Analysis of the regulatory motifs in eukaryotic initiation factor 4E-binding protein Vivian H Y Lee1, Timothy Healy1, Bruno D Fonseca1, Amanda Hayashi2 and Christopher G Proud1 Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada Institute of Food Nutrition and Human Health, Massey University and Food, Metabolism and Microbiology, AgResearch Limited, Palmerston North, New Zealand 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 March 2008) doi:10.1111/j.1742-4658.2008.06372.x Mammalian target of rapamycin complex (mTORC1) phosphorylates proteins such as eukaryotic initiation factor 4E-binding protein (4E-BP1) and the S6 kinases These substrates contain short sequences, termed TOR signalling (TOS) motifs, which interact with the mTORC1 component raptor 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 phosphorylation 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 Mutational 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 Signalling through the mammalian target of rapamycin complex (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 rapamycin, 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 protein kinase domain related to lipid kinases, and raptor, a scaffold protein that interacts with proteins that are phosphorylated by mTOR [3–8] mTORC1 also comprises 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 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 Regulatory motifs in 4E-BP1 V H Y Lee et al Rheb.GTP to its inactive GDP-bound form For example, 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 GTPbound form and to switch on mTORC1 Recent data have suggested that RhebỈGTP 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 rapamycin-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 proteins were shown to contain TOS motifs: hypoxiainducible 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 regulate cellular functions in addition to mRNA translation 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 phosphorylation 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 phosphorylation 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 phosphorylation 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 phosphorylation 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 2186 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 phosphorylation 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 phosphorylation of the N-terminal threonines in both 4E-BP1 and 4E-BP2 (which is not very prone to inhibition by rapamycin) In contrast, the TOS motif is required for the insulin-induced phosphorylation of Ser65 (and, in some cell types, Thr70) Phosphorylation of Ser65 is generally completely blocked by rapamycin 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 functional 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 understood 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], suggesting that the RAIP motif does not itself bind raptor In contrast, another study [31] found that, although wild-type 4E-BP1 could be coimmunoprecipitated 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 demonstrates 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 (mammalian) protein For simplicity, we refer to the binding seen as ‘raptor binding’.] Because of substantial problems of nonspecific binding, we have been unable to FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al Regulatory motifs in 4E-BP1 A C B D Fig 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 (farwestern) assay (see Experimental procedures) 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 running 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 Student’s t-test (two-sample unequal variance, two-tailed distribution) was used to determine the probability that raptor binds wildtype 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) E 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 phenylalanine 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 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2187 Regulatory motifs in 4E-BP1 V H Y Lee et al that 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-terminally 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 ‘farwestern’ 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 fulllength 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 example, 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 (outside 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-terminus 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 2188 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 interaction (compare with the D6 truncation 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 raptor; (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 possible 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 complicating feature of the human protein (in which this antiserum recognizes