Myogenic Differentiation Is Dependent on Both the Kinase Function and the N-terminal Sequence of Mammalian Target of Rapamycin*

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase known to control initiation of translation through two downstream pathways: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic initiation factor 4E and ribosomal p70 S6 kinase (S6K1). We previously showed in C2C12 murine myoblasts that rapamycin arrests cells in G1phase and completely inhibits terminal myogenesis. To elucidate the pathways that regulate myogenesis, we established stable C2C12 cell lines that express rapamycin-resistant mTOR mutants (mTORrr; S2035I) that have N-terminal deletions (Δ10 or Δ91) or are full-length kinase-dead mTORrr proteins. Additional clones expressing a constitutively active S6K1 were also studied. Our results show that Δ10mTORrr signals 4E-BP1 and permits rapamycin-treated myoblasts to differentiate, confirming the mTOR dependence of the inhibition of myogenesis by rapamycin. C2C12 cells expressing either Δ91mTORrr or kinase-dead mTORrr(D2338A) could not phosphorylate 4E-BP1 in the presence of rapamycin and could not abrogate the inhibition of myogenesis. Taken together, our results indicate that both the kinase function of mTOR and the N terminus (residues 11–91, containing part of the first HEAT domain) are essential for myogenic differentiation. In contrast, constitutive activation of S6K1 does not abrogate rapamycin inhibition of either proliferation or myogenic differentiation.

A rapamycin-sensitive pathway is required for differentiation of C2C12 and L6 myoblasts (10 -12). This finding is consistent with reports that wortmannin, an inhibitor of phos-phatidylinositol 3-kinase upstream of the mammalian target of rapamycin (mTOR, 1 FRAP), inhibits IGF-I-stimulated differentiation. Conversely, however, Jayaraman and Marks (13) reported that rapamycin induces terminal differentiation. This discrepancy may arise partially from the use of different clones and different end points for assessing differentiation. Whereas several reported studies have used myoblast fusion as the marker of differentiation, the single study that showed rapamycin induction of differentiation used ␣-actin expression in a nonfusing C3H clone to identify differentiation (13). To date, the mechanism by which rapamycin inhibits myogenesis has not been established.
The rapamycin target, mTOR (14), links mitogen stimulation to translation through control of ribosomal S6K1 and 4E-BP1, the suppressor of eukaryotic initiation factor 4E (eIF4E) (15). The S6K1 pathway controls synthesis of proteins, such as IGF-II and ribosomal proteins, whereas the 4E-BP1 pathway controls many proteins involved in cell cycle regulation. The two pathways regulate the initiation of translation of distinct classes of mRNA. Mitogen-induced phosphorylation and activation of S6K1 appear to play an important role during the G 1 phase of the cell cycle (16,17). Phosphorylation of the S6 protein, the small ribosomal subunit, by S6K1 permits efficient translation of mRNAs containing terminal oligopyrimidine tracts in their 5Ј-untranslated regions (18). Phosphorylation of 4E-BP1 controls cap-dependent translation of mRNAs with extensive secondary structure. Growth factors stimulate phosphorylation of 4E-BP1, thereby reducing its affinity for the cap-binding protein eIF4E and releasing the blockade of capdependent translation (19). mTOR phosphorylates at least two residues of 4E-BP1: Thr 37 and Thr 46 (20). mTOR-dependent phosphorylation of these residues blocks 4E-BP1 association with eIF4E in vitro, and phosphorylation of Thr 46 appears to be the main regulator of the 4E-BP1-eIF4E interaction in vivo (21). By preventing the phosphorylation of specific residues on S6K1 and 4E-BP1, rapamycin inhibits mitogen-stimulated activation of S6K1 and the resultant phosphorylation of S6 (22,23) and prevents dissociation of 4E-BP1 from eIF4E (24,25). Rapamycin negates mitogen-induced activation of S6K1 by preventing the acute phosphorylation of a specific subset of sites, including Thr 229 , Thr 389 , Ser 404 , and Ser 411 . Thr 389 , which resides in the linker region coupling the catalytic and autoinhibitory domains, has been identified as the principal site of rapamycin-induced dephosphorylation that leads to S6K1 inactivation (26).
