorylation of the
4E-BP reduces the affinity between the 4E-BP and eIF4E, thus allowing
eIF4G to compete more effectively for binding to eIF4E. This means that
the active eIF4F complex can assemble on the mRNA, promoting
cap-dependent translation initiation. The relationship
between eIF4E-4E-BP formation and interactions between eIF4G and eIF4A
(and thus eIF4B) are not understood. B, the
human 4E-BP1 sequence and the seven known phosphorylation sites of
4E-BP1. The sites functionally studied in this work are marked with
p. The phosphorylation of Ser101, described in
this work, is not known to occur in vivo, while the
phosphorylation of Ser112 is not thought to be relevant to
modulation of 4E-BP1 activity in vivo (36).
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We assessed the effects of these changes initially using a yeast growth
assay. Previous work established a system whereby the synthesis of
human 4E-BPs in yeast causes inhibition of translation and consequent
reduction in growth (24, 29). This allows us to study translational
regulation by 4E-BP1 in vivo. The system is dependent on
replacement of the endogenous yeast eIF4E gene (CDC33) by
the human eIF4E gene, which complements cdc33. Human, but
not yeast, eIF4E is able to bind 4E-BP1 tightly, therefore only the
strain with the eIF4E substitution is sensitive to 4E-BP1 expression;
expression of the human 4E-BP1 gene in an unmodified yeast strain has
no inhibitory effect on growth.
The 4E-BP1 gene sequences were introduced into the test strain on a
plasmid that allows conditional induction. We used a colony growth
assay to provide semiquantitative estimates of growth inhibition and
thus first indications as to the impact of the amino acid substitutions
on 4E-BP1 repressive activity. When synthesis of "wild-type" 4E-BP1
was induced, colony growth was inhibited, the cell doubling time being
increased to over 15 h at 30 °C (Fig. 2, WT), compared with the
control, in which no 4E-BP1 was synthesized and the doubling time was
about 3 h (Fig. 2, CON). When the modified 4E-BP1 genes
were induced, the phenotypes of most of the substituted forms were
indistinguishable from that of the wild type and showed strong growth
inhibition. Two of the forms, however, involving substitution of Asp
for Thr at position 46 and of Glu for Ser at position 65, showed
partial relief of the growth inhibition (Fig. 2, S65E and T46D; Fig.
3). All of the yeast strains grew normally when the 4E-BP1 genes were repressed (Fig. 2,
Repressed). Moreover, in an unmodified control strain
expressing the endogenous yeast eIF4E, none of the modified 4E-BP1
genes caused any inhibitory effects (data not shown). The fact that
acidic side chain substitution has effects at positions 46 and 65 suggests that these two sites are of primary importance in terms of
their potential influence on the affinity of 4E-BP1 for eIF4E.

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Fig. 2.
4E-BP1 synthesis in a S. cerevisiae strain in which human eIF4E has been substituted
for yeast eIF4E leads to the inhibition of translation and growth.
Mutation of selected phosphorylation sites to Asp or Glu can partially
suppress the inhibitory potential of 4E-BP1 in this system. The plates
show the growth of the yeast host strain which constitutively expresses
the human eIF4E gene instead of the yeast eIF4E gene. Each of the
plated transformants contained a galactose-inducible expression vector
carrying the 4E-BP1 gene or a derivative of the 4E-BP1 gene. The site
of modification is indicated in each case. The CON strain contained the
expression plasmid lacking an insert. The plate medium contained either
galactose (Induced) or glucose (Repressed). The fast-growing colonies
visible among some of the induced transformants are attributable to
spontaneous mutations which cause loss of the ability to synthesize
functional 4E-BP1.
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Fig. 3.
Asp and Glu substitutions at 4E-BP1
phosphorylation sites change the affinity of 4E-BP1 for eIF4E and the
ability of 4E-BP1 to act as a translational inhibitor. The
approximate relative rate of yeast growth in the plate assay (Fig. 2)
during 4E-BP1 synthesis (from multiple trials) is represented on a
two-point scale as + (slight) or ++ (moderate). The relative rates of
synthesis of luciferase in the reticulocyte lysate assay are given as
percentages of the control experiment value obtained in the absence of
4E-BP1. The KD estimates obtained via SPR are
represented on a bar chart and given as numbers on the
right-hand side. All experiments were performed at least
three times and the white portions of the bars
represent the standard deviations.
