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Genes to Cells (2006) 11, 757-766. doi:10.1111/j.1365-2443.2006.00977.x
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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

Satoshi Eguchi1,2, Chiharu Tokunaga1,2, Sujuti Hidayat1,a, Noriko Oshiro1,2, Ken-ichi Yoshino1,2, Ushio Kikkawa1,2,* and Kazuyoshi Yonezawa1,2,b

1 Biosignal Research Center, Kobe University, Kobe 657-8501, Japan
2 CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Experimental procedures
 References
 
The translational regulator protein 4E-BP1, that binds to eukaryotic initiation factor-4E (eIF4E) to prevent the formation of the active translation complex, dissociates from eIF4E by phosphorylation through the mammalian target of rapamycin (mTOR) in the cells stimulated by amino acids. 4E-BP1 has been shown to associate with the scaffold protein raptor through its TOS and RAIP motifs to be recognized by mTOR. We revealed that the TOS motif mutant was phosphorylated by mTOR only at the priming sites of Thr37/46 but the RAIP motif mutant was phosphorylated not only at the priming sites but also at the subsequent site of Thr70 in vitro and in response to amino acid treatment in HEK293 cells. Analysis using the phosphorylation site mutants indicated that phosphorylation of the priming and subsequent sites of 4E-BP1 was required for dissociation from raptor as well as for the release of eIF4E. The expression of the 4E-BP1 mutants replacing the TOS motif and phosphorylation sites, that are poor substrates for mTOR and have no or little dissociation ability from raptor and eIF4E, respectively, significantly reduced the size of K562 cells. These results indicate that the the TOS motif has an essential function whereas the RAIP motif has an accessory role in the association with raptor and mTOR-mediated phosphorylation of 4E-BP1 to dissociate it from raptor and release eIF4E in response to amino acid stimulation leading to the control of cell size.


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Experimental procedures
 References
 
It is well known that the mammalian target of rapamycin (mTOR) integrates the signals of growth factors and nutrients to regulate cell growth through the control of mRNA translation (Schmelzle & Hall 2000). TOR proteins are evolutionary conserved serine/threonine protein kinases that were first identified in Saccharomyces cerevisiae through the analysis of the mutants that conferred resistance to growth inhibition induced by rapamycin (Kunz et al. 1993), and yeast TOR1 and TOR2 control translational initiation and early G1 progression in response to nutrient availability (Barbet et al. 1996; Di Como & Arndt 1996). In mammalian cells, mTOR catalyzes the phosphorylation of eukaryotic initiation factor 4E binding protein 1 (4E-BP1) and p70 S6 kinase (p70S6k) under the control of insulin and amino acids and rapamycin blocks the phosphorylation of these translational regulators (Hara et al. 1998; Jacinto & Hall 2003). Genetic studies of Drosophila melanogaster revealed that loss of function mutants such as insulin receptor (dinr), insulin receptor substrate-1 (dIRS-1), phosphatidylinositol 3-kinase (dPI3k) and Akt protein kinase (dAkt) in the growth factor-signaling pathway decrease cell number and cell size (Jacinto & Hall 2003), whereas p70S6k mutant (dS6k) in the downstream of TOR does not lower cell number but reduces cell and body size (Montagne et al. 1999). Inhibition of the mTOR signaling pathway by rapamycin results in cell size reduction in U2O2 osteosarcoma cells (Schalm et al. 2003) and HEK293T cells (Kim et al. 2002) and the conditional knockout of mTOR decreases cell size in mouse ES cells (Murakami et al. 2004). These results indicate that the TOR signaling contributes specifically to the cell size control.

Recently, the regulatory associated protein of mTOR (raptor) has been identified from various species (Hara et al. 2002; Kim et al. 2002). Raptor serves as an indispensable scaffold protein for the mTOR-catalyzed phosphorylation of its downstream target proteins: raptor binds to 4E-BP1 and p70S6k, and the association of raptor with mTOR is required for the mTOR-catalyzed phosphorylation of these proteins (Beugnet et al. 2003; Choi et al. 2003; Nojima et al. 2003; Schalm et al. 2003). Application of rapamycin causes dissociation of raptor from mTOR in vivo, and the treatment of the mTOR/raptor complex with detergent in vitro separates them to prevent the mTOR-mediated phosphorylation reaction (Hara et al. 2002; Oshiro et al. 2004). Consistently, short interfering RNA of raptor induces cell size reduction mimicking the phenotype of the mTOR knockdown in HEK293T cells (Kim et al. 2002).