Although the molecular mechanisms involved in rapamycin inhibition of cell proliferation are coming to light, the precise mechanisms by which rapamycin inhibits myogenesis have remained elusive. A recently reported study by Erbay and Chen (27) concluded that the kinase function of mTOR is not required for myogenic differentiation. In that study, rapamycin treatment of C2C12 myoblasts prevented differentiation and inhibited S6K1, but it did not induce significant hypophosphorylation of 4E-BP1. Here we confirm the finding that rapamycin inhibits myogenesis through inhibition of mTOR. However, unlike Erbay and Chen (27), we show that rapamycin induces hypophosphorylation of 4E-BP1 and that the kinase function of mTOR is required for differentiation. Our results indicate that the extreme N terminus of mTOR (residues 1-10) is not required for differentiation, whereas residues 11-91, which include part of the first HEAT sequence, are essential.

EXPERIMENTAL PROCEDURES
Cell Line and Cultures-Mouse C2C12 myoblasts were purchased from American Type Culture Collection (Manassas, VA) and routinely grown in antibiotic-free Dulbecco's modified Eagle's medium with 15% fetal calf serum (growth medium (GM)). Cells were induced to differentiate by growth in differentiation medium (DM; Dulbecco's modified Eagle's medium with 2% horse serum supplemented with 4 mM Lglutamine) at 37°C and 5% CO 2 .
Antibodies and Reagents-Anti-4E-BP1 was purchased from Zymed Laboratories Inc.. Phospho-specific antibodies to the Thr 37 and Thr 46 residues of 4E-BP1 and the Thr 389 residue of S6K1 were from Cell Signaling Technology (Beverly, MA). M2 anti-FLAG, anti-myosin heavy chain, and antibodies to laminin, ␣-actin, and ␤-tubulin were from Sigma. Anti-Au1 was from BabCO (Richmond, CA). Anti-MyoD and anti-myogenin were from BD PharMingen, and anti-S6K1, anti-Myc, and protein A/G plus-agarose were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
The S6 kinase assay kit was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Horseradish peroxidase-conjugated anti-mouse IgG and anti-rabbit IgG were from Amersham Biosciences. The Supersignal chemiluminescent substrate was from Pierce. Transfection reagent TransIT TM -LT1 was from PanVera Corp. (Madison, WI). All other chemicals were purchased from Sigma.
Plasmids-The plasmid pcDNAmTORrr containing the entire mTOR gene with the S2035I rapamycin resistance mutation was used as a template for all additional mutations. To make truncated mTORrr mutants, we synthesized the forward primers TorF11 and TorF92 to delete N-terminal amino acids 1-10 or 1-91 of mTORrr, respectively ( Table I). The HindIII restriction site and FLAG sequence were added in-frame to the 5Ј end of each forward primer. One reverse primer (TorR2549) beginning at the stop codon and containing a BamHI site was used to amplify truncated mTORrr fragments. It should be noted that a high-fidelity PCR system was used. PCR products were digested with HindIII and BamHI and cloned into the pcDNA3 vector. The fidelity of cloned inserts was confirmed by sequencing.
Establishment of a Stable C2C12 Cell Line Expressing Constitutively Active S6K1(D3E-E389)-C2C12 cells were co-transfected with Myc-S6K1-D3E-E389 and pcDNA3 by using the TransIT TM LT1 kit as instructed by the manufacturer. After selection in medium containing G418, the single clones were isolated and expanded. Protein expression was evaluated by immunoprecipitation with anti-Myc antibody and by Western blot detection with anti-S6K1. These clones are designated C2C12(D3E-E389).
Establishment of Stable C2C12 Cell Lines Expressing ⌬10mTORrr, ⌬91mTORrr, or Kinase-dead mTORrr-C2C12 cells were transfected with plasmids expressing ⌬10, ⌬91, or kinase-dead mTORrr (SIDA) or with pcDNA3 control vector by using the TransIT TM LT1 kit. Cells were selected for G418 resistance and cloned. Individual clones were screened for expression of mutant proteins by immunoprecipitation with M2 anti-FLAG or anti-Au1 antibodies and by Western blot with anti-mTOR (26E3) mouse monoclonal antibody.