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Effects on Translational Repression of Glu and Asp
Substitutions--
If the effects on yeast growth caused by the T46D
and S65E substitutions are due to reduced binding to eIF4E, then it
should be possible to observe these effects on translation more
directly. The effects of the acidic residue substitutions in 4E-BP1 on
translation were therefore investigated using a reticulocyte lysate
translation assay. The Asp- or Glu-substituted 4E-BP1 proteins were
synthesized in, and purified from, E. coli (an N-terminal
poly-His "tag" was used to facilitate purification). We then
investigated their effects on the translation of a capped, firefly
luciferase mRNA in the assay system. The optimal ratio of 4E-BP1 to
capped mRNA to give maximal inhibition of cap-dependent
translation was established prior to the experiment by determining
dose-response curves, which were found to follow a saturation profile
(data not shown). The effects, expressed as the proportion of
luciferase activity compared with the reaction in the absence of added
4E-BP1, are illustrated in Fig. 3. As observed previously (19),
wild-type 4E-BP1 imposes partial, rather than complete, inhibition on
translation in a reticulocyte lysate system. This inhibition is
specifically attributable to 4E-BP1 binding to eIF4E since, in control
experiments, the addition of an equimolar amount of recombinant eIF4E
together with 4E-BP1 was found to reverse the effect. In the case of
the Glu substitutions, just as is observed in the yeast growth assay, only substitution of Glu for Ser at position 65 has any significant effect in alleviating the inhibition of translation (Fig. 3,
BP1-S65E). In the case of the Asp substitutions, T46D and
S65D have the most significant effects, and of these, as observed in
the yeast growth assay, the effect of T46D appears to be the strongest
(Fig. 3, BP1-T46D and -S65D). These results are
therefore broadly consistent with those of the yeast growth assay in
showing that the presence of an acidic side chain at either positions
46 or 65 causes the greatest reduction in the repressive potential of
4E-BP1.
Asp and Glu Substitutions Reduce 4E-BP1:eIF4E Binding
Affinity--
We then asked whether these effects of acidic side chain
substitutions on translation and cell growth correlate with changes in
the affinity of eIF4E binding. The affinities of the substituted forms
of 4E-BP1 for eIF4E were estimated by means of surface plasmon resonance (SPR) analysis. This technique was performed using 4E-BP1 as
the ligand (immobilized on the sensor surface) and eIF4E as the analyte
(passed in solution over the sensor surface). The substituted forms of
4E-BP1 were polyhistidine tagged at the N terminus to facilitate
binding to the sensor chip. These were the same forms as those analyzed
in the reticulocyte lysate assay and were therefore known to inhibit
translation. Changes in the resonance response observed upon the
introduction and then removal of eIF4E were measured, and estimates of
the kinetic constants based on the respective response curves allowed
us to derive apparent dissociation constants for each of the
substituted 4E-BP1·eIF4E complexes (Fig. 3). Typical response
curves for the Glu-substituted and Asp-substituted 4E-BPs at a single
eIF4E concentration are shown in Fig. 4,
A and B, respectively.

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Fig. 4.
SPR analysis reveals changes in the binding
characteristics of 4E-BP1-eIF4E as a function of Asp and Glu
substitutions at known phosphorylation sites. The interaction data
for wild-type 4E-BP1 (WT) and BP1-S65E (A) and BP1-T46D
(B) are compared. The plots depict the resonance unit levels
detected by a BIAcore 3000 instrument as a function of time. In the
first phase of each experiment, eIF4E was present in the buffer (eIF4E
present). eIF4E was omitted in the second phase.