It is known that the translational regulator 4E-BP1 binds to a 7-methyl-guanosine mRNA cap binding protein named eukaryotic initiation factor-4E (eIF4E) to prevent the formation of the active translation complex of this initiation factor with eIF4G and that phosphorylation of 4E-BP1 predominantly by mTOR induces the dissociation of eIF4E from 4E-BP1 (Beretta et al. 1996; Proud 2002). Five phosphorylation sites have been identified in 4E-BP1: Thr37, Thr46, Ser65, Thr70 and Ser83 (Fadden et al. 1997). mTOR phosphorylates 4E-BP1 in an ordered manner at these multiple sites in the cells stimulated with growth factors: initially mTOR enhances the phosphorylation of Thr37 and Thr46 as the priming sites for the subsequent phosphorylation, then phosphorylates Thr70 and lastly Ser65 (Gingras et al. 2001). A basal level of phosphorylation of Thr37 and Thr46 is, however, observed in serum starved cells (Gingras et al. 2001; Proud 2002). Phosphorylation of Ser83 has been reported to be independent of the release of eIF4E (Yang et al. 1999), and it is unknown which protein kinase recognizes Ser83. Therefore, the precise role of the phosphorylation of this residue is not clear. 4E-BP1 thus appears as three distinct bands according to the order of the mobility on SDS-PAGE, that roughly correspond to the protein phosphorylated at Thr37/46 alone, further phosphorylated at Thr70 and then at Ser65 (Gingras et al. 2001). It is well established that rapamycin blocks 4E-BP1 phosphorylation and the cap-dependent translation (Beretta et al. 1996). The combination of the phosphorylation reaction on these residues is considered necessary for 4E-BP1 to dissociate from eIF4E (Gingras et al. 2001) and the expression of the mutant replacing Thr37 and Thr46 by Ala in U2O2 osteosarcoma cells reduces their size (Schalm et al. 2003).

On the other hand, a short amino acid segment, the TOR signaling motif (TOS motif), is identified in 4E-BP1 and p70S6k, through which these TOR substrates bind to raptor. The TOS motif was first found as a five amino acid sequence required for phosphorylation by mTOR in these two translational regulators (Schalm & Blenis 2002) and this motif was then revealed to mediate the binding of 4E-BP1 to raptor for the multisite phosphorylation by the mTOR/raptor complex (Nojima et al. 2003). The human 4E-BP1 mutant F114A and its rodent version F113A have been employed to show the essential function of the TOS motif (Schalm & Blenis 2002; Beugnet et al. 2003; Choi et al. 2003; Nojima et al. 2003; Schalm et al. 2003). It has been shown that the F114A mutant failed to associate with raptor, and that insulin- and serum-induced phosphorylation of Thr37/46 was heavily reduced and the subsequent modification of Thr70 and Ser65 was almost abolished in this TOS motif mutant (Schalm & Blenis 2002; Beugnet et al. 2003; Schalm et al. 2003). However, the role of the TOS motif is not clear for amino acid induced phosphorylation in 4E-BP1. Later, Tee & Proud (2002) proposed that another four amino acid sequence named the RAIP motif according to its primary structure (Arg13, Ala14, Ile15, Pro16) is required for the sufficient phosphorylation of 4E-BP1. They found this motif during the analysis of the truncated molecule lacking the amino-terminal 24 residues generated by caspase-catalyzed cleavage of 4E-BP1. The mutants replacing the residue(s) in this motif by Ala have been employed to analyze the role of this motif designated I15A (Tee & Proud 2002), RAAA (Choi et al. 2003) and AAAA (Beugnet et al. 2003). Choi et al. (2003) concluded that this motif is essential for the binding of 4E-BP1 with raptor, as the mutation in the RAIP motif abolishes the association of the protein with raptor. In contrast, Beugnet et al. (2003) have reported that the RAIP motif does not associate with raptor based on the result that the amino-terminal fragment of 4E-BP1 containing the RAIP motif does not bind to raptor. In both cases, however, the phosphorylation of the RAIP motif mutants was reduced in the cells. Therefore, opposing results for the role of the RAIP motif have been reported, and the precise function of this motif for the association with raptor is still unclear. The relationship between the TOS and RAIP motifs thus remains unknown.

In this study, we analyzed the roles of the TOS and RAIP motifs in 4E-BP1 for its mTOR-catalyzed phosphorylation in vitro and then in HEK293 cells in response to the amino acid treatment using the point mutants replacing the critical residues in these two motifs, Phe114 and Ile15, respectively. The involvement of mTOR-mediated phosphorylation of 4E-BP1 for the dissociation from raptor and for the release of eIF4E was studied using the phosphorylation site mutants. Furthermore, the roles of these two motifs and phosphorylation sites of 4E-BP1 in the cell size regulation were also analyzed by measuring the diameters of the cells expressing the mutants of these motifs and phosphorylation sites.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Experimental procedures
 References
 