For immunoprecipitation, cell lysates were precleared with 0.25 g/ml normal rabbit IgG and 30 l of protein A/G plus-agarose at 4°C for 30 min and then centrifuged at 1000 ϫ g for 5 min. Primary antibody (2 g/ml) was added to the supernatant, and samples were rotated at 4°C for 1 h. Thirty l of protein A/G plus-agarose were then added, and samples were rotated at 4°C overnight. After centrifugation, the collected beads were washed three times with lysis buffer, 30 l of 1ϫ protein loading buffer were added, and samples were boiled for 5 min and centrifuged for 5 min at 1000 ϫ g.
Electrophoresis was performed under conditions that do not resolve 4E-BP isoforms, as described previously (28). Isoforms of 4E-BP1 were separated in parallel experiments that used 15% Tris-HCl denaturing gel. Electrophoresis was performed at a constant 100 V at 4°C for 2 h. The separated proteins were transferred to an Immobilon-P membrane by electrophoresis at 4°C for 1 h. Nonspecific binding was blocked by incubation with 5% nonfat milk at room temperature for 1 h, and the membrane was incubated overnight with primary antibody at 4°C. The membrane was washed three times with phosphate-buffered saline with 0.1% Tween 20, incubated with secondary antibody conjugated to horseradish peroxidase at room temperature for 1 h, washed three times in PBS-T, incubated with Supersignal substrate, and exposed to Kodak BioMax.
Growth Inhibition Assay-C2C12 cells (1 ϫ 10 4 cells/well) were plated in triplicate in 35-mm 6-well plates (Falcon; Becton Dickinson Labware, Franklin, NJ). The following day, medium was removed, and 2 ml of medium containing serial concentrations of rapamycin (0 -1000 ng/ml) were added to each well. Cells were incubated at 37°C for 5 days and lysed under hypotonic conditions. The nuclei were counted by using a Coulter counter.
S6K1 Kinase Assay-C2C12 cells (2.5 ϫ 10 6 ) expressing Myc-S6K1-D3E-E389 were seeded in a 100-mm dish and allowed to attach overnight. Cells were serum-starved for 24 h, exposed to rapamycin (100 ng/ml) for 15 min, and then washed extensively and incubated for 1 h with IGF-I (10 ng/ml). Untreated cells and cells treated only with IGF-I were used as controls. The activity of S6K1 was assayed by using an S6 kinase assay kit as described previously (29,30).
Detection of Myosin Heavy Chain by Immunofluorescence-C2C12 cells were seeded on 35-mm plates and grown to 80 -90% confluence in GM. The next day, cells were washed once, and the medium was replaced with DM with or without rapamycin (0 -100 ng/ml). After 72 h, cells were fixed in Buffered Formalde-Fresh solution (Fisher Scientific) for 30 min, permeabilized with 0.25% Triton X-100 in PBS for 30 min, and incubated with 10% swine serum in PBS for 30 min to block nonspecific antibody binding. Cells were rinsed thoroughly, incubated with mouse monoclonal anti-myosin heavy chain antibody (Sigma; 15 g/ml in 1% swine serum-PBS) for 2 h, rinsed with PBS, and incubated with fluorescein isothiocyanate-conjugated anti-mouse IgG (Santa Cruz Biotechnology, Inc.; 4 g/ml in 1% swine serum-PBS) for 2 h. All procedures were carried out at room temperature. The cells were examined under an inverted fluorescence microscope. Both phase-contrast and fluorescence images (at least 50 fields) were recorded by a digital camera. Nonfluorescent immunohistochemical detection of myosin heavy chain was performed as described previously (31).