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The pattern of variation in the KD values of the
respective complexes broadly reflects the differences observed in the
effects on yeast growth and on translation in vitro (Figs. 2
and 3). Thus, of the Glu substitutions, only the BP1-S65E·eIF4E complex manifests a significant elevation in KD
(2-3-fold compared with wild-type), and S65E is the only Glu
substitution that allows increased yeast growth and an increase in
translation in the reticulocyte lysate system, compared with wild-type
4E-BP1. Of the Asp substitutions, both the BP1-T46D·eIF4E and
BP1-S65D·eIF4E complexes have elevated KD values
(again 2-3-fold compared with wild-type 4E-BP1). BP1-T46D and BP1-S65D
also allow the highest levels of translation in reticulocyte lysate
and, of these two, T46D causes the most prominent increase in
KD and translation in vitro, and also
allows an increased rate of yeast growth.
We conclude that substitution of an acidic side chain at either of
positions 46 or 65 within the 4E-BP1 molecule can cause a 2-3-fold
reduction in the binding affinity for eIF4E, and that this reduction in
affinity is sufficient to cause detectable changes in
cap-dependent translational activity and in cell growth
rate. Because acidic side chain substitutions are potentially capable of partially mimicking the effects of phosphorylation, these findings provided a good starting point for a comparative analysis of the effects of phosphorylation at the respective (T/S)P sites.
Generating Single-site Phosphorylated Forms of 4E-BP1--
In
order to investigate the effects of phosphorylation at individual
sites, a series of 4E-BP1 proteins were synthesized in which alanine
had been substituted at four of the five (T/S)P sites, leaving in each
case one of the sites unchanged and susceptible to phosphorylation
("4A"-substitutions). An additional protein (BP1-5A),
in which all five sites were replaced by Ala, was also synthesized as a
control. Each protein was phosphorylated in vitro using
recombinant activated ERK2 mitogen-activated protein kinase. ERK2 is a
useful tool in these studies because it is known to phosphorylate the
(T/S)P sites of PHAS-I in vitro (35)
The 32P-labeled proteins, resolved electrophoretically in a
denaturing polyacrylamide gel, are shown in Fig.
5A. Wild type 4E-BP1 was
phosphorylated at multiple positions, but primarily at sites Thr46 and Ser65 (Fig. 5B). It is
apparent that BP1-5A underwent a low level of phosphorylation,
indicating that in this protein at least some phosphorylation of an
additional site or sites occurs. Isolation from phosphorylated BP1-5A
of the phosphopeptide after tryptic digestion, followed by analysis
using mass spectrometry and solid-phase sequencing, revealed that the
phosphorylated residue was Ser101 (data not shown), which
is not a site where phosphorylation has been previously reported. Like
the five known phosphorylation sites upstream of it, Ser101
is also followed by proline. To examine the possibility that the other
Ala-substituted forms were significantly phosphorylated at
Ser101, we employed two different techniques: separation of
the phosphopeptides by HPLC and two-dimensional mapping. The
phosphorylated proteins were analyzed after cleavage either with
trypsin alone or in combination with chymotrypsin. The HPLC analyses
yielded single phosphopeptide peaks (see, for example, Fig. 5,
C and D), indicating that the proteins are indeed
phosphorylated almost exclusively at the expected sites. A few cases
where the HPLC resolution was not clear were investigated further, as
outlined below.

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Fig. 5.
Analysis of phosphorylated forms of
4E-BP1. A, SDS-polyacrylamide gel electrophoresis of
the wild-type and mutant forms of 4E-BP1 after incubation with
activated recombinant ERK2 in the presence of
[ -32P]ATP. Comparison of the 4E-BP1 bands indicates
different levels of incorporation. HPLC analysis was performed on the
phosphorylated forms of wild-type 4E-BP1 (B), S65(4A)
(C), and S83(4A) (D). The resulting radioactivity
traces are shown. The inset in the wild-type profile
(B) represents an enlarged part of the trace between 12.5 and 14.5 min, showing elution predominantly of
phospho-Ser65 and phospho-Thr46 peptides.
E, phosphorylated T37(4A) and T46(4A) were digested with
trypsin and analyzed by two-dimensional. "x" marks the
origin where the sample was loaded onto the TLC plate. The
arrows indicate the two main phosphopeptides as discussed in
the text.