Phosphorylation of 4E-BP1 mutants by mTOR in vitro

The mutants replacing the critical residues of Phe114 and Ile15 in the TOS and RAIP motifs, respectively, were employed to study the mTOR-catalyzed phosphorylation. These TOS and RAIP mutants generated as GST-fusion proteins were phosphorylated by mTOR in vitro (Fig. 1). The wild-type 4E-BP1 protein was phosphorylated by the mTOR/raptor complex immunoprecipitated from HEK293 cells as shown by autoradiography, and immunoblot analysis revealed that Thr37/46 and Thr70 were efficiently phosphorylated. When mTOR was immunoprecipitated from the cell lysates prepared in the presence of Nonidet P-40 to dissociate raptor from mTOR as shown in the lower two panels of Fig. 1, the phosphorylation of the wild-type 4E-BP1 protein by the immunoprecipitate was heavily reduced as reported previously (Nojima et al. 2003). The I15A/F114A mutant was not phosphorylated significantly as revealed by autoradiography and immunoblot analysis. The phosphorylation of the F114A mutant was reduced compared to the wild-type protein as reported previously (Nojima et al. 2003). The TOS motif mutant was revealed to be phosphorylated at Thr37/46 in a raptor-dependent manner, but the phosphorylation of Thr70 was not detected as in the case of the I15A/F114A mutant. In contrast, the I15A mutant was recognized by mTOR and the phosphorylation not only of Thr37/46 but also of Thr70 was detected. Namely, the mutations in the TOS and RAIP motifs show distinct effects on the 4E-BP1 protein: the replacement in the TOS motif heavily reduces the ability of the protein to associate with raptor, whereas the 4E-BP1 mutant with a substitution in the RAIP motif still interacts with raptor to be phosphorylated by mTOR not only at the primary but also at the subsequent sites.


Figure 1
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  		Figure 1  Phosphorylation of 4E-BP1 motif site mutants by mTOR in vitro. Each GST-fusion protein expressed in E. coli was incubated with the immunoprecipitate isolated from HEK293 cells by the anti-mTOR antibody in the presence of [{gamma}-32P]ATP. In the left end lane, the normal mouse immunoglobulin was employed instead of the anti-mTOR antibody. Where indicated, the immunoprecipitate was prepared in the presence of Nonidet P-40. The top panel shows autoradiography and lower five panels show immunoblot by using each antibody. The radioactivity incorporated into 4E-BP1 proteins is shown with that into the wild-type (WT) in the absence of Nonidet P-40 as 100%. These results are representative of three independent experiments.

 
mTOR-catalyzed phosphorylation of 4E-BP1 mutants in vivo

Next, the TOS and RAIP motif mutants were expressed in HEK293 cells to study the mTOR catalyzed phosphorylation in response to treatment with amino acids (Fig. 2). Immunoblot analysis using the anti-phospho-Thr37/46 antibody indicated that the wild-type 4E-BP1 is phosphorylated at these sites even after deprivation of amino acids. The treatment of the cells with amino acids enhanced the phosphorylation as well as induced an upshift in the mobility of the wild-type protein, suggesting that the protein is phosphorylated at the subsequent residues. Rapamycin prevented the amino acid-induced enhancement of phosphorylation, and thus the modification reaction of 4E-BP1 was confirmed to be dependent on raptor as previously described (Nojima et al. 2003). In contrast, the phosphorylation of Thr37/46 was neither detected in amino acid starved cells nor induced by amino acid treatment in the I15A/F114A mutant. The basal phosphorylation of Thr37/46 was not found in the F114A mutant either as the double mutant, but the amino acid treatment stimulated the phosphorylation in this mutant in a manner sensitive to rapamycin as in the insulin-stimulated cells (Beugnet et al. 2003; Schalm et al. 2003). Amino acid–induced phosphorylation of Thr37/46 was also observed for the I15A mutant, whereas the basal phosphorylation was not evident in the RAIP motif mutant. Thus, the in vivo phosphorylation detected by the phosphorylation site specific antibody was similar between these two mutants, although they are recognized differently by mTOR in vitro (Fig. 1). The RAIP motif mutant, however, showed a slight upshift on the blot in Fig. 2, suggesting that this mutant protein is phosphorylated at Thr70 in vivo as shown in vitro even less efficiently than the wild-type. These results indicate that the 4E-BP1 molecule with mutation to both the TOS and RAIP motifs does not interact with raptor, but the mutants having at least one of these two motifs intact still associate with raptor to be phosphorylated by mTOR. Importantly, the results in vivo and in vitro indicate that the TOS and RAIP motifs have the different properties for the recognition by mTOR.