RESULTS
Rapamycin Inhibits Myogenic Differentiation of C2C12 Myoblasts but not ⌬10mTORrr-expressing C2C12 Cells-Although the inhibition of C2C12 differentiation by rapamycin has been reported previously, relatively high concentrations of rapamycin were used in those studies, and myotube formation was only partially inhibited. We used C2C12 cells to examine the cellular role of rapamycin in terminal differentiation. Cells were grown in DM with rapamycin (0, 1, 10, or 100 ng/ml). After 3 days, cells were fixed and examined by immunofluorescence for expression of myosin heavy chain (Fig. 1A). The results showed that C2C12 cells differentiated to myotubes when shifted to DM for 3 days and that rapamycin (10 ng/ml) completely inhibited myotube formation. To determine the effect of rapamycin on expression of muscle-specific proteins, we cultured C2C12 cells in DM in the presence or absence of rapamycin for 1, 2, and 3 days. Rapamycin either suppressed or delayed the expression of MyoD, myogenin, laminin, and ␣-actin (Fig. 1B).
To determine whether rapamycin inhibition of differentiation is mTOR-dependent, we derived a stable C2C12 cell line expressing a ⌬10mTORrr. We first tested whether ⌬10mTORrr protein conferred resistance to rapamycin-induced inhibition of proliferation. C2C12 parental cells, clones expressing ⌬10mTORrr, and vector control cells were grown in GM for 5 days in the presence of increasing concentrations of rapamycin. The IC 50 was Ͻ1 ng/ml for parental C2C12 cells and vector control cells but was Ͼ1000 ng/ml for the C2C12⌬10mTORrr clone (Fig. 1C). To detect whether rapamycin inhibited the differentiation of C2C12⌬10mTORrr, we cultured C2C12 cells expressing ⌬10mTORrr in DM in the absence or presence of rapamycin (1, 10, or 100 ng/ml) for 3 days. Cells were fixed and examined by immunofluorescence for expression of myosin heavy chain (Fig. 1D). The results showed that cells expressing ⌬10mTORrr differentiated in the presence or absence of rapamycin. These findings indicate that myogenesis is blocked through rapamycin inhibition of mTOR.
Expression of Myc-tagged Constitutively Active S6K1(D3E-E389)-The target of rapamycin, mTOR, is a serine/threonine kinase that signals to S6K1 and 4E-BP1. To further understand which downstream pathway is necessary for differentiation, we derived stable C2C12 cell lines expressing the Myc-tagged, constitutively active S6K1 mutant D3E-E389 by co-transfecting C2C12 with S6K1 (D3E-E389) and pcDNA3 plasmids. The proteins expressed in the presence or absence of rapamycin were immunoprecipitated with anti-Myc monoclonal antibody and detected by Western blot with polyclonal anti-S6K1 ( Fig. 2A). High-level expression of S6K1-D3E-E389 protein was found in five clones and was not altered by treatment of cells with rapamycin (100 ng/ml).
Rapamycin Does Not Inhibit the S6 Kinase Activity of Myctagged S6K1(D3E-E389)-To determine whether rapamycin inhibits IGF-I-induced stimulation of S6K1, we assayed S6 kinase activity in vector control cells (C2C12pcDNA3) and in C2C12 cells expressing S6K1 mutant D3E-E389. The D3E-E389 clone had a higher basal level of S6K1 activity than the vector control clone (Fig. 2B) and had 50% greater total S6K1 activity than the control clone after IGF-I stimulation. Importantly, the basal kinase activity (derived from endogenous and mutant S6K1) was only slightly inhibited (ϳ30%) by rapamycin (100 ng/ml). These results suggest that the constitutively active D3E-E389 mutant functioned in C2C12 cells. If rapamycin inhibits differentiation through inhibition of the S6K1 pathway, the C2C12(D3E-E389) clones would be expected to continue to differentiate in the presence of rapamycin.