both Ser65 and another site, Ser101 [33]) We have shown previously that the behaviour of FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al Regulatory motifs in 4E-BP1 A B C D Fig 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 nM insulin for 25 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 the 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 substitutions, 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 especially true for the basal phosphorylation at Thr36 ⁄ 45, which is maintained by the amino acids in the medium FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2189 Regulatory motifs in 4E-BP1 V H Y Lee et al [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 (notably 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 importance 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 insulinstimulated 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 substantial basal phosphorylation at Thr36 ⁄ 45 (which was increased somewhat by insulin; Fig 2A,D), the AAIV mutant did not undergo any detectable phosphorylation at Thr36 ⁄ 45 under basal or insulin-stimulated conditions (Fig 2B,D) As even the relatively conservative replacement of proline by valine almost completely 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 proline to alanine (to give the RAIA mutant) causes a defect in the basal and insulin-stimulated phosphorylation of 4E-BP1 [25] We also tested the proline to valine mutation in wild-type 4E-BP1 The phosphorylation of the resulting RAIV mutant was more severely impaired than that of the RAIA variant (Fig 2D) 2190 We then turned our attention to the arginine residue within the RAIP motif, making mutations at this position within the RAIA variant, which already shows a reduction in basal and insulin-stimulated phosphorylation 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 important than Pro16 for the function of the RAIP motif, and that several different types of residue can be tolerated here with only small, if any, effects on 4E-BP1 phosphorylation For reasons that remain to be clarified, 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 phosphorylation (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 demonstrating 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 containing 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 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al Regulatory motifs in 4E-BP1 Table 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 Homo sapiens) S6K1 S6K2 4E-BP1 4E-BP2 4E-BP3 HIF1a PRAS40 PKCd PKCe STAT3 Sequence Residue numbers FDIDL FDLDL FEMDI FEMDI FEMDI FVMVL FVMDE FVMEF FVMEY FPMEL FDMDL 5–9a 5–9a 114–118 116–120 86–90 99–103 129–133 425–429 484–488 26–30 756–760 a Numbering is based on the shorter splice variants of these proteins proteins Here, we employed two approaches to study this: (a) the ability of 4E-BP1 variants to bind to raptor; 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 performed 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 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 altering the TOS motif, we mainly examined the phosphorylation 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, phosphorylation 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 AEMDI 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 phosphorylation 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 phenylalanine to alanine change is clearly major, and we therefore tested whether the more conservative mutation 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 phosphorylation 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 interaction 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 binding (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 discernibly 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 phosphorylation of 4E-BP1 when compared with the wildtype 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 actually appear to be very important for the regulation of FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2191 Regulatory motifs in 4E-BP1 V H Y Lee et al A B C D Fig Binding of raptor to the TOS motif in wild-type 4E-BP1 (FEMDI) and variants thereof (A) An overlay assay (see Experimental procedures) 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 alanine spacer (Ala4) 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 controls 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 antiGST (but not bind raptor) (D) The binding of bacterially expressed native wild-type GST-4E-BP1 or variants to raptor was tested using a dot blot as described in Experimental procedures 4E-BP1 or for raptor binding Interestingly, the TOSlike 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 residue in promoting the phosphorylation of 4E-BP1 at Ser64 (Fig 4D) In contrast with the tolerance for variations in the second