Figure 2
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  		Figure 2  Phosphorylation of 4E-BP1 motif site mutants in HEK293 cells. HEK293 cells transfected with FLAG-4E-BP1 (WT) or its mutants were incubated without amino acids, and stimulated with amino acids in the presence or absence of rapamycin. Immunoblot was carried out with each antibody after immunoprecipitation with the anti-FLAG antibody. These results are representative of three independent experiments.

 
Cell size reduction by 4E-BP1 motif site mutants

Next, the TOS and RAIP motif site mutants were introduced into K562 cells to examine the effects on cell size. The cells were transfected with each expression vector at high expression efficiencies using Nucleofector 3, and were directly applied to the particle size determination by using Coulter Counter with Multisizer 3 without sorting the transfectants (Fig. 3, upper panel). The amounts of the wild-type and each mutant of 4E-BP1 expressed in the cells were similar (Fig. 3, lower panel). The expression of the wild-type 4E-BP1 did not affect cell size significantly (data not shown). The introduction of the I15A/F114A mutant, which is not phosphorylated by mTOR, reduced cell size compared to that of the wild-type protein as expected. The expression of the F114A mutant reduced cell size as reported by Shalm et al. (2003). In contrast, the I15A mutant, which is phosphorylated not only at the priming sites of Thr37/46 but also at the subsequent residue of Thr70 in vitro and presumably in vivo, did not affect cell size as the wild-type 4E-BP1. These results agree with the phosphorylation of Thr70 in the TOS and RAIP motif site mutants as revealed by in vitro analysis in Fig. 1.


Figure 3
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  		Figure 3  Cell size reduction by expressing 4E-BP1 motif site mutants. K562 cells expressing FLAG-4E-BP1 (WT) or its mutants were subjected to cell size determination (upper panel). Shown are size distributions of cells expressing 4E-BP1-WT (black line) and of cells transfected with 4E-BP1-I15A, 4E-BP1-F114A or 4E-BP1-I15A/F114A (red line). Cell size as particle diameter in µm (the means ± SD) of four independent experiments are shown with immunoblot of FLAG-tagged proteins in lysates of the typical experiment (lower panel). Statistical significance was determined by t-test. *P < 0.05.

 
Association of 4E-BP1 mutants with raptor

As the analysis of the motif site mutants indicated that the phosphorylation of 4E-BP1 after the association with raptor through the motif sites is important for the control of this translational regulator, the contribution of the phosphorylation reaction of 4E-BP1 was studied by using a series of mutants replacing the phosphorylation sites (Fig. 4). When the wild-type FLAG-4E-BP1 was co-expressed with the myc-raptor in HEK293 cells, FLAG-4E-BP1 was detected in the immunoprecipitate with anti-myc antibody. The 5A mutant replacing Thr37, Thr46, Ser65, Thr70 and Ser83 with Ala, which mimics the dephosphorylated form of 4E-BP1, associated with myc-raptor more efficiently that the wild-type 4E-BP1, whereas the 5E mutant, resembling the fully phosphorylated form by mutating these five residues to Glu, did not bind to raptor as reported previously (Hara et al. 2002). These results indicate that raptor binds preferentially to the nonphosphorylated or poorly phosphorylated form of 4E-BP1, and thus the binding with raptor is critical for the mTOR-catalyzed phosphorylation of 4E-BP1. Therefore, the role of each residue was investigated using the mutants replacing them with Ala or Glu. The 37/46A mutant associated with raptor strongly as the 5A mutant, confirming that the phosphorylation the priming sites of Thr37 and Thr46 is essential for the dissociation of 4E-BP1 from raptor. The 37/46/83E-65/70A mutant, in which the priming sites as well as the additional phosphorylation site of Thr83 are replaced with Glu to mimic the phosphorylated state whereas the subsequent sites of Thr70 and Ser65 are mutated to the unphosphorylatable residue, still associated with raptor. These results indicate that the phosphorylation of Thr37 and Thr46 is not enough, but the phosphorylation of the further site(s) is required for the dissociation of 4E-BP1 from raptor. The association with raptor was not found in the 37/46/70/83E-65A mutant, in which Thr70 of the 37/46/83E-65/70A mutant was further replaced by Glu, indicating that the phosphorylation of Thr70 is critical for the release of 4E-BP1 from raptor. Consistently, the 70/83E-37/46/65A mutant, with the acidic residue at the position of Thr70, associated with raptor potently, even though the priming phosphorylation sites are replaced by Ala. These results indicate that the phosphorylation of Thr70 following that of Thr37/46 is required for the dissociation of 4E-BP1 from raptor.


Figure 4
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  		Figure 4  Association of 4E-BP1 with raptor. HEK293 cells were transfected with myc-raptor with FLAG-tagged 4E-BP1 (WT) or its mutants. Immunoblot was carried out with each antibody after immunoprecipitation with the anti-FLAG antibody. The expression of FLAG-tagged proteins was shown by immunoblot of the lysates. These results are representative of three independent experiments.

