We have analysed the interaction of 4E-BP1 with raptor and the amino acid residues required for functional RAIP and TOS motifs, as assessed by raptor binding and the phosphoryla-tion of
Trang 1factor 4E-binding protein 1
Vivian H Y Lee1, Timothy Healy1, Bruno D Fonseca1, Amanda Hayashi2and
Christopher G Proud1
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 for insulin, growth factors and mitogens, activates mTORC1 through the inactivation of the tuberous sclerosis complex (TSC), which comprises TSC1 and TSC2 [11–15] In association with TSC1, TSC2 acts as
a GTPase activator protein (GAP) which converts
Keywords
4E-BP1; mTOR; mTORC1; RAIP motif; TOS
motif
Correspondence
C G Proud, Department of Biochemistry
and Molecular Biology, University of British
Columbia, Life Sciences Centre, 2350
Health Sciences Mall, Vancouver V6T 1Z3,
BC, Canada
Fax: +1 604 822 5227
Tel: +1 604 827 3923
E-mail: cgpr@interchange.ubc.ca
Website: http://www.biochem.ubc.ca/
fac_research/faculty/proud.html
(Received 10 December 2007, revised 22
February 2008, accepted 3 March 2008)
doi:10.1111/j.1742-4658.2008.06372.x
Mammalian target of rapamycin complex 1 (mTORC1) phosphorylates proteins such as eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and the S6 kinases These substrates contain short sequences, termed TOR signalling (TOS) motifs, which interact with the mTORC1 component rap-tor Phosphorylation of 4E-BP1 requires an additional feature, termed the RAIP motif (Arg–Ala–Ile–Pro) We have analysed the interaction of 4E-BP1 with raptor and the amino acid residues required for functional RAIP and TOS motifs, as assessed by raptor binding and the phosphoryla-tion of 4E-BP1 in human cells Binding of 4E-BP1 to raptor strongly 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.
Trang 2Rheb.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
(TOS) motifs found in proteins whose phosphorylation
is positively regulated by mTORC1 [4,5,7,18,19] The
first proteins shown to contain functional TOS motifs
were the ribosomal protein S6 kinases (S6Ks) and the
eukaryotic initiation factor (eIF) 4E-binding proteins
(4E-BPs; Fig 1A), each of which is subject to
rapamy-cin-sensitive phosphorylation at multiple sites The
interaction of these proteins with raptor, via their TOS
motifs, promotes their phosphorylation by mTOR
in vitro Both of these types of protein are implicated
in controlling the translational machinery [20]
mTORC1 also controls other cellular functions,
although the mTORC1 targets involved in these effects
largely remain to be identified [1] Very recently, whilst
our manuscript was in preparation, two further
pro-teins were shown to contain TOS motifs:
hypoxia-inducible factor 1a (HIF1a [21]) and the proline-rich
Akt-substrate 40 kDa (PRAS40 [22–24])
Although the TOS motifs in these proteins resemble
one another, there are a number of differences between
them, and it is not clear what are the real requirements
for a functional TOS motif Defining a ‘consensus’
TOS motif would help to identify such motifs in other
proteins that may be controlled by mTORC1 and
reg-ulate cellular functions in addition to mRNA
transla-tion It is also not clear whether the TOS motif is
sufficient for the interaction with raptor, or whether
other features are also required
It is of particular interest that the in vivo
phosphory-lation of 4E-BP1, the best-understood 4E-BP, requires
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 phosphoryphosphory-lation 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
Similarly, the (sequence) requirements for a func-tional RAIP motif remain to be defined The roles of the RAIP and TOS motifs in the interaction of 4E-BP1 with raptor also remain incompletely under-stood In this article, we address these issues and the requirements for a functional TOS motif We show that several regions of 4E-BP1, including both the TOS and RAIP motifs, plus other features, play roles
in its binding to raptor We also analyse the amino acid sequence requirements for functional TOS and RAIP motifs in 4E-BP1
Results and Discussion
Regions of 4E-BP1 involved in binding to raptor The two known regulatory motifs in 4E-BP1 are located at opposite ends of the polypeptide chain (Fig 1A) We have previously reported that the extreme C-terminus of 4E-BP1 (the final 20 amino acids) can bind raptor in an overlay (far-western) assay, whereas the N-terminal portion cannot [19], sug-gesting that the RAIP motif does not itself bind rap-tor In contrast, another study [31] found that, although wild-type 4E-BP1 could be coimmunoprecipi-tated with raptor, variants with mutations in the TOS, RAIP or both motifs could not This implies a role for the RAIP motif in binding to raptor [It should be noted, however, that neither protocol definitively dem-onstrates that raptor binds directly to any part of 4E-BP1, as raptor is expressed in mammalian cells, and the interaction could be mediated by another (mamma-lian) protein For simplicity, we refer to the binding seen as ‘raptor binding’.] Because of substantial prob-lems of nonspecific binding, we have been unable to