position, mutation of the third residue (methionine: 2192 an uncharged, relatively nonpolar amino acid) to alanine 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 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al Regulatory motifs in 4E-BP1 A B C D E F G H I Fig 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, subsequently treated with 100 nM insulin for 25 Where indicated, cells were also incubated with 100 nM rapamycin for 30 prior to insulin 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 levels (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 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2193 Regulatory motifs in 4E-BP1 V H Y Lee et al The 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 phosphorylation 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 similarly has a glutamate at this position, other residues are tolerated, even if, as for arginine, they carry a positive charge The latest reported TOS motif, in HIF1a, has an aliphatic, nonpolar residue in the fourth position (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 leucine 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 (e.g the FEMRI and FEMDR variants) It is possible that the use of denatured 4E-BP1 in the far-western 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 analyses, 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 so to an identical extent to GST 2194 itself, indicating nonspecific that this residual binding is 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 phosphorylation of STAT3 has been reported to be controlled 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 isoform, 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 introduce 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 phosphorylation of 4E-BP1) As shown in Fig 5A, in the far-western assay, all of these variant 4E-BP1 proteins, except one, bound raptor 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 wildtype 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 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al A Regulatory motifs in 4E-BP1 promoting phosphorylation of 4E-BP1 within cells The latter approach is probably more informative in terms of motif function Conclusions B C Fig Ability of (putative) TOS motifs from other proteins to support the binding of 4E-BP1 to raptor or its phosphorylation at Ser64 (A) Wild-type 4E-BP1 or variants containing the indicated (putative) TOS motifs from other proteins (as noted) were expressed as GST fusions in Escherichia coli Binding to raptor was assessed using the overlay assay (top section) The bottom section shows the amount of each protein used, as assessed by western blot with anti-GST Arrowheads with asterisks denote degradation products (cleaved at the C-terminus) that react with anti-GST (but not bind raptor) GST and GST-tagged wild-type 4E-BP1 served as negative and positive controls, respectively (B, C) Wild-type Myc-tagged 4E-BP1 or the indicated variants were expressed in HEK293 cells Twenty-four hours following transfection, the cells were starved of serum for 16 h and then treated with 100 nM insulin for 25 min, where indicated (see Experimental procedures for details) Samples were analysed with the indicated phosphospecific antibody for Ser64 (top section of each panel) or anti-Myc (bottom section of each panel; to assess the expression levels of 4E-BP1 variants) The differentially phosphorylated a–c species are indicated Only the slowest migrating species is phosphorylated at Ser64 This study represents the first systematic attempt to define the sequence requirements of the TOS and RAIP motifs It provides new information on the features of 4E-BP1 that are required for its interaction with raptor and ⁄ or for its phosphorylation at different sites in living cells Firstly, our findings demonstrate that, although the TOS motif is essential for interaction with raptor and for phosphorylation of specific sites in cells, other features of 4E-BP1 are necessary for this interaction Indeed, the five-amino-acid TOS motif is not sufficient to confer binding to raptor Our findings show that the RAIP motif also plays a role in binding raptor (although it is not essential for this), and that other regions of the 4E-BP1 polypeptide are involved in this interaction (especially parts of the C-terminal half of the molecule) The present data help 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 consistent with the recent observation that short interfering 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 raptor in the far-western assay were able to support phosphorylation of 4E-BP1 within cells Although mutants that bind raptor in vitro effectively support phosphorylation within cells, the converse is not true: for example, the LEMDI mutant does not bind raptor but is as effective as the wild-type sequence at