 
Association of 4E-BP1 mutants with eIF4E

It is known that 4E-BP1 associates with eIF4E to inhibit its translational initiation activity, and that the phosphorylation of 4E-BP1 promotes the disruption of the complex of 4E-BP1 and eIF4E (Gingras et al. 2001; Proud 2002). As the studies using the phosphorylation site mutants of 4E-BP1 revealed the involvement of the phosphorylation reaction in the association of 4E-BP1 with raptor, the interaction of 4E-BP1 with eIF4E was next studied (Fig. 5). The cell extract prepared from HEK293 cells over-expressing each phosphorylation site mutant was incubated with 7-methyl-GTP Sepharose, which mimics the 5' mRNA cap structure, and the proteins bound to the resin were analyzed by immunoblot for the endogenous eIF4E and the 4E-BP1 mutants. In agreement with the association to raptor, the 5A mutant efficiently associated with eIF4E, whereas the binding of the 5E mutant was heavily attenuated. The 37/46A mutant associated with eIF4E efficiently as the 5A mutant, and the 37/46/83E-65/70A and 70/83E-37/46/65A mutants were detected in complex with eIF4E in the affinity resin. On the other hand, only a faint binding of the 37/46/70/83E-65A mutant to eIF4E was detected as in the case of its the association with raptor. These results indicate that the phosphorylation of the priming sites of Thr37/46 and the secondary site of Thr70 is required for the dissociation of 4E-BP1 from eIF4E.


Figure 5
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  		Figure 5  Association of 4E-BP1 with eIF4E. HEK293 cells were transfected with FLAG-4E-BP1 (WT) or its mutants. Immunoblot was carried out with each antibody after affinity purification using 7-methyl-GTP Sepharose. The expression of FLAG-tagged proteins was shown by immunoblot of the lysates. These results are representative of three independent experiments.

 
Cell size reduction by 4E-BP1 phosphorylation site mutants

The phosphorylation site mutants were introduced into K562 cells, and cell size was examined as described above (Fig. 6, upper panel). The amounts of the proteins expressed in the cells were similar among the wild-type and mutants of 4E-BP1 (Fig. 6, lower panel). The size of the cells expressing the 5E mutant, which does not interact with raptor or eIF4E, was similar to those expressing the wild-type 4E-BP1. In contrast, the 5A mutant, which binds to raptor and takes hold of eIF4E, reduced cell size. The 37/46A mutant, which binds to raptor and eIF4E, also reduced cell size as reported by Schalm et al. (2003). Other mutants that can associate with raptor and eIF4E such as the 37/46/83E-65/70A and 70/83E-37/46/65A mutants decreased cell size, whereas the 37/46/70/83E-65A mutant that can dissociate from raptor and eIF4E did not affect cell size. These results indicate that the expression of the 4E-BP1 mutants having no or little dissociation ability from raptor and eIF4E induces cell size reduction.


Figure 6
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  		Figure 6  Cell size reduction by expressing 4E-BP1 phosphorylation site mutants. K562 cells expressing FLAG-4E-BP1 (WT) or its mutants were subjected to cell size determination (upper panel). Shown are size distributions of cells expressing 4E-BP1-WT (black line) and of cells transfected with 4E-BP1-5E, 4E-BP1-5A, 4E-BP1-37/46A, 4E-BP1-37/46/83E-65/70A, 4E-BP1-37/46/70/83E-65A and 4E-BP1-70/83E-37/46/65A (red line). Cell size as particle diameter in µm (the means ± SD) of four independent experiments are shown with immunoblot of FLAG-tagged proteins in lysates of the typical experiment (lower panel). Statistical significance was determined by t-test. *P < 0.05.

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Experimental procedures
 References
 