Rapamycin Inhibits Differentiation of C2C12 Clones Expressing Constitutively Active S6K1-D3E-E389 -To determine whether C2C12(D3E-E389) clones could terminally differentiate, we cultured the cells in DM in the presence or absence of rapamycin (100 ng/ml). After 3 days, cells were fixed and incubated with a monoclonal antibody to myosin heavy chain (Fig. 2C). The C2C12(D3E-E389) cells differentiated into myotubes, which stained positively for myosin heavy chain. However, differentiation was completely inhibited by rapamycin. Similar results were obtained with four additional clones that we examined (data not shown). These results indicate that the S6K1 pathway downstream of mTOR may not be crucial in the regulation of C2C12 myoblast differentiation. Therefore, we suspect that rapamycin inhibits myogenesis through the mTOR/4E-BP1 pathway. In support of this conjecture, S6K1 activity decreased in C2C12 myoblasts undergoing normal differentiation. In vector control and D3E-E389 clones, S6K1 activity decreased by 55% and 61%, respectively, when cells were cultured in differentiation medium without rapamycin (data not shown).
FIG. 1. Rapamycin inhibits the myogenic differentiation of C2C12 myoblasts but not of C2C12⌬10mTORrr cells. C2C12 cells were seeded in triplicate in 35-mm wells of 6-well plates and kept in GM to attach overnight. The cells were then washed once with serum-free Dulbecco's modified Eagle's medium and cultured in DM in the absence or presence of rapamycin (1, 10, or 100 ng/ml). A, after 3 days, C2C12 cells were examined by immunofluorescence for expression of myosin heavy chain. The photomicrographs show phase-contrast and fluorescence images of the same microscopic field. Results of a representative experiment are shown. B, C2C12 cells were cultured in DM in the presence or absence of rapamycin (100 ng/ml) for up to 3 days. At the times shown, cell lysates were prepared, and the expression of skeletal muscle-specific proteins was determined by Western blot analysis. C, C2C12 cells expressing ⌬10mTORrr are resistant to the growth-inhibitory effect of rapamycin. Parental C2C12 cells (OE) or cell lines stably transfected with empty vector (pcDNA3) (q) or ⌬10mTORrr (f) were grown in GM in the presence or absence of increasing concentrations of rapamycin. Cells were counted after 5 days. The IC 50 value was Ͻ1 ng/ml for parental C2C12 and vector control cells and Ͼ1000 ng/ml for C2C12⌬10mTORrr cells (n ϭ 3, error bars Ͻ symbol size). D, C2C12⌬10mTORrr cells were grown for 3 days in DM in the presence or absence of rapamycin. Cells were fixed and examined by immunofluorescence for myosin heavy chain. The treated and untreated cells differentiated equally to form multinuclear myotubes and expressed myosin heavy chain at rapamycin concentrations up to 100 ng/ml. The photomicrographs show phase-contrast and fluorescence images of the same microscopic field. Results of a representative experiment are shown.

Rapamycin Induces Dephosphorylation of 4E-BP1 in C2C12
Cells-Erbay and Chen (27) reported that whereas rapamycin suppressed activation of S6K1, it had a minor effect on dephosphorylation of 4E-BP1 in C2C12 cells. We examined the effect of rapamycin on 4E-BP1 phosphorylation and function by using three approaches. C2C12 cells were cultured in DM with or without rapamycin (100 ng/ml) for up to 48 h. First, we determined the phosphorylation state of 4E-BP1 by Western blot analysis under electrophoretic conditions that separate isoforms. Rapamycin-induced dephosphorylation of 4E-BP1 was observed in the parental C2C12 cells at all times tested (3-48 h of culture; Fig. 3A); this result is inconsistent with those of Erbay and Chen (27). Second, because mTOR has been reported to phosphorylate 4E-BP1 at residues Thr 37 and Thr 46 (20), we examined the phosphorylation status of Thr 37 and Thr 46 of 4E-BP1 by using phospho-specific antibodies. The phosphorylation of Thr 37 and Thr 46 was slightly decreased (ϳ20 -30%) in rapamycin-treated C2C12 cells (Fig. 3A). Third, we independently determined whether rapamycin affected the binding activity of 4E-BP1 to eIF4E protein. We used the 7-methyl-GTP-Sepharose binding assay of Gingras et al. (32) to detect 4E-BP1 associated with eIF4E. Under control conditions, virtually no 4E-BP1 was associated with eIF4E (Fig. 3B). In contrast, 4E-BP1 was associated with eIF4E in rapamycintreated cells. These results indicate that rapamycin inhibits mTOR signaling to 4E-BP1 under conditions in which it inhibits differentiation.