facilitating phosphorylation (Figs 3B and 4C) Studying the phosphorylation 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: mutation 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 residues here, a range of other residues support the phosphorylation of 4E-BP1 in cells The diversity of these FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2195 Regulatory motifs in 4E-BP1 V H Y Lee et al ‘functional’ residues (valine, alanine, proline) indicates that a range of side-chains are tolerated here Interestingly, 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 residue (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 carboxyl groups, whereas, in PRAS40, this is not the C-terminal residue and thus carries only one negative charge Our data thus suggest that the nature of the first and third residues in the motif is particularly important, at least for the phosphorylation of 4E-BP1: hydrophobic residues are required at both positions (phenylalanine or leucine in the first position, and methionine, isoleucine or probably similar residues in the third) The requirements at the other positions are less strict: in the second and fourth positions, a variety of residues seem to be tolerated; based on our data and the recent information from newly discovered TOS motifs, there is no strict need for an acidic residue (as found in the ‘original’ TOS motifs) Although a positively charged residue seems to be detrimental at position two (FRMDI; Fig 4F), this is not so at position four (FEMRI; Fig 4I) The situation for the final residue is less clear, as an acidic residue is detrimental in 4E-BP1, but functions in PRAS40 (as noted already) [22–24] Mutational analysis within the RAIP motif demonstrates that the isoleucine residue is the most important: 2196 even its replacement by a very closely related amino acid, leucine, substantially impairs the intracellular phosphorylation of 4E-BP1 Proline also plays an important role: its replacement by the most similar amino acid (valine) abolishes phosphorylation (although alanine is tolerated slightly better) The substitution of alanine by other residues with relatively small sidechains (valine or aspartate) markedly impairs phosphorylation In contrast, a number of different residues can be tolerated at the first position: in particular, methionine 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] provides an efficiently functional motif There are currently 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 further refine them Interestingly, although 4E-BP3 contains 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 phosphorylation (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 probably make it difficult to identify proteins containing such motifs by computational methods, such as blast searches, alone Functional approaches, such as that recently described by Oshiro et al [23] (which identified PRAS40 as a target for mTORC1), may be much more useful for this The present data will nonetheless be valuable in identifying potential TOS and RAIP motifs in proteins linked to mTORC1 signalling Experimental procedures Chemicals and other reagents General laboratory chemicals were obtained from SigmaAldrich (Oakville, Canada), Fisher Scientific (Ottawa, FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al Canada) or EMD Chemicals ⁄ Calbiochem (La Jolla, CA, USA) Rapamycin and recombinant human insulin were purchased from Calbiochem and Sigma-Aldrich, respectively Protein G-Sepharose and glutathione-Sepharose 4B were purchased from Amersham (GE Healthcare, Piscataway, NJ, USA) Tissue culture reagents were purchased from Invitrogen (Burlington, Canada) Vectors, cloning and site-directed mutagenesis pRK5 Myc-tagged human raptor was a generous gift from D Sabatini (Massachusetts Institute of Technology, Boston, MA, USA) pcDNA3.1 Myc ⁄ His-tagged rat 4E-BP1 for mammalian expression and the pGEX-3X human 4E-BP1 for bacterial expression have been described previously [19,25] N-terminally truncated human 4E-BP1 mutants were generated by PCR amplification using the forward primers (5¢-CGGGATCCCCCCAGGGGTCACTAGCCC TAC-3¢; 5¢-CGGGATCCCCCTGATGGAGTGTCGGA 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) QuikchangeÒ system, according to the manufacturer’s guidelines Regulatory motifs in 4E-BP1 cleared by centrifugation at 13 000 g for 10 at °C Typically, 20 lg of total protein lysate was used for analysis by SDS-PAGE ⁄ western blotting Myc immunoprecipitates were prepared by incubating mg of total lysate with antiMyc and 50 lL of protein G-Sepharose 50% (w ⁄ v) slurry for h at °C Expression and purification of GST fusion proteins in Escherichia coli GST-tagged versions of wild-type 4E-BP1 and various mutants were expressed in E coli and purified on glutathione-Sepharose 4B as described previously [40] SDS-PAGE and western blotting SDS-PAGE and