Trang 3successfully 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-dures) The top sections of each panel show
the blots for Myc-tagged raptor; the bottom
sections show the blots with anti-GST to
allow a comparison of the amounts of GST
fusion proteins used in each case Some
degradation of the GST fusion proteins is
evident from the presence of products
run-ning at the position of GST itself (E) Binding
of raptor to different amounts of wild-type
4E-BP1 or the AAAA mutant, assessed
using the overlay (far-western) assay The
graph shows the quantification of the data
from three independent experiments Error
bars indicate the standard deviation
Stu-dent’s t-test (two-sample unequal variance,
two-tailed distribution) was used to
deter-mine the probability that raptor binds
wild-type 4E-BP1 and AAAA mutant equally In
all instances, the P-value was 0.01 or lower
(*0.002; §0.002; ‡0.01; †0.002; #0.00004).
Trang 4that the TOS motif is essential for detectable stable
binding of raptor to 4E-BP1, but does not tell us
whether it is sufficient
To assess the contribution of other regions of
4E-BP1 to raptor binding, we created a series of
N-ter-minally truncated mutants We reasoned that such
truncations cannot perturb the higher order structure
in 4E-BP1, as 4E-BP1 is apparently unstructured in
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
4E-BP1 also contribute to stable binding to raptor
The first 17 residues of 4E-BP1 contain the RAIP
motif To assess whether removal of the RAIP motif
accounts for the reduced binding of raptor to the 18–
117 fragment, we compared the binding of raptor to
this truncated protein and to full-length 4E-BP1 in
which the RAIP motif was altered to AAAA The
phosphorylation of this mutant within cells was
severely impaired ([25]; see also Fig 2A) The binding
of raptor to these two variants was similar (Fig 1D),
implying that the loss of raptor binding on removal of
the first 17 residues may be accounted for simply by
the loss of the RAIP motif We therefore also tested
the binding of raptor to full-length 4E-BP1 and to the
RAIP⁄ AAAA variant A marked and reproducible
decrease was seen for the RAIP⁄ AAAA mutant, when compared with wild-type 4E-BP1 (Fig 1E) The RAIP motif clearly makes a substantial contribution to the binding of 4E-BP1 to raptor However, in contrast with the TOS motif, it is not essential for this interac-tion (compare with the D6 truncainterac-tion in Fig 1B, which displays no binding to raptor) The finding that the RAIP motif is important for the binding of 4E-BP1 to raptor is consistent with earlier observations showing that an intact RAIP motif is required for the efficient
in vitro phosphorylation of the N-terminal threonines
in 4E-BP1 by mTOR raptor [5]
Taken together, these data show the following: (a) that the TOS motif plays a critical role in binding rap-tor; (b) that the region containing the RAIP motif also 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 RAIP motif [25] The use of the rat protein also has the advantage that there is no cross-reactivity of the (P)Ser64 antibody with other sites, which is a compli-cating feature of the human protein (in which this anti-serum recognizes both Ser65 and another site, Ser101 [33]) We have shown previously that the behaviour of
Trang 5the rat and human 4E-BP1 proteins expressed in
HEK293 cells is very similar [33]
To assess the functional consequences of mutations
in the RAIP motif, we studied the phosphorylation of
4E-BP1 in HEK293 cells, focusing on Thr36⁄ 45, as
these sites are involved earlier in the hierarchy of
phosphorylation and depend absolutely on the RAIP
motif [25,26] Clearly, making a full range of
substitu-tions, even within a four-residue motif, would be an
enormous undertaking We therefore created and
tested a set of mutants, selected as described below
Given the diversity of mutants tested, we are unable
to show data for each one relative to all relevant
variants within the same panel in Fig 2; however, each panel contains wild-type 4E-BP1 (‘RAIP’) as a reference
Our earlier data [25] indicated that isoleucine within the RAIP motif (Ile15) plays a particularly important role in the phosphorylation of 4E-BP1 in HEK293 cells [25] This is also clearly seen in the data in Fig 2, where the phosphorylation of the RAAP variant (Fig 2A) is more severely reduced relative to wild-type 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.