In this study, we investigated the regulation mechanisms of 4E-BP1 by amino acids focusing on its association with raptor and phosphorylation by mTOR, and the role of this translational regulator in the control of cell size was examined by expressing the mutants replacing the binding motifs and phosphorylation sites. 4E-BP1 has been shown to contain the TOS and RAIP motifs as the binding sites with raptor. It is established that the TOS motif is important for the binding with raptor and mTOR-catalyzed phosphorylation of 4E-BP1 in response to growth-factor stimulation (Beugnet et al. 2003; Choi et al. 2003; Nojima et al. 2003; Schalm et al. 2003). Concerning the RAIP motif, however, different results have been reported: the RAIP motif is essential for the binding of 4E-BP1 with raptor (Choi et al. 2003), whereas the RAIP motif does not associate with raptor (Beugnet et al. 2003). Here, we compared the roles of these two motif sites by expressing the TOS and RAIP motif mutants in the cells stimulated by amino acids. It is known that 4E-BP1 has a basal level phosphorylation of Thr37 and Thr46 in serum starved cells (Gingras et al. 2001), but the phosphorylation of these priming sites was not detected in both of the TOS and RAIP motif mutants when cultured in the absence of amino acids. Furthermore, the amino acid treatment after cell starvation induced phosphorylation of these priming sites in each motif site mutant in a rapamycin-sensitive manner. The double motif mutant was not phosphorylated at all in the cells. Consistently, the in vitro phosphorylation of the RAIP motif and TOS motif mutants by mTOR was heavily reduced in the absence of raptor. These results show that the phosphorylation of Thr37 and Thr46, which is fairly resistant to serum starvation (Schmelzle & Hall 2000; Proud 2002), is mediated by mTOR and that both of these two motifs are involved in the association of 4E-BP1 with raptor for its basal phosphorylation. It seems that a basal activity of mTOR catalyzes the ground level of phosphorylation of 4E-BP1 even in the starved cells. The TOS motif mutant did not associate with raptor by GST-pull down assay when this mutant was expressed as a GST-fusion protein, whereas the wild-type protein showed a good binding with raptor (Nojima et al. 2003). The RAIP motif mutant did not associate with raptor apparently either when expressed in the cells as a GST-fusion protein (data not shown). Thus, the GST-pull down assay is not sensitive enough or appropriate to detect the association of 4E-BP1 with raptor through each binding motif, and it is therefore necessary to be careful to evaluate the roles of these short amino acid sequences for the protein–protein interaction.

mTOR phosphorylates 4E-BP1 sequentially: initially at Thr37 and Thr46, then at Thr70 (Gingras et al. 2001). Proud (2002) has reported that the phosphorylation of Thr70 was important for the release of eIF4E. Therefore, the phosphorylation of Thr70 was examined after in vitro phosphorylation by mTOR. The TOS motif mutant was not phosphorylated at Thr70, whereas the RAIP motif mutant was clearly phosphorylated at this site as was the wild-type. It has been reported that the I15A mutant is similarly phosphorylated at Thr70 after insulin stimulation in vivo and that rapamycin blocks the phosphorylation of Thr70 (Tee & Proud 2002; Choi et al. 2003). Presumably, this RAIP motif mutant is phosphorylated by amino acid stimulation in the cell. Taken together, the results indicate that the TOS and RAIP motifs have different properties for the association with raptor: the former is critical for the association of 4E-BP1 with raptor to mediate its full phosphorylation, but the latter is not essential and may have an accessory role for the association with raptor. Wang et al. (2005) have proposed that the RAIP motif of 4E-BP1 interacts with mTOR through a protein distinct from raptor in a manner dependent on amino acids. A precise analysis is necessary to elucidate the role of the RAIP motif in 4E-BP1.

As the two binding motifs showed distinct phosphorylation levels, we focused on the role of 4E-BP1 phosphorylation and examined whether the phosphorylation of 4E-BP1 affects the binding of raptor to 4E-BP1. Previously, we demonstrated that raptor binds preferentially to poorly or nonphosphorylated form of 4E-BP1 (Hara et al. 2002). As expected, the 5A mutant tightly associated with raptor in contrast to the 5E mutant which did not bind to raptor. Similar results were obtained for the dissociation of eIF4E. Interestingly, the 37/46/70/83E-65A mutant dissociated from raptor as in the case of the 5E mutant. These results suggest that phosphorylation of Ser65 is not essential for the dissociation from raptor. Proud (2002) has proposed that phosphorylation of Ser65 may prevent the reassociation of 4E-BP1 with eIF4E. It is necessary to study the physiological role of Ser65 phosphorylation for the interaction between endogenous proteins. On the other hand, we employed the 70/83E-37/46/65A mutant to evaluate the role of the phosphorylation of Thr70. As this mutant could not dissociate from raptor, it seems that phosphorylation of Thr70 alone is not sufficient for the release of 4E-BP1 from raptor. Namely, the phosphorylation of Thr37 and Thr46 is a prerequisite for the regulation of 4E-BP1. In addition, Gingras et al. (2001) have reported that the 37/46A mutant is not phosphorylated at subsequent sites by mTOR in vitro and consistently the 37/46A mutant associated with raptor intensely. These results indicate that the sequential phosphorylation of Thr37/46 and then of Thr70 by mTOR is required for the dissociation of 4E-BP1 from raptor and the release of eIF4E. It seems that the conformational change of 4E-BP1 induced by mTOR-mediated phosphorylation has a critical role in the binding between mTOR and 4E-BP1.

