Analysis of the regulatory motifs in eukaryotic initiation
factor 4E-binding protein 1
Vivian H. Y. Lee
1
, Timothy Healy
1
, Bruno D. Fonseca
1
, Amanda Hayashi
2
and
Christopher G. Proud
1
1 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada
2 Institute of Food Nutrition and Human Health, Massey University and Food, Metabolism and Microbiology, AgResearch Limited,
Palmerston North, New Zealand
Signalling through the mammalian target of rapamycin
complex 1 (mTORC1) plays a key role in the control
of a number of cellular functions [1,2]. These roles
have largely been revealed through the use of rapamy-
cin, an immunosuppressant drug that interferes with
signalling through mTORC1.
mTORC1 is a complex comprising several proteins.
These include mammalian target of rapamycin
(mTOR), a multidomain protein that possesses a pro-
tein kinase domain related to lipid kinases, and raptor,
a scaffold protein that interacts with proteins that are
phosphorylated by mTOR [3–8]. mTORC1 also com-
prises Rheb, a small G-protein that appears to activate
mTOR when it is in its GTP-bound form [9,10].
Signalling from cell surface receptors, such as those

depends on an intact TOS motif, but the RAIP motif and additional
C-terminal features of 4E-BP1 also contribute to this interaction. Muta-
tional analysis of 4E-BP1 reveals that isoleucine is a key feature of the
RAIP motif, that proline is also very important and that there is greater
tolerance for substitution of the first two residues. Within the TOS motif,
the first position (phenylalanine in the known motifs) is most critical,
whereas a wider range of residues function in other positions (although an
uncharged aliphatic residue is preferred at position three). These data
provide important information on the structural requirements for efficient
signalling downstream of mTORC1.
Abbreviations
4E-BP1, eukaryotic initiation factor 4E-binding protein 1; ECL, enhanced chemiluminescence; eIF, eukaryotic initiation factor; GAP, GTPase
activator protein; GST, glutathione S-transferase; HIF1a, hypoxia-inducible factor 1a; mTOR, mammalian target of rapamycin; mTORC1,
mTOR complex 1; PKB, protein kinase B (also termed Akt); PKC, protein kinase C; PRAS40, proline-rich Akt-substrate 40 kDa; PVDF,
poly(vinylidene difluoride); RAIP motif, Arg–Ala–Ile–Pro motif; S6K, S6 kinase; TOS motif, TOR signalling motif; TSC, tuberous sclerosis
complex.
FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2185
Rheb
.
GTP to its inactive GDP-bound form. For exam-
ple, agents that activate protein kinase B (PKB, also
termed Akt) induce the phosphorylation of TSC2. This
is believed to inactivate its GAP function [9,16],
thereby allowing Rheb to accumulate in its GTP-
bound form and to switch on mTORC1. Recent data
have suggested that RhebÆGTP activates mTORC1 by
bringing about the release of FKBP38, an inhibitor of
mTORC1 activity [17].
Raptor appears to promote signalling downstream
of mTORC1 by binding to short TOR signalling

an additional motif with the sequence Arg–Ala–Ile–
Pro (hence ‘RAIP motif’ [25]; Fig. 1A). The phosphor-
ylation of the two N-terminal sites in 4E-BP1
(Thr37 ⁄ 46 in the human protein; Thr36 ⁄ 45 in rat
4E-BP1) requires the RAIP motif [19], and their phos-
phorylation is needed for the subsequent modification
of two sites (Thr70 ⁄ Ser65) close to the eIF4E-binding
motif [19,26–29]. The mTOR-dependent control of
4E-BP1 is thus an example of hierarchical phosphory-
lation. It is the phosphorylation of Thr70 ⁄ Ser65 that
controls the binding of 4E-BP1 to eIF4E, and thus the
availability of eIF4E to form functional translation
initiation complexes (as 4E-BP1 competes with the
scaffolding factor eIF4G for binding to eIF4E [30]).
Our earlier work revealed that the RAIP and TOS
motifs play distinct roles in regulating the phosphory-
lation of 4E-BP1 within cells. The phosphorylation of
4E-BP1 is regulated by amino acids and by stimuli
such as insulin. The RAIP motif appears to mediate
the amino acid input [25,29] that promotes the phos-
phorylation of the N-terminal threonines in both
4E-BP1 and 4E-BP2 (which is not very prone to inhi-
bition by rapamycin). In contrast, the TOS motif is
required for the insulin-induced phosphorylation of
Ser65 (and, in some cell types, Thr70). Phosphoryla-
tion of Ser65 is generally completely blocked by rapa-
mycin. Although TOS motifs have now been identified
in a number of proteins, no systematic analysis of the
sequence requirements for a functional TOS motif has
been performed.