Trang 6[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
as the c species
As shown in Fig 2A, the basal phosphorylation of
Thr36⁄ 45 in the Ile15Val (RAVP) variant was identical
to that of the wild-type protein and, likewise, was only
slightly stimulated by insulin In contrast, replacement
of Ile15 by leucine caused a very marked decrease in
basal phosphorylation at Thr36⁄ 45 and impaired the
insulin-stimulated phosphorylation of these sites
Next, we studied the importance of the arginine and
proline residues within the RAIP motif In order to
help us discern the effects of the substitutions more
clearly, we used a 4E-BP1 mutant (AAIP) which
already contained one mutation in the RAIP motif,
the rationale being that using a mutant with a partially
defective RAIP motif would probably enhance any
effects of other mutations Thus, we tested the
impor-tance of the proline residue in a variant of 4E-BP1 in
which the arginine was mutated to alanine (AAIP,
which shows modestly decreased basal and
insulin-stimulated phosphorylation relative to wild-type
4E-BP1; Fig 2A,D) Proline is an imino, not an
amino, acid: arguably the most closely related amino
acid is valine Although the AAIP mutant showed
sub-stantial basal phosphorylation at Thr36⁄ 45 (which was
increased somewhat by insulin; Fig 2A,D), the AAIV
mutant did not undergo any detectable
phosphoryla-tion at Thr36⁄ 45 under basal or insulin-stimulated
conditions (Fig 2B,D) As even the relatively
conser-vative replacement of proline by valine almost
com-pletely abolished the phosphorylation of 4E-BP1
(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-fied, such deficits are often more apparent for basal than for insulin-induced phosphorylation One possible explanation is that, when the function of the RAIP motif is impaired, the phosphorylation of Thr36⁄ 45 becomes more dependent on the rapamycin-sensitive input provided by the TOS motif
Lastly, we tested the effect of selected mutations of the alanine residue in the RAIP motif Mutation to valine markedly reduced the basal phosphorylation of 4E-BP1 and slightly impaired the effect of insulin (Fig 2B), whereas replacement by a negatively charged residue, aspartate, had no effect on basal phosphoryla-tion (Fig 2B)
Overall, these data indicate that isoleucine is the most important single residue within the RAIP motif This is in accordance with our earlier data [25]: the present findings extend those observations by demon-strating that replacing this residue with valine, but not leucine, permits retention of RAIP motif function
Defining what constitutes a functional TOS motif The data presented above (Fig 1B) further confirm the key role played by the TOS motif in the binding of 4E-BP1 to raptor in the far-western analysis employed here Two further proteins were described as contain-ing TOS motifs whilst this paper was in the final stages
of preparation (HIF1a [21] and PRAS40 [22–24]; Table 1) However, so far, no detailed analysis has been performed to define which residues are actually required for a functional TOS motif: such data would
be helpful in identifying potential TOS motifs in other
Trang 7proteins 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-Ala4-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,
and hence ‘integrates’ the effects of phosphorylation of
other sites in 4E-BP1 In HEK293 cells,
phosphoryla-tion at this site is stimulated by insulin [19,29], and this
is entirely dependent on the TOS motif [19,25]
(Fig 4A) The level of phosphorylation of Ser64 in
insulin-treated cells is therefore especially informative
We have shown previously that mutation of Phe113
to alanine in the 4E-BP1 TOS motif markedly impairs
the phosphorylation of Ser64 [19] The present data also showed that this mutation (which yields the AE-MDI mutant) completely blocks the ability of 4E-BP1
to bind raptor in a far-western blot (Fig 3A) and almost eliminates the phosphorylation of 4E-BP1 at Ser64 [19] (Fig 4A,B) As reported previously [19], this mutation can decrease the basal level of phosphoryla-tion of the N-terminal threonines in 4E-BP1 in HEK293 cells This mutation also impairs the in vitro phosphorylation of Thr36⁄ 45 by mTOR [5] The phen-ylalanine to alanine change is clearly major, and we therefore tested whether the more conservative muta-tion of the aromatic phenylalanine to a bulky aliphatic residue (leucine) also affected function The LEMDI mutant underwent insulin-stimulated phosphorylation
at Ser64 to a similar degree to the wild-type protein (Fig 4C): in this and all other cases, this phosphoryla-tion was blocked by rapamycin, confirming that it requires mTORC1 However, the LEMDI variant failed to bind raptor in the far-western assay (Fig 3B) The simplest explanation for this is that the mutation weakens the TOS–raptor interaction to the extent that
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-ally appear to be very important for the regulation of
Table 1 Known potential TOS motifs in selected proteins (italics
indicate putative TOS motifs; the other TOS motifs have been
shown to function in their respective proteins).