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The HPLC profile for BP1-T37(4A) indicated incomplete digestion by one
of the two enzymes, while mass spectrometric analysis of the major
tryptic phosphopeptides derived from BP1-T46(4A) resolved two products
(not resolved by HPLC) resulting from alternative cleavages at adjacent
arginines (Arg19 and Arg20). Therefore, to
resolve the phosphorylation patterns of BP1-T37(4A) and BP1-T46(4A) in
more detail, the tryptic digests were analyzed by two-dimensional
mapping (Fig. 5E). The two different phosphopeptides, one
containing and one lacking an N-terminal arginine, were resolved by
this method (marked by arrows in Fig. 5E). Amino
acids 37 and 46 are contained in the same tryptic peptide, thus
yielding almost identical peptide maps. In conclusion, these data
confirm that BP1-T37(4A) and BP1-T46(4A) are phosphorylated by ERK2
primarily at Thr37 and Thr46.
Due to the failure of cleavage at Arg73, as revealed by
analysis of the peptides by mass spectrometry (data not shown),
Ser83 occurs within a large peptide spanning residues
70-99. The HPLC profile of the BP1-5A protein digested with trypsin
and chymotrypsin showed phosphopeptides containing phosphorylated
Ser101 with retention times of 11.9 and 14.5 min (data not
shown); these can also be seen as minor peaks in the profile of
BP1-S83(4A) (Fig. 5D). We were unable to incorporate
phosphate at position Thr70 in BP1-T70(4A) using ERK2. This
suggests that prior phosphorylation at one or more other sites might be
necessary. We note that others have encountered the same difficulty
using a similar protein as substrate (37, 41).
Taken together, these data show that the 4A-substituted proteins were
indeed phosphorylated predominantly at the expected single sites and
that the relative degree of phosphorylation at Ser101 was
very low. The two major peaks seen in the HPLC profile of the wild-type
protein (Fig. 5B) have retention times corresponding to
those of the Ser65 and Thr46 containing
phosphopeptides (13.1 and 13.7 min, respectively). In conclusion,
Ser65 and Thr46 are the major ERK2
phosphorylation sites in the wild-type protein in vitro.
Site-dependent Differential Effects of Phosphorylation
on 4E-BP1-eIF4E Binding Affinity--
How do individual
phosphorylations affect the affinity of 4E-BP1 for eIF4E? We analyzed
the effects of single-site phosphorylations on eIF4E binding using two
different techniques. First, we tested binding in a semiquantitative
manner by determining the proportion of 32P-labeled 4E-BP1
eluting with eIF4E from m7GTP-Sepharose (to which eIF4E
binds directly). This was achieved by preincubating
32P-labeled 4E-BP1 preparations with a molar excess of
eIF4E, allowing the eIF4E to bind to m7GTP-Sepharose, then
resolving the unbound and elutable fractions by SDS-polyacrylamide gel
electrophoresis and measuring the proportion of labeled protein in each
fraction (Fig. 6A). The
distribution of each phosphoprotein between the bound and unbound
fractions provides an indication of their relative affinities for
eIF4E. Thus, the wild-type phosphoprotein remains predominantly (more than 80%) unbound (Fig. 6A, WT + P), and therefore has a
relatively low affinity for eIF4E, as expected. Of the four singly
phosphorylated proteins, BP1-S65(4A) + P has the lowest affinity
for eIF4E (over 75% remains in the unbound fraction), BP1-T46(4A) + P and BP1-T37(4A) + P each have moderate affinity (50 and 46% unbound,
respectively), and BP1-S83(4A) + P retains relatively high affinity
(35% unbound; Fig. 6A). These results therefore indicate
that phosphorylation at different sites affects 4E-BP1-eIF4E binding
affinity to varying degrees, and that phosphorylation at
Ser65 has the single most pronounced effect on binding
affinity.

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Fig. 6.