seen as ‘raptor binding’.] Because of substantial prob-
lems of nonspecific binding, we have been unable to
Regulatory motifs in 4E-BP1 V. H. Y. Lee et al.
2186 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS
successfully use coimmunoprecipitation approaches to
study raptor–4E-BP1 binding (A. Beugnet, B. D. Fonseca
& C. G. Proud, unpublished data; see also [19]).
Previous work has shown that mutation of the phen-
ylalanine to alanine in the TOS motif eliminates the
binding of raptor to the C-terminal fragment of human
4E-BP1 in the overlay assay [19] (see also Fig. 1B). We
have also observed no binding of raptor to a truncated
4E-BP1 molecule lacking the final six residues that
harbour the TOS motif (D6; Fig. 1B). This confirms
A
B
D
E
C
Fig. 1. Analysis of the binding of raptor to
variants based on 4E-BP1. (A) Schematic
diagram of 4E-BP1 showing the RAIP and
TOS motifs, the region that binds eIF4E and
the four phosphorylation sites discussed in
this report. Numbering is based on human
4E-BP1; for the rodent proteins, adjust by
)1. Schematic diagram is not to scale.
(B–D) Binding of raptor to wild-type 4E-BP1
or variants, assessed using the overlay (far-
western) assay (see Experimental proce-

solution (as assessed by NMR spectroscopy [32]). A
second potential concern is that, in this type of ‘far-
western’ analysis, 4E-BP1 is denatured (by SDS). This
concern is also lessened by the fact that 4E-BP1 lacks
a folded structure.
We created variants in which the first 17, 37, 57,
77 or 97 residues of 4E-BP1 were removed. The first
of these, ‘4E-BP1 (18–117)’, already lacks the RAIP
motif. As shown in Fig. 1C, each of these truncated
proteins bound to raptor less efficiently than full-
length wild-type 4E-BP1 (1–117) in the overlay assay.
Reproducibly, two regions appeared to be involved in
assisting the binding to raptor: the first 17 amino
acids [compare the signal for full-length 4E-BP1 (1–
117) with that for the ‘18–117’ variant] and sections
of the C-terminal half of 4E-BP1 [compare, for exam-
ple, the 4E-BP1 (98–117) variant with full-length
4E-BP1 (1–117)], in agreement with our earlier data
[19]. This suggests that the N-terminus, containing
the RAIP motif, and a more C-terminal region (out-
side the final 20 residues, i.e. other than the TOS
motif) are involved in binding to raptor. Although
the TOS motifs in 4E-BP1 and 4E-BP2 are identical,
other parts of their C-terminal regions are poorly
conserved, and it is not obvious which other features
contribute to raptor binding. We have not therefore
attempted to define further the features in the C-ter-
minus of 4E-BP1 that are involved in its binding to
raptor. The data for the other truncation mutants
shown in Fig. 1C indicate that other regions of