western blotting were carried out as described previously [19,33], with the modification that, for all experiments using 4E-BP1, proteins were cross-linked to the poly(vinylidene difluoride) (PVDF) membrane using 0.2% (v ⁄ v) glutaraldehyde in NaCl ⁄ Pi containing 0.02% (v ⁄ v) Tween-20 Cross-linking was carried out (after transfer but prior to blocking) for 30 at room temperature with constant agitation Blots were visualized by enhanced chemiluminescence (ECL) Far-western analysis of raptor binding 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, immunoprecipitations and related procedures HEK293 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% (v ⁄ v) fetal bovine serum, mm l-glutamine, 100 lgỈmL)1 streptomycin sulfate and 100 mL)1 penicillin G Transient transfections 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 indicated, cells were also treated with 100 nm rapamycin for 30 min, followed by stimulation with 100 nm insulin for 25 Cells were lysed in 400 lL of extraction buffer containing 50 mm b-glycerophosphate (pH 7.5), mm EGTA, mm EDTA, 1% (v ⁄ v) Triton X-100, mm Na3VO4, 0.1% (v ⁄ v) b-mercaptoethanol, protease inhibitors (leupeptin, pepstatin and antipain, each lgỈmL)1) and phenylmethylsulfonyl fluoride (200 lm) Lysates were pre- 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 bacterially expressed, GST-tagged, wild-type 4E-BP1 (or 4E-BP1 mutants) on nitrocellulose membrane (0.45 lm) from BioRad Laboratories (Hercules, CA, USA) The membranes were blocked with 5% (w ⁄ v) fat-free milk in NaCl ⁄ Pi– Tween-20 for 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 the University of Dundee (School of Life Sciences) Alumni Fund FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2197 Regulatory motifs in 4E-BP1 V H Y Lee et al References Wullschleger S, Loewith R & Hall MN (2006) TOR signaling in growth and metabolism Cell 124, 471– 484 Sabatini DM (2006) mTOR and cancer: insights into a complex relationship Nat Rev Cancer 6, 729–734 Kim DH, Sarbassov D, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P & Sabatini DM (2002) mTOR interacts with raptor to form a nutrientsensitive complex that signals to the cell growth machinery Cell 110, 163–175 Hara K, Maruki Y, Long X, Yoshino K, Oshiro N, Hidayat S, Tokunaga C, Avruch J & Yonezawa K (2002) Raptor, a binding partner of target of rapamycin (TOR), mediates TOR action Cell 110, 177–189 Choi K-M, McMahon LP & Lawrence JC (2003) Two motifs in the translational repressor PHAS-I required for efficient phosphorylation by mammalian target of rapamycin and for recognition by raptor J Biol Chem 278, 19667–19673 Nojima H, Tokunaga C, Eguchi S, Oshiro N, Hidayat S, Yoshino K, Hara K, Tanaka J, Avruch J & Yonezawa K (2003) The mTOR partner, raptor, binds the mTOR substrates, p70 S6 kinase and 4E-BP1, through their TOS (TOR signaling) motifs J Biol Chem 278, 15461– 15464 Schalm SS, Fingar DC, Sabatini DM & Blenis J (2003) TOS motif-mediated raptor binding regulates 4E-BP1 multisite phosphorylation and function Curr Biol 13, 797–806 Kim DH & Sabatini DM (2004) Raptor and mTOR: subunits of a nutrient-sensitive complex Curr Top Microbiol Immunol 279, 259–270 Manning BD & Cantley LC (2003) Rheb fills a GAP between TSC and TOR Trends Biochem Sci 28, 573– 576 10 Long X, Lin Y, Ortiz-Vega S, Yonezawa K & Avruch J (2005) Rheb binds and regulates the mTOR kinase Curr Biol 15, 702–713 11 McManus EJ & Alessi DR (2002) TSC1–TSC2: a complex tale of PKB-mediated S6K regulation Nat Cell Biol 4, E214–E216 12 Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC & Blenis J (2002) Tuberous sclerosis complex-1 and -2 gene products function together to inhibit mammalian target of rapamycin (mTOR)-mediated downstream signaling Proc Natl Acad Sci USA 99, 13571–13576 13 Inoki K, Li Y, Xu T & Guan KL (2003) Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling Genes Dev 17, 1829–1834 14 Tee AR, Anjum R & Blenis J (2003) Inactivation of the tuberous sclerosis complex-1 and -2 gene products occurs by phosphoinositide 3-kinase ⁄ Akt-dependent 2198 15 16 17 18 19 20 21 22 23 24 25 26 27 and -independent phosphorylation of tuberin J Biol Chem 278, 37288–37296 Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA & Pan D (2003) Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins Nat Cell Biol 5, 578–581 Cai SL, Tee AR, Short JD, Bergeron JM, Kim J, Shen J, Guo R, Johnson CL, Kiguchi K & Walker CL (2006) Activity of TSC2 is inhibited by AKT-mediated phosphorylation and membrane partitioning J Cell Biol 173, 279–289 Bai X, Ma D, Liu A, Shen X, Wang QJ, Liu Y & Jiang Y (2007) Rheb activates mTOR by antagonizing its endogenous inhibitor, FKBP38 Science 318, 977–980 Schalm SS & Blenis J (2002) Identification of a conserved motif required for mTOR signaling Curr Biol 12, 632–639 Beugnet A, Wang X & Proud CG (2003) The TOR-signaling and RAIP motifs