Protein (all
Residue numbers
FDMDL
26–30 756–760
a Numbering is based on the shorter splice variants of these
proteins.
Trang 84E-BP1 or for raptor binding Interestingly, the
TOS-like motifs in PRAS40 and HIF1a each lack an acidic
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-GST (but do not bind raptor) (D) The binding of bacterially expressed native wild-type anti-GST-4E-BP1 or variants to raptor was tested using a dot blot as described in Experimental procedures.
Trang 9levels (Fig 4E) It seems probable that the presence of an
aliphatic residue with a side-chain larger than a methyl
group is required for function This is in accordance
with the sequences of known TOS motifs (Table 1), which have methionine (4E-BPs; PRAS40; HIF1a), isoleucine (S6K1) or leucine (S6K2) at this position
A
Fig 4 Phosphorylation of 4E-BP1 variants expressed in HEK293 cells (A–I) Wild-type Myc-tagged 4E-BP1 or variants of 4E-BP1 were expressed in HEK293 cells Twenty-four hours following transfection, the cells were starved of serum for 16 h and, in some instances, sub-sequently treated with 100 n M insulin for 25 min Where indicated, cells were also incubated with 100 n M rapamycin for 30 min prior to insu-lin stimulation (see Experimental procedures for details) Samples were analysed using the indicated phosphospecific antibodies for 4E-BP1 (top sections of each panel) or anti-Myc (bottom section in each panel; to assess the expression levels of 4E-BP1 variants) The differentially phosphorylated a–c species are indicated.
Trang 10The fourth residue in the 4E-BP1 TOS motif is
acidic: aspartate This was mutated to alanine
(Fig 4H), asparagine (Fig 4H) or arginine (Fig 4I)
The substitution by alanine very substantially
decreased the phosphorylation at Ser64, but the
phos-phorylation of the FEMRI variant was similar to that
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 isoleuleu-cine 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
(e.g the FEMRI and FEMDR variants) It is
possi-ble that the use of denatured 4E-BP1 in the
far-wes-tern assay led to misleading results (although this
does not seem likely, as 4E-BP1 reportedly has little
if any folded structure [32]) Therefore, we also
performed dot blot overlay assays in which GST–
4E-BP1 was applied to the membrane without prior
denaturation on an SDS-polyacrylamide gel This
yielded very similar results to the far-western
analy-ses, i.e all of the variants that were negative in that
assay (including the two just mentioned) were also
negative in the dot blot assay, whereas wild-type
4E-BP1 and FAMDI variants bound raptor in both
assays (Fig 3C,D) It should be noted that, although
the other 4E-BP1 variants appear to interact weakly
with raptor in the ‘dot blot far-western’ assay
(Fig 3D), they do so to an identical extent to GST
itself, indicating that this residual binding is nonspecific
Analysis of TOS-like motifs from other proteins reported to be controlled by mTOR signalling
A number of other proteins have been reported to be regulated in a rapamycin-sensitive manner The phos-phorylation of STAT3 has been reported to be con-trolled by mTOR [35,36], as has the phosphorylation
of the atypical protein kinase C isoforms (PKCd⁄ e) [37,38] As shown in Table 1, there are two putative TOS motifs in STAT3, i.e FDMDL and (with less similarity to the known TOS motifs) FPMEL PKCd (one of the forms studied by Parekh et al [37,38]) has 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) (Fig 5B,C) The exception, surprisingly, in view of its good ability to bind raptor (Fig 5A), was the FVMEF motif (from PKCd) Conversely, although the FVMEY variant bound poorly to raptor (Fig 5A), it became quite strongly phosphorylated in response to insulin (Fig 5B) As already observed for other variants tested here, there is imperfect correspondence between raptor binding (in the far-western assay) and function in