Schalm et al. (2003) have demonstrated that the expression of the F114A or 37/46A mutant in U2OS cells induces size reduction by measuring forward scatter using flow cytometer. Here, we employed Coulter Counter for the direct analysis of particular diameters of K562 cells expressing 4E-BP1 mutants replacing the binding motifs and phosphorylation sites. The introduction of the TOS motif mutant lowered cell size significantly, and phosphorylation site mutants lacking the modification reaction at Thr70 as in the case of the TOS motif mutant, that have little or no ability for the dissociation with raptor and eIF4E, cause cell size reduction, whereas the RAIP motif mutant did not show a significant effect on cell size. The procedures for cell size determination developed in this study will be useful for the analysis of the roles of the proteins involved in the signaling pathway of growth factors and nutrients. The I15A mutation in the RAIP motif brought a substantial decrease in in vivo phosphorylation similar to the TOS motif mutant, but the RAIP motif and TOS motif mutants had different effects on cell size. This cell size analysis seems to be sensitive enough to differentiate the roles of these binding motifs. Further studies of the binding motif sites in combination with the analysis of the phosphorylation of 4E-BP1 will contribute to the understanding of the translational regulation of cell growth.


   Experimental procedures
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
References
 
cDNAs

The expression vector of the wild-type 4E-BP1 with the FLAG epitope tag at the amino terminus designated FLAG-4E-BP1 (WT) was generated in pCMV vector as described (Hara et al. 2002), and the insert was mutated using the QuickChange site-directed mutagenesis kit (Stratagene). The mutants having Ala in the residues of Ile15 (I15A), Phe114 (F114A) and Ile15 and Phe114 (I15A/F114A) were cloned into pCMV, and the resulting expression vectors were designated as FLAG-4E-BP1-I15A, FLAG-4E-BP1-F114A and FLAG-4E-BP1-I15A/F114A, respectively. These three mutants were cloned into pGEX as the expression vectors of GST-fusion proteins in Escherichia coli, and designated as GST-4E-BP1-I15A, GST-4E-BP1-F114A and GST-4E-BP1-I15A/F114A, respectively. The plasmid GST-4E-BP1 (WT) encoding the wild-type 4E-BP1 fused with GST was prepared as described (Nojima et al. 2003). The pCMV vectors with the FLAG epitope tag designated FLAG-4E-BP1-5A and FLAG-4E-BP1-5E replacing Thr37, Thr46, Ser65, Thr70 and Ser83 by Ala and Glu, respectively, were constructed as described (Hara et al. 2002). The phosphorylation sites were further replaced to make the following mutants: having Ala at the positions of Thr37 and Thr46 (FLAG-4E-BP1-37/46A); having Glu at the positions of Thr37, Thr46 and Ser83, and Ala at the positions of Ser65 and Thr70 (FLAG-4E-BP1-37/46/83E-65/70A); having Glu at the positions of Thr37, Thr46, Thr70 and Ser83, and Ala at the position of Ser65 (FLAG-4E-BP1-37/46/70/83E-65A); having Glu at the positions of Thr70 and Ser83, and Ala at the positions of Thr37, Thr46 and Ser65 (FLAG-4E-BP1-70/83E-37/46/65A). The myc-tagged expression vector of raptor named myc-raptor was generated in pcDNA3 as described (Hara et al. 2002). The expression vector of green-fluorescent protein (GFP) in pEGFP was purchased from BD Clontech.

Antibodies

The anti-FLAG, anti-myc, anti-GST and anti-eIF4E antibodies were purchased from Sigma, Roche Molecular Biochemicals, Upstate Biotechnology and Transduction Laboratories, respectively. The anti-phospho-4E-BP1 antibodies against Thr37/46 and Thr70 were purchased from Cell Signaling Technology. The monoclonal anti-mTOR antibody and anti-raptor were produced as previously described (Nishiuma et al. 1998; Hara et al. 2002). The horseradish peroxidase-conjugated anti-mouse and anti-rabbit antibodies were purchased from Bio-Rad.

Cell culture and size analysis

HEK293 cells were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% fetal bovine serum (FBS, Gibco BRL) at 37 °C in a 5% CO2 incubator. The cells were transfected with each expression vector by lipofection using lipofectAMINE according to the manufacturer's protocol (Life Technologies). For starvation of amino acids, cells were cultured in DMEM with FBS for 24 h and then without FBS for 16 h, and further incubated in DMEM without amino acids for 1.5 h. Then the medium was replaced with DMEM containing amino acids for 30 min (Hara et al. 2002). Where indicated, 200 nM rapamycin was added with amino acids. When the association of the 4E-BP1 proteins with raptor or eIF4E was analyzed, the cells were cultured in DMEM with FBS for 48 h after transfection (Nojima et al. 2003). For cell size analysis, K562 cells cultured in RPMI 1640 medium with 10% FBS (HyClone Laboratories) were employed. K562 cells (2 x 107 cells) transfected with 5 g of each plasmid using Nucleofector 3 (AMAXA Biosystems) were incubated for 48 h, washed 2 times with Dulbecco's phosphate-buffered saline (PBS, Gibco BRL), fixed with 70% ethanol, and were stored at –20 °C until the time of analysis. The stored cells were centrifuged at 3000 g for 10 min to remove ethanol, washed once with PBS, and diluted with Isoton 2 (Beckman Coulter). The cell size was determined as particle diameters using Coulter Counter with Multisizer 3 (Beckman Coulter). The efficiency of transfection was approximately 90% as judged by the expression of GFP by the introduction of the pEGFP vector.