Rapamycin-resistant mTOR Signaling to 4E-BP1 Requires mTOR Kinase Function and N-terminal Sequences-To further explore how mTOR signaling regulates C2C12 myogenesis, we derived stable C2C12 cell lines expressing mTORrr constructs with N-terminal truncations of 10 (⌬10mTORrr) or 91 S6K1 mutant (D3E-E389). A, C2C12 cells were transfected with expression vectors encoding S6K1 mutant D3E-E389 and selected in G418. Expression of mutant S6K1 was analyzed in six independent clones. Clones were cultured in the presence or absence of rapamycin for 3 days. The expression of mutant S6K1 was detected by immunoprecipitation with anti-Myc monoclonal antibody and by Western blot with rabbit anti-S6K1 antibody. B, S6K1 activity of mutant S6K1 is not inhibited by rapamycin. C2C12(D3E-E389) (clone 36, black bars) and vector control (C2C12pcDNA3, hatched gray bars) cells were seeded in 100-mm dishes (2 ϫ 10 6 cells/dish) and serum-starved for 24 h. Basal kinase activity was determined with and without rapamycin treatment, or cells were stimulated for 1 h with IGF-I (10 ng/ml) in the presence or absence of rapamycin (100 ng/ml). S6K1 was immunoprecipitated with rabbit anti-S6K1 antibody, and its activity was assayed. Results show a representative experiment. C, expression of a constitutively activated S6K1 (D3E-E389) does not overcome rapamycin-induced inhibition of myogenesis. C2C12(D3E-E389) cells were grown in DM in the presence or absence of rapamycin (100 ng/ml) for 3 days. Expression of myosin heavy chain was detected by immunostaining. Representative microscopic fields are shown.

FIG. 3. Rapamycin induces dephosphorylation of 4E-BP1 and association of 4E-BP1 with eIF4E in parent C2C12 cells. A, C2C12
cells were cultured in DM in the presence or absence of rapamycin (100 ng/ml) for 3, 6, 24, and 48 h. Whole-cell lysates were prepared as described under "Experimental Procedures." Equal aliquots of extracts were separated by SDS-PAGE under conditions that did (top panel) or did not (bottom panels) resolve 4E-BP isoforms. Proteins were transferred to polyvinylidene difluoride membranes and incubated with anti-4E-BP1 with phospho-specific antibodies that detect phosphorylated Thr 37 or Thr 46 of 4E-BP1 or with ␤-tubulin (loading control, bottom panel). B, C2C12 cells were cultured in the presence or absence of rapamycin as described above. Cell lysates were prepared at the times shown and incubated overnight at 4°C with 30 l of 7-methyl-GTP-Sepharose beads. After centrifugation, the precipitate was washed with cold PBS, resolved by SDS-PAGE, and transferred to Immobilon-P membranes. Membranes were incubated with anti-4E-BP1 and anti-eIF4E rabbit polyclonal antibodies, as described under "Experimental Procedures." (⌬91mTORrr) amino acids or expressing a kinase-dead (mTOR-rrSIDA) mutant. As shown in Fig. 4A, rapamycin did not cause hypophosphorylation of 4E-BP1 or decrease phosphorylation of Thr 37 or Thr 46 in C2C12⌬10mTORrr cells. In contrast, 4E-BP1 was hypophosphorylated in rapamycin-treated clones expressing C2C12⌬91mTORrr or kinase-dead mTORrrSIDA. Similarly, rapamycin induced only a weak association of 4E-BP1 with eIF4E in C2C12⌬10mTORrr cells, whereas 4E-BP1 associated with eIF4E in the presence of rapamycin in the cells expressing C2C12⌬91mTORrr or kinase-dead mTORrrSIDA (Fig. 4B). These results further indicate that both the N-terminal 11-91 amino acids of mTOR and the kinase domain of mTOR are crucial for phosphorylation of 4E-BP1.