Functional changes in 4E-BP1 associated with
phosphorylation. A, binding of phosphorylated 4E-BP1 to
eIF4E on an m7GTP-Sepharose affinity matrix. The gel shows
the amounts of 32P-labeled 4E-BP1 that are bound
(B), and thus elutable using m7GDP, and not
bound (unbound, U) together with eIF4E on the matrix. The
bar chart depicts the results obtained with the respective
4E-BP1 mutant proteins after phosphorylation in vitro
(compare Fig. 8). B, inhibitory effect of 4E-BP1
phosphorylated at Ser65 in a reticulocyte lysate reporter
gene assay. The inhibition of luciferase synthesis is expressed as a
percentage of the rate obtained in the absence of 4E-BP1 (100%). All
experiments were performed at least three times and the white
portions of the bars represent standard
deviations.
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We tested the effects of phosphorylation of Ser65 on
translation directly using the rabbit reticulocyte lysate assay. First it was necessary to purify the phosphoprotein from any
nonphosphorylated protein, and we achieved this by resolving the two
forms by native polyacrylamide gel electrophoresis and by eluting the
phosphoprotein from the gel (see "Materials and Methods"). In a
comparative investigation, we observed that both unphosphorylated
wild-type and BP1-S65(4A) inhibited translation to about the same
degree, whereas phosphorylation of each of the proteins caused
pronounced alleviation of the inhibition; phosphorylation of the
wild-type somewhat more so than of BP1-S65(4A) (Fig. 6B).
These results correlate well with the effects of phosphorylation on
eIF4E binding (Fig. 6A), indicating that the effects on
translation are almost certainly the direct result of changes in
4E-BP1-eIF4E affinity. They also confirm that phosphorylation of
Ser65 strongly reduces the ability of 4E-BP1 to inhibit translation.
We measured quantitatively the effects of individual phosphorylations
on 4E-BP1-eIF4E binding affinity by means of SPR analysis. The purified
4E-BP1 phosphoproteins were used as chip ligands and eIF4E as analyte,
as before. Sample sets of response curves for the phosphorylated and
nonphosphorylated BP1-WT and BP1-S65(4A) are illustrated in Fig.
7, A and B,
respectively, while the apparent dissociation constants for the
4E-BP1-eIF4E complexes derived from the complete analysis are listed in
Fig. 8.

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Fig. 7.
SPR analysis reveals changes in the binding
of 4E-BP1 to eIF4E as a function of phosphorylation at multiple sites
or only at Ser65. Interaction curves compare
nonphosphorylated wild-type (WT) 4E-BP1 and phosphorylated
wild-type 4E-BP1 (WT + P) (A) and nonphosphorylated
BP1-S65(4A) and phosphorylated BP1- S65(4A) + P (B). The
phases of the experiments are as described in the legend to Fig.
4.
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Fig. 8.
Summary of the SPR analysis results for the
respective 4E-BP1 proteins in their unphosphorylated and phosphorylated
forms, respectively. The table shows the position of the unchanged
amino acid in each case (the remaining four positions being mutated to
Ala). The estimated KD values are represented as
horizontal columns and numerically on the right-hand
side. All experiments were performed at least three times and the
white portions of the bars represent standard
deviations.
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Phosphorylation of BP1-WT caused the greatest change in affinity: the
apparent KD increased by more than 3 orders of
magnitude. Of the single site phosphorylations, phosphorylation of
BP1-S65(4A) had the greatest effect, causing a change in affinity of
nearly 2 orders of magnitude. Phosphorylation of either BP1-T46(4A) or
BP1-T37(4A) caused only a 3-5-fold change in affinity, and phosphorylation of BP1-S83(4A) caused a less than 2-fold change in
affinity. These differences are represented graphically in Fig.
9A, in which the area of each
circle is proportional to the relative magnitude of the apparent
dissociation constant (KD) for each of the
phosphoproteins.

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Fig. 9.
Effects of phosphorylation on the molecular
environment at the binding surfaces. A, the relative
impact of phosphorylation at each 4E-BP1 site on the eIF4E binding
affinity and inhibitory function of this translational repressor. The
relative areas of the circles are proportional to the
changes in estimated affinity between eIF4E and 4E-BP1 observed with
phosphorylation at the respective sites (indicated by P
followed by the amino acid position). The largest
circle represents the impact of phosphorylation of
wild-type 4E-BP1 (P-WT). Thr46 and
Ser65 are closest to the N-terminal limit and the
C-terminal limit, respectively, of the known eIF4E-binding domain.