Protein purification

Immunoprecipitation was carried out at 0–4 °C essentially as described (Hara et al. 1997). Briefly, the cells were lyzed with a lysis buffer (20 mM Tris-HCl at pH 7.5, 20 mM NaCl, 1 mM EDTA, 20 mM ß-glycerophosphate, 5 mM EGTA, 1 mM dithiothreitol 1 mM phenylmethylsulfonyl fluoride [PMSF], 2 µg/mL aprotinin and 1 µM leupeptin). Where indicated, 1% Nonidet P-40 was added in the lysis buffer (Hara et al. 2002). After centrifugation at 18 000 g for 10 min, the lysate was incubated with each antibody and Protein G-Sepharose (Amersham Biosciences) for 2 h. The immunoprecipitate was collected by centrifugation and washed 3 times with the lysis buffer containing 500 mM NaCl. GST-pull down was carried out by the procedures similar to immunoprecipitation using GST-Sepharose (Amersham Biosciences) instead of the antibodies and Protein G-Sepharose. The proteins bound to the resin were eluted by an elution buffer (100 mM Tris-HCl pH 8.0, 150 mM NaCl, 20 µM glutathione, 5 mM dithiothreitol) (Nojima et al. 2003). Affinity purification using 7-methyl-GTP Sepharose was performed as previously described (Hara et al. 2002). Briefly, the cell extracts by lysis buffer containing 1% Nonidet P-40 was incubated with 15 µL of 7-methyl-GTP Sepharose beads for 2 h at 4 °C. The beads were washed 3 times with lysis buffer containing 500 mM NaCl, and bound proteins were eluted by boiling with SDS-sample buffer.

Immunoblot

The lysates, immunoprecipitates and eluates were separated by SDS-PAGE and the proteins were transferred on to a polyvinylidene difluoride (PVDF) membrane and subjected to immunoblot using each primary antibody (Hara et al. 2002). After incubation the horseradish peroxidase-conjugated secondary antibody, the chemiluminescence reaction was carried out as described (Nishiuma et al. 1998). When the 4E-BP1 proteins were detected in cell lysates, the cell lysates were heated at 100 °C for 5 min and centrifuged at 18 000 g for 30 min, and the heat-stable proteins were separated were subjected to immunoblot (Shigemitsu et al. 1999).

mTOR kinase assay

The mTOR kinase assay was performed as previously described (Hara et al. 1998; Isotani et al. 1999; Hara et al. 2002). The GST fusion proteins of the wild-type 4E-BP1 and its mutants were expressed and purified from E. coli as described as the substrates for mTOR (Nojima et al. 2003). The immunoprecipitate by the anti-mTOR antibody from HEK293 cells were incubated with each GST fusion protein and [{gamma}-32P]ATP in the reaction buffer (10 mM HEPES, 50 mM NaCl, 50 mM ß-glycerophosphate, 10 mM MnCl2, 200 µM ATP) (Hara et al. 2002). Where indicated, the normal mouse immunoglobulin was employed instead of the anti-mTOR antibody. After incubation at 30 °C for 30 min, the reaction was terminated by the addition of the SDS-sample buffer. Proteins were separated by SDS-PAGE, transferred to a PVDF membrane, and the incorporation of the radioactivity was visualized and quantitated by Bioimaging analyzer (Fuji). The membrane was then subjected to successive immunoblot analysis by using different primary antibodies after stripping the membrane each time.


   Acknowledgements
 
We thank H. Miyamoto and K. Fujii for technical assistance and R. Kato for skillful secretarial assistance. This work was supported in part by research grants from the Scientific Research Funds of the Ministry of Education, Culture, Sports, Science and Technology of Japan.


   Footnotes
 
Communicated by: Kozo Kaibuchi

aPresent address: Biochemistry Department, Faculty of Medicine, Brawijaya University, Malang 65145, Indonesia. Back

bDeceased on 8 July 2005 Back

* Correspondence: E-mail: ukikkawa{at}kobe-u.ac.jp


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Experimental procedures
 References
 
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Received: 19 November 2005
Accepted: 5 April 2006




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