contributes to this interaction, but is not absolutely
required; and (c) that other regions of 4E-BP1 are also
involved in binding raptor. Interestingly, as noted
above, mutating the RAIP and TOS motifs separately
has qualitatively distinct effects on the phosphorylation
of 4E-BP1 within cells [19], revealing that they serve
different, rather than additive, functions. Interestingly,
Eguchi et al. [31] have shown that the introduction of
acidic residues at the positions of the phosphorylation
sites in 4E-BP1 decreases the interaction of 4E-BP1
with raptor. This implies that the regions of 4E-BP1
containing these residues also influence the interaction
with raptor, and is in accordance with our data
(Fig. 1C), which indicate that it is not only the TOS
and (to a lesser extent) RAIP motifs that are needed
for raptor–4E-BP1 binding.
Further definition of the RAIP motif in the
N-terminus of 4E-BP1
So far, very little information is available on what
actually constitutes a RAIP-type motif, i.e. what are
the sequence requirements. To learn more about the
nature of the RAIP motif and, in particular, to define
better what residues constitute this type of motif, we
created a range of further mutations in this region of
4E-BP1. It is important to note that, in the vector used
here, the Myc tag is at the C-terminus, i.e. at the
opposite end from the RAIP motif, to avoid any possi-
ble interference with the function of the N-terminal
RAIP motif. The vector encodes rat 4E-BP1, which
was used extensively in our earlier studies to define the

4E-BP1 than the phosphorylation of either the AAIP
(Fig. 2A) or RAIA (Fig. 2B) variants. This is espe-
cially true for the basal phosphorylation at Thr36 ⁄ 45,
which is maintained by the amino acids in the medium
A
B
C
D
Fig. 2. Assessment of the phosphorylation of 4E-BP1 mutants containing variants of the RAIP motif. (A–D) Wild-type 4E-BP1 (RAIP) or the
indicated mutants were expressed in HEK293 cells. Twenty-four hours following transfection, the cells were starved of serum for 16 h and,
where indicated, treated with 100 n
M insulin for 25 min. The top sections of each panel show the results from western blots using the
phosphospecific antibody for Thr36 ⁄ 45; the bottom sections show the data from anti-Myc blots (to assess the relative levels of expression
of the 4E-BP1 variants). With this gel system, 4E-BP1 runs as up to three bands (a–c, in order of increasing phosphorylation) as indicated.
V. H. Y. Lee et al. Regulatory motifs in 4E-BP1
FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2189
[29], but is also true for the increased phosphorylation
induced by insulin.
We therefore first replaced the isoleucine by the
other branched-chain residues, valine and leucine.
These 4E-BP1 variants were expressed in HEK293
cells. Their phosphorylation was analysed using a
phosphospecific antiserum that recognizes both
(P)Thr36 and (P)Thr45 in rat 4E-BP1. 4E-BP1
migrates as three distinct species (a–c) under these
conditions of SDS-PAGE. The slowest moving species
(c) is the most highly phosphorylated form, and is only
evident after insulin stimulation. This is because insulin
induces the phosphorylation of additional sites (nota-
bly Ser64, see below), which causes the protein to run

(compared with the AAIP variant; Fig. 2A,D), we did
not test any other mutations at this position in this
study. Earlier work has shown that mutating the pro-
line to alanine (to give the RAIA mutant) causes a
defect in the basal and insulin-stimulated phosphoryla-
tion of 4E-BP1 [25]. We also tested the proline to
valine mutation in wild-type 4E-BP1. The phosphory-
lation of the resulting RAIV mutant was more severely
impaired than that of the RAIA variant (Fig. 2D).
We then turned our attention to the arginine residue
within the RAIP motif, making mutations at this posi-
tion within the RAIA variant, which already shows a
reduction in basal and insulin-stimulated phosphoryla-
tion at Thr36 ⁄ 45 (Fig. 2A). Mutation of the arginine
to lysine in the RAIA variant (to create KAIA) did
not discernibly affect the basal or insulin-stimulated
phosphorylation of Thr36 ⁄ 45 (Fig. 2A). Mutation of
the arginine to methionine (no charge, bulky side-chain
similar to arginine; Fig. 2B) also did not impair the
phosphorylation of Thr36 ⁄ 45. Mutation to glutamate
(negative charge; Fig. 2C) diminished the basal level of
phosphorylation, but still permitted some induction of
phosphorylation by insulin. Mutation of the arginine
to glutamine (QAIP; Fig. 2B), threonine or asparagine
(both Fig. 2C) in wild-type 4E-BP1 had similar partial
effects. It therefore appears that Arg13 is less impor-
tant than Pro16 for the function of the RAIP motif,
and that several different types of residue can be toler-
ated here with only small, if any, effects on 4E-BP1
phosphorylation. For reasons that remain to be clari-