B, a three-dimensional ribbon diagram of the structure of
m7GDP:eIF4E[28-217]-4E-BP1[51-67] reported by
Marcotrigiano et al. (25). The peptide backbone structure
for 4E-BP1 is not resolved beyond position 64 because no electron
density data were obtainable. C, the surface structure of
eIF4E in the region of interactions with the 4E-BP1 peptide shown in
B. A potential position of the phosphate group
of Ser65 (P(S65)) in the 4E-BP1 binding motif peptide is
shown as a circle defined by a broken line. This
could potentially interact with Glu70, Asp71,
and other amino acids on the dorsal surface of eIF4E. The eIF4E residue
Trp73 interacts with the helical region of the 4E-BP1
peptide used in the crystal structure (25), and is essential for eIF4E
binding to eIF4G and 4E-BP1 (10, 23, 24).
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DISCUSSION |
We have characterized the modulatory 4E-BP1-eIF4E interactions in
order to be able to ascribe relative levels of functional significance
to phosphorylation events at the respective (T/S)P sites on the
regulator protein (Fig. 9). Our SPR analyses yield an estimated change
in affinity of wild-type 4E-BP1 for eIF4E of approximately 3 orders of
magnitude as a result of phosphorylation. Since ERK2 phosphorylates
primarily Thr46 and Ser65 in the wild-type
protein (Fig. 5), it is likely that even greater reductions in the
affinity of 4E-BP1 for eIF4E would be observed if full phosphorylation
of all sites were achieved. However, the large reduction in affinity
seen here clearly demonstrates the wide range over which affinity can
be varied in response to phosphorylation. It follows that
phosphorylation of all sites on each 4E-BP1 molecule is not a
requirement for strong regulation to be achievable.
The results obtained with the Asp and Glu substitutions have provided
useful comparative information about the roles of the respective
phosphorylation sites in vitro and in vivo. The
changes in affinity associated with these substitutions were found to be comparatively small (Fig. 3), but gave a first indication that Ser65 is of particular significance in terms of modulating
4E-BP1 affinity for eIF4E and thus its ability to inhibit translation.
Thr46 was also identified as potentially playing an
important role. Yet, it is interesting to note that the Asp and Glu
substitutions fall short of being good mimics of phosphorylated Ser or
Thr, at least in a quantitative sense (compare Figs. 3 and 8). The resolution of differences in binding affinity associated with the
respective Asp and Glu mutations is limited, indicating overall a clear
distinction between the role of Ser65 and the other
phosphorylation sites, but not differentiating as convincingly between
the effects of mutations at positions 37, 46, 70, and 83.
The distinction between the roles of the respective sites on 4E-BP1 is
made fully apparent by the results obtained using the single site
phosphorylated forms of this protein. Phosphorylation of
Ser65 reduces the estimated affinity of 4E-BP1 for eIF4E
~100-fold (Fig. 8). A second group of sites (Thr37 and
Thr46) shows a 3-5-fold decrease in apparent binding
affinity in response to single phosphorylation events. Finally,
phosphorylation of Ser83 has little effect on binding
affinity. For technical reasons, we were not able to study 4E-BP1 with
a single phosphorylation at position 70. However, the results of the
Asp and Glu substitution experiments suggest that the effect of
phosphorylation of Thr70 will fall somewhere in the range
between Ser83 and Thr37. Overall, the
relationship between phosphorylation at each site and its influence on
binding affinity is consistent with a model in which the proximity of
the added phosphate to the eIF4E-binding motif plays a major role in
determining the impact of the modification on the interaction between
the two proteins (Fig. 9A). It should, however, be
emphasized that while the above data are useful indicators of how the
competitiveness of 4E-BP1 as an eIF4E-binding partner is likely to
change in response to phosphorylation at each of the major sites, they
are not equivalent to absolute estimates of the real affinity values in
the cell.