play distinct roles in the mTOR-dependent phosphorylation of initiation factor 4E-binding protein in vivo J Biol Chem 278, 40717–40722 Wang X & Proud CG (2006) The mTOR pathway in the control of protein synthesis Physiology (Bethesda) 21, 362–369 Land SC & Tee AR (2007) Hypoxia inducible factor 1alpha is regulated by the mammalian target of rapamycin (mTOR) via an mTOR-signalling motif J Biol Chem 282, 20534–20543 Wang L, Harris TE, Roth RA & Lawrence JC (2007) PRAS40 regulates mTORC1 kinase activity by functioning as a direct inhibitor of substrate binding J Biol Chem 282, 20036–20044 Oshiro N, Takahashi R, Yoshino KI, Tanimura K, Nakashima A, Eguchi S, Miyamoto T, Hara K, Takehana K, Avruch J et al (2007) The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mTOR complex J Biol Chem 282, 20329–20339 Fonseca BD, Smith EM, Lee VH, MacKintosh C & Proud CG (2007) PRAS40 is a target for mammalian target of rapamycin complex and is required for signaling downstream of this complex J Biol Chem 282, 24514–24524 Tee AR & Proud CG (2002) Caspase cleavage of initiation factor 4E-binding protein yields a dominant inhibitor of cap-dependent translation and reveals a novel regulatory motif Mol Cell Biol 22, 1674–1683 Gingras A-C, Gygi SP, Raught B, Polakiewicz RD, Abraham RT, Hoekstra MF, Aebersold R & Sonenberg N (1999) Regulation of 4E-BP1 phosphorylation: a novel two-step mechanism Genes Dev 13, 1422–1437 Mothe-Satney I, Yang D, Fadden P, Haystead TAJ & Lawrence JC (2000) Multiple mechanisms control phosphorylation of PHAS-I in five (S ⁄ T)P sites that govern translational repression Mol Cell Biol 20, 3558–3567 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS V H Y Lee et al 28 Mothe-Satney I, Brunn GJ, McMahon LP, Capaldo CT, Abraham RT & Lawrence JC (2000) Mammalian target of rapamycin-dependent phosphorylation of PHAS-1 in four (S ⁄ T)P sites detected by phospho-specific antibodies J Biol Chem 275, 33836– 33843 29 Wang X, Beugnet A, Murakami M, Yamanaka S & Proud CG (2005) Distinct signaling events downstream of mTOR cooperate to mediate the effects of amino acids and insulin on initiation factor 4E-binding proteins Mol Cell Biol 25, 2558–2572 30 Mader S, Lee H, Pause A & Sonenberg N (1995) The translation initiation factor eIF-4E binds to a common motif shared by the translation factor eIF-4gamma and the translational repressors 4E-binding proteins Mol Cell Biol 15, 4990–4997 31 Eguchi S, Tokunaga C, Hidayat S, Oshiro N, Yoshino K, Kikkawa U & Yonezawa K (2006) Different roles for the TOS and RAIP motifs of the translational regulator protein 4E-BP1 in the association with raptor and phosphorylation by mTOR in the regulation of cell size Genes Cells 11, 757–766 32 Fletcher CM, McGuire AM, Gingras A-C, Li H, Matsuo H, Sonenberg N & Wagner G (1998) 4E binding proteins inhibit the translation factor eIF4E without folded structure Biochemistry 37, 9–15 33 Wang X, Li W, Parra J-L, Beugnet A & Proud CG (2003) The C-terminus of initiation factor 4E-binding protein contains multiple regulatory features that influence its function and phosphorylation Mol Cell Biol 23, 1546–1557 Regulatory motifs in 4E-BP1 34 Gingras A-C, Raught B, Gygi SP, Niedzwieka A, Miron M, Burley SK, Polakiewicz RD, Wyslouch-Cieczyska A, Aebersold R & Sonenberg N (2001) Hierarchical phosphorylation of the translation inhibitor 4E-BP1 Genes Dev 15, 2852–2864 35 Yokogami K, Wakisaka S, Avruch J & Reeves SA (2000) Serine phosphorylation and maximal activation of STAT3 during CNTF signaling is mediated by the rapamycin target mTOR Curr Biol 10, 47–50 36 Fang P, Hwa V & Rosenfeld RG (2006) Interferongamma-induced dephosphorylation of STAT3 and apoptosis are dependent on the mTOR pathway Exp Cell Res 312, 1229–1239 37 Le Good JA, Ziegler WH, Parekh DB, Alessi DR, Cohen P & Parker PJ (1998) Protein kinase C isotypes controlled by phosphoinositide 3-kinase through the protein kinase PDK1 Science 281, 2042–2045 38 Parekh D, Ziegler W, Yonezawa K, Hara K & Parker PJ (1999) Mammalian TOR controls one of two kinase pathways acting upon PKCdelta and epsilon J Biol Chem 274, 34758–34764 39 Scheper GC, Parra J-L, Wilson ML, van Kollenburg B, Vertegaal ACO, Han Z-G & Proud CG (2003) The N and C termini of the splice variants of the human mitogen-activated protein kinase-interacting kinase Mnk2 determine activity and localization Mol Cell Biol 23, 5692–5705 40 Waskiewicz AJ, Flynn A, Proud CG & Cooper JA (1997) Mitogen-activated kinases activate the serine ⁄ threonine kinases Mnk1 and Mnk2 EMBO J 16, 1909–1920 FEBS Journal 275 (2008) 2185–2199 ª 2008 The Authors Journal compilation ª 2008 FEBS 2199 ... 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;... 4E-BP1 (1? ? ?11 7) 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? ?? 11 7)... define further the features in the C-terminus 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