Regulatory motifs in 4E-BP1 V. H. Y. Lee et al.
2190 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS
proteins. Here, we employed two approaches to study
this: (a) the ability of 4E-BP1 variants to bind to rap-
tor; and (b) the ability of a given TOS-like motif to
promote the phosphorylation of 4E-BP1 in cells.
The first type of analysis could, in principle, be per-
formed using the TOS motif segment alone, provided
that this motif is sufficient to confer binding to raptor.
To test this, we added the sequence FEMDI (the TOS
motif found in the C-termini of mammalian 4E-BP1–
3) to the C-terminus of glutathione S-transferase
(GST). To obviate possible issues of steric hindrance,
we provided a spacer (four alanine residues) between
the C-terminus of GST and the TOS motif, to create
‘GST-Ala
4
-TOS’. As shown in Fig. 3A, the addition of
the TOS motif to GST did not allow raptor binding.
Thus, the five-residue TOS motif is incapable, by itself,
of binding raptor in this assay. This is consistent with
the data in Fig. 1 and [19], which show that additional
features in 4E-BP1 are required for raptor binding
(but that the TOS motif is nonetheless essential).
We therefore elected to examine the effects of
altering the TOS motif in 4E-BP1. Phosphorylation of
4E-BP1 involves multiple sites and a rather complex
hierarchy [19,26,27,33,34]. To assess the effects of alter-
ing the TOS motif, we mainly examined the phosphory-
lation state of Ser64, as this site is late in the hierarchy,

it is insufficiently stable to ‘survive’ the washes of the
far-western procedure, but can still support an interac-
tion in vivo. These data imply that merely examining
raptor binding in, for example, a far-western method
does not indicate what constitutes a functional TOS
motif. In contrast with the LEMDI variant, the
IEMDI mutant underwent only a small degree of
phosphorylation at Ser64 (Fig. 4B). This variant did
not bind to raptor in the overlay assay (Fig. 3A). It is
notable that all the currently known TOS motifs have
phenylalanine in the first position (Table 1).
We then created a systematic set of other variants
based on the FEMDI sequence found in the 4E-BPs.
Mutation of the second residue (glutamate) to another
acidic residue (aspartate) had no effect on raptor bind-
ing (FDMDI; Fig. 3B), and we did not therefore
examine its effect on the phosphorylation of 4E-BP1.
Changing the second residue to valine (FVMDI;
Fig. 4D) or alanine (FAMDI; Fig. 4D) did not dis-
cernibly affect the phosphorylation of 4E-BP1 in
HEK293 cells. Replacement by proline slightly
impaired the phosphorylation of Ser64 (FPMDI;
Fig. 4E). Mutation to arginine (carries positive charge,
FRMDI; Fig. 4F) substantially decreased the phos-
phorylation of 4E-BP1 when compared with the wild-
type protein. Raptor binding to all of these variants
was similar to that of the wild-type protein
(Fig. 3B,C). Thus, although an acidic residue is present
at this position in both the 4E-BPs (glutamate) and
S6Ks (aspartate) (Table 1), this feature does not actu-