How can we envisage the molecular basis of the effects of 4E-BP1
phosphorylation? The dorsal face binding site on eIF4E and the
eIF4E-binding motif on 4E-BP1 are the key sites of interaction between
these two proteins, although it seems likely that further intermolecular contacts play a role (23, 24). The respective motifs
bind via a combination of hydrophobic, van der Waals, hydrogen bonding,
and salt bridge interactions (25). Presumably, differences in the three-dimensional structure of the dorsal face motif, and most
likely of other residues on S. cerevisiae eIF4E, prevent a
sufficiently tight association with the human 4E-BP1 (24), which is why
4E-BP1 does not function as a translational regulator in wild-type
yeast cells (29).
It has been proposed that electrostatic repulsion between like-charged
groups on the surfaces of 4E-BP1 and eIF4E plays a dominant role in the
regulatory interplay between 4E-BP1 phosphorylation and inhibition of
eIF4E function. More specifically, the acidic side chains of
Glu70 and Asp71 in eIF4E have been identified
as potential sources of electrostatic forces repelling a phosphate
group on Ser65 (25). The relative position of the
Ser65 phosphate in the eIF4E·4E-BP1 complex has not been
determined (25), but it might be sufficiently close to
Glu70 and Asp71 for electrostatic effects to be
significant (Fig. 9C). The observation of a very large
change in binding affinity upon phosphorylation of Ser65 is
consistent with this model.
However, there are also other potential explanations of the large
quantitative impact of Ser65 phosphorylation reported here.
First of all, it should be noted that Glu70 and
Asp71 are in salt-bridge interactions with
His37 and Lys36, respectively (Fig.
9C), which may reduce the significance of the repulsive
charge effects between Glu70, Asp71, and the
phosphate group. Second, the presence of a phosphate group in the near
vicinity could affect the pKa of Glu70,
thus influencing the stability of the Glu70 to
His37 side chain and main chain hydrogen-bond network. This
could mean that at least the local structure of eIF4E could be altered
in the presence of 4E-BP1 bearing a phosphorylated Ser65,
thus destabilizing the eIF4E·4E-BP1 complex. Third, while
-helix N-terminal NH groups can provide effective solvation for a negatively charged phosphate group (50), the CO groups available at the C terminus
of an
-helical region are expected to be incompatible with phosphate
solvation. This means that the helical structure of the binding motif
region of 4E-BP1 may be destabilized by phosphorylation of
Ser65, which in turn would be expected to destabilize the
set of interactions seen with the nonphosphorylated peptide (Ref. 25;
Fig. 9C). In summary, analysis of the available structural
data reveals that the marked destabilization of the eIF4E·4E-BP1
complex by Ser65 phosphorylation may be attributable to
electrostatic repulsion, conformational effects, or steric effects, or
a combination of these. The greater distance of the other
phosphorylation sites on 4E-BP1 from this interaction region may mean
that they are unable to influence this core set of interactions as
significantly as Ser65. Further work is needed to elucidate
the consequences of phosphorylation at this molecular interface.
In this study, we have characterized the consequences of
phosphorylation at the respective sites on 4E-BP1. The resulting data
provide a first approximation quantitative framework that should
contribute to more precise interpretations of the significance of the
action of specific kinases on this important regulatory protein, as
well as to the design of new experiments that should yield insight into
the molecular mechanism underlying regulation of eIF4E-4E-BP1 binding.
It should be pointed out that the presence of phosphorylation sites
which can impose different degrees of influence on eIF4E binding raises
the intriguing possibility that the cell can impose fine or coarse
control by targeting different sites on 4E-BP1. This might be achieved
via kinases of distinct specificities, providing considerable
flexibility in terms of control, perhaps at the individual tissue
level. Finally, to what extent the principles of quantitative control
characterized here for 4E-BP1 apply to the other human 4E-BPs remains
to be determined. This is a particularly interesting question because
of the reported evidence that 4E-BP1 and 4E-BP2 are subject to distinct
modulatory pathways in human myeloid cell differentiation (51).