residue at the second position (FVMDE and FVMVL,
respectively [23,24]). They have valine in this position
instead, which is clearly as effective as an acidic resi-
due in promoting the phosphorylation of 4E-BP1 at
Ser64 (Fig. 4D).
In contrast with the tolerance for variations in the sec-
ond position, mutation of the third residue (methionine:
an uncharged, relatively nonpolar amino acid) to ala-
nine or glutamate abolished raptor binding (Fig. 3C).
The methionine to alanine mutation also strongly
decreased the phosphorylation of Ser64 (FEADI;
Fig. 4F), and the phosphorylation of Ser64 was also
decreased by placing glutamate or, to a lesser extent,
arginine at this position (Fig. 4G). Mutation of the
methionine to isoleucine (also a nonpolar, aliphatic
residue) maintained Ser64 phosphorylation at wild-type
A
C
D
B
Fig. 3. Binding of raptor to the TOS motif in wild-type 4E-BP1 (FEMDI) and variants thereof. (A) An overlay assay (see Experimental proce-
dures) was used to assess the binding of raptor to the TOS motif (FEMDI) tagged at its N-terminus with GST and also containing a four ala-
nine spacer (Ala
4
) between the GST tag and the TOS motif. Wild-type GST–4E-BP1 and GST–4E-BP1 (AEMDI) served as positive and
negative controls, respectively. The top sections of each panel show raptor overlays, developed with anti-Myc. The bottom sections show
western blots for GST to assess the levels of each protein. (B,C) The overlay assay was used to detect binding of raptor to wild-type GST–
4E-BP1 (FEMDI) or mutants with the indicated sequences in place of the TOS motif. GST and GST–4E-BP1 (AEMDI) served as negative con-
trols. The top sections of each panel show the Myc-tagged raptor overlay. The bottom sections show western blots with anti-GST to assess
the amounts of each protein used. Arrowheads with asterisks denote degradation products (cleaved at the C-terminus) that react with anti-

of wild-type 4E-BP1, and that of the FEMNI protein
was intermediate between the other two mutants
(Fig. 4H). None of these three variants was able to
bind raptor (Fig. 3C). Thus, although the first TOS
motifs to be discovered contained a negatively charged
aspartate at this position (4E-BPs; S6K1 and S6K2),
and the recently reported TOS motif in PRAS40 simi-
larly has a glutamate at this position, other residues
are tolerated, even if, as for arginine, they carry a posi-
tive charge. The latest reported TOS motif, in HIF1a,
has an aliphatic, nonpolar residue in the fourth posi-
tion (valine; Table 1).
Mutation of the final residue from isoleucine to
arginine or alanine reduced raptor binding (FEMDR ⁄
FEMDA; Fig. 3C), but had little effect on Ser64
phosphorylation (Fig. 4C,I). We also tested the effect
of an acidic residue at this position, i.e. the FEMDE
variant. Phosphorylation of this mutant at Ser64 was
slightly reduced compared with the wild-type protein
(Fig. 4D). It was still able to bind raptor, albeit less
well than wild-type 4E-BP1 (Fig. 3C). In view of this
tolerance for a variety of residues at position five, we
did not create further mutations here. Although
almost all of the known TOS motifs have either leu-
cine or isoleucine at this position, residues that are not
branched-chain amino acids can clearly function in
this position.
It seems surprising that several variants failed to
bind raptor in the overlay assay, but still underwent
substantial phosphorylation within HEK293 cells

the motif FVMEF. Interestingly, the classical PKC iso-
form, PKCc, also contains a similar motif, FVMEY.
To test whether motifs with these sequences could
actually bind raptor, and to learn more about the
requirements for raptor binding, we decided to intro-
duce these motifs into 4E-BP1 (in place of its own
TOS motif), as the mTOR regulation of 4E-BP1 is
much better characterized than the control of STAT3
or PKC isoforms. We therefore created a range of
mutants of 4E-BP1, in both the GST fusion protein
(to test raptor binding) and the vector for mammalian
expression (to check their effect on the phosphoryla-
tion of 4E-BP1).
As shown in Fig. 5A, in the far-western assay, all of
these variant 4E-BP1 proteins, except one, bound rap-
tor to a similar extent to wild-type 4E-BP1. (The
exception is the variant with the FVMEY motif, which
did bind raptor, but less well than the others). As each
variant contains at least two changes from the wild-
type FEMDI sequence, it is inappropriate to try to
interpret these data in terms of the roles of individual
residues, except to say that placing a tyrosine in the
last position has a deleterious effect on raptor binding
(compare FVMEY with FVMEF in Fig. 5A).
These findings suggested that it was probable that
these motifs would support the phosphorylation of
4E-BP1 when the variant proteins were expressed in
HEK293 cells. Indeed, all but one of the variants
underwent substantial insulin-induced phosphorylation
at all the sites tested (Thr36 ⁄ 45 ⁄ 69 and Ser64)

to resolve earlier discrepancies concerning the role of
the RAIP motif: although this motif is not sufficient
by itself to bind stably to raptor [19], it plays an
‘accessory’ role in raptor binding, provided that a TOS
motif is also present [5,31]. This interpretation is con-
sistent with the recent observation that short interfer-
ing RNA (siRNA)-mediated knock-down of raptor
expression impairs the phosphorylation of Thr37 ⁄ 46 in
4E-BP1 (which depends on the RAIP motif) [24].
Our analysis of the TOS motif demonstrates that its
ability to bind raptor in vitro is not a reliable index of
function: a number of variants that failed to bind rap-
tor in the far-western assay were able to support phos-
phorylation of 4E-BP1 within cells. Although mutants
that bind raptor in vitro effectively support phosphory-
lation within cells, the converse is not true: for exam-
ple, the LEMDI mutant does not bind raptor but is as
effective as the wild-type sequence at facilitating phos-
phorylation (Figs 3B and 4C). Studying the phosphor-
ylation of 4E-BP1 is thus a more reliable method than
in vitro raptor binding to assess TOS motif function.
The first position in the TOS motif is critical: muta-
tion to another closely similar residue (isoleucine)
almost abolishes the phosphorylation of Ser64
(Fig. 4B). Consistent with this, all the known TOS
motifs have phenylalanine at this position. Our data
indicate that the nature of the second residue in the
motif is less critical: although the first motifs to be
identified (in S6Ks and 4E-BPs) contained acidic resi-
dues here, a range of other residues support the phos-

ingly, the least effective residue tested (arginine) is a
positively charged residue, which is not found in any
known TOS motif. Indeed, the known TOS motifs
have either an acidic residue or valine at this position
(which works well in 4E-BP1; Fig. 4D).
In the third position (methionine in 4E-BPs, HIF1a
and PRAS40; Table 1), we tested another aliphatic resi-
due (isoleucine), and positively or negatively charged
residues (arginine, glutamate). Isoleucine worked as
well as methionine (Fig. 4E), consistent with the fact
that this residue is isoleucine in S6K1, and suggesting
that a bulky side-chain is needed here. It is therefore
interesting that the only other known TOS motif, in
S6K2, has a similar residue (leucine) at this position
(Table 1). Although almost all the known TOS motifs
have an acidic residue (glutamate or aspartate; Table 1)
in the fourth position, arginine (which carries a positive
charge) also functions well in this position (FEMRI;
Fig. 4I). The only exception (HIF1a; Table 1; reported
very recently) has valine at this position.
Finally, in the fifth position (leucine or isoleucine in
all known TOS motifs except one, PRAS40; Table 1),
alanine or arginine worked well (Fig. 4C,I). Glutamate
was less effective, although the PRAS40 motif contains
glutamate at this position (Table 1). The observation
that the FEMDE motif was not fully effective in
4E-BP1 may reflect the fact that the final glutamate is
also the C-terminal residue of the entire mutant protein,
and thus actually carries two negatively charged car-
boxyl groups, whereas, in PRAS40, this is not the C-ter-

be tolerated at the first position: in particular, methio-
nine allows phosphorylation to at least the same extent
as arginine (Fig. 2B). This shows that a positive charge
is not required here: rather, the important factor may be
a bulky polar side-chain, although even alanine works
reasonably well at this position.
Our study is consistent with the concept that the
sequence [bulky side-chain]–[Ala]–[Ile ⁄ Val]–[Pro] pro-
vides an efficiently functional motif. There are cur-
rently no other known examples of proteins that
contain functional RAIP-type motifs (apart from
4E-BP2, which contains an identical sequence that
also functions to mediate the amino acid input to the
protein’s phosphorylation [29]). Therefore, we cannot
draw upon further information (as for the TOS motif)
to help define the functional requirements. As pointed
out above, further mutational analysis may help to fur-
ther refine them. Interestingly, although 4E-BP3 con-
tains a RAIP-like motif (CPIP), it is phosphorylated
only modestly even after treatment of cells with insulin
[25]. Replacing its N-terminus with the N-terminal part
of 4E-BP1 led to a marked increase in its phosphoryla-
tion (even in the absence of insulin). Thus, it seems
that the CPIP motif is inferior to the RAIP motif in
supporting phosphorylation in vivo, perhaps because
the first residue is not a large aliphatic residue. Further
work is needed to study this.
The rather ‘tolerant’ nature of the functional
requirements for the TOS and RAIP motifs will proba-
bly make it difficult to identify proteins containing

ACTC-3¢;5¢-CGGGATCCCCGGCGGCACGCTCTTCA
GC-3¢;5¢-CGGGATCCCCCGCCGCGTAGCCCTCGG-3¢)
and reverse primer (5¢-GATGAATTCTAAATGTCCAT
CTCAAACTGTG-3¢). 4E-BP1 PCR fragments were cloned
into the pGEX-3X vector (BamHI, EcoRI) for bacterial
expression. Site-directed mutagenesis was carried out using
the Stratagene (La Jolla, CA, USA) Quikchange
Ò
system,
according to the manufacturer’s guidelines.
Sources of Antisera
Antisera specific to Myc and GST were purchased from
Sigma-Aldrich and Roche Applied Science (Laval, Canada),
respectively. Antisera specific to phosphorylated 4E-BP1
(Thr36 ⁄ 45, Thr69 and Ser64) were purchased from Cell
Signaling (Danvers, MA, USA).
Cell culture, transfections, lysis, immuno-
precipitations and related procedures
HEK293 cells were grown in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% (v ⁄ v) fetal
bovine serum, 2 mml-glutamine, 100 lgÆmL
)1
streptomy-
cin sulfate and 100 UÆmL
)1
penicillin G. Transient trans-
fections were carried out by calcium phosphate
precipitation, as detailed previously [39]. Cells were
starved of serum for 16 h and, in some instances, of
amino acids for 90 min, as detailed previously [24]. Where

0.2% (v ⁄ v) glutaraldehyde in NaCl ⁄ P
i
containing 0.02%
(v ⁄ v) Tween-20. Cross-linking was carried out (after trans-
fer but prior to blocking) for 30 min at room temperature
with constant agitation. Blots were visualized by enhanced
chemiluminescence (ECL).
Far-western analysis of raptor binding
This was performed as described previously using lysates
from HEK293 cells expressing Myc-tagged raptor [24].
Blots were developed with anti-Myc and visualized by
ECL.
Dot blot far-western analysis of raptor binding
Dot blots were performed by spotting 0.8 lg of bacteri-
ally expressed, GST-tagged, wild-type 4E-BP1 (or 4E-BP1
mutants) on nitrocellulose membrane (0.45 lm) from Bio-
Rad Laboratories (Hercules, CA, USA). The membranes
were blocked with 5% (w ⁄ v) fat-free milk in NaCl ⁄ P
i
–
Tween-20 for 1 h at room temperature, and subsequently
incubated with lysates from HEK293 cells
expressing Myc-tagged raptor, as detailed previously [24].
Dot blots were developed with anti-Myc and visualized
by ECL.
Acknowledgements
This work was funded through support from the
Wellcome Trust (UK), the Canadian Institutes for
Health Research and the University of British
Columbia. BDF acknowledges generous support from

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