Rapamycin Inhibits Proliferation and Differentiation of C2C12 Cells Expressing ⌬91mTORrr or Kinase-dead mTORrr-SIDA-To determine whether C2C12⌬91mTORrr or C2C12mTORrrSIDA were resistant to the proliferation-inhibitory effects of rapamycin, vector control and clones expressing mTORrr mutants were grown in GM for 5 days with or without rapamycin, and the IC 50 was calculated for each cell line (Fig.  5A). Vector control cells and C2C12 cells expressing ⌬91mTORrr or mTORrrSIDA were equally sensitive to rapamycin. Thus, neither mutant signals to 4E-BP1 in the presence of rapamycin or confers resistance to the growth-inhibitory action of this agent. To determine whether the expression of ⌬91mTORrr or the kinase-dead mTORrrSIDA allowed myogenic differentiation in the presence of rapamycin, we cultured clones in DM with or without rapamycin (1, 10, or 100 ng/ml) for 3 days and used immunofluorescence to detect myosin heavy chain. C2C12 cells that expressed ⌬91mTORrr or mTORrrSIDA differentiated normally in the absence of rapamycin, although cell death was slightly increased in clones expressing the kinase-dead construct (Fig. 5B). Differentiation of both clones was markedly suppressed by 1 ng/ml rapamycin and completely inhibited by 10 ng/ml rapamycin. These results indicate that both the N-terminal 11-91 amino acids and the kinase function of mTOR are essential for the regulation of myogenesis.

DISCUSSION
Myogenic differentiation is a highly complex process regulated by the balance between positive and negative effectors. The insulin-like growth factors (IGF-I and IGF-II) are unique among growth factors in that they stimulate both proliferation and differentiation of muscle cells in culture (10,33). We have shown previously (29,34) that the macrolide antibiotic rapamycin inhibits one signaling pathway downstream of the IGF-I receptor. A rapamycin-sensitive pathway is increasingly thought to be required for myogenic differentiation. Whereas rapamycin potently inhibits cell growth and induces G 1 arrest, it also prevents myogenesis in both L6 and C2C12 myoblasts. However, the precise mechanism by which rapamycin inhibits myogenesis has not been elucidated. Rapamycin, when bound to its cytosolic receptor FK506-binding protein 12, potently inhibits signaling by the serine/threonine kinase mTOR. Although it has been established that inhibition of mTOR is responsible for the growth-inhibitory effect of rapamycin (29), there are only two reports that rapamycin inhibits myogenesis via inhibition of mTOR (12,27). The investigators in the more recent study (27) concluded that the kinase function of mTOR was not essential for myogenic differentiation of C2C12 myoblasts and that rapamycin did not cause hypophosphorylation of 4E-BP1. If these findings are substantiated, they will have identified the first kinase-independent function of mTOR.
To further understand which domains and functions of mTOR are required for myogenesis, we investigated the ability of a rapamycin-resistant mutant mTOR (S2035I) with N-terminal deletions to support downstream signaling and myogenesis in the presence of rapamycin. In parental C2C12 myoblasts, rapamycin potently inhibited differentiation and suppressed or delayed the expression of muscle-specific proteins, confirming previously reported results (10,12,27). C2C12 myoblasts that expressed ⌬10mTORrr were highly resistant to inhibition of proliferation by rapamycin under growth conditions (GM), and they differentiated normally when shifted to DM in the absence of rapamycin. However, FIG. 4. Effect of expression of kinase-dead (mTORrrSIDA), ⌬10mTORrr, or ⌬91mTORrr rapamycin-resistant mutants on rapamycin-induced dephosphorylation of 4E-BP1 in C2C12 myoblasts. Clones of C2C12 that stably expressed rapamycin-resistant mTOR mutants were grown and processed as described in Fig. 3. A, changes in phosphorylation were assessed by electrophoretic mobility and by phospho-specific antibodies (␤-tubulin is shown as a loading control). B, changes in phosphorylation were assessed by binding of 4E-BP1 to 7-methyl-GTP-Sepharose as described in Fig. 3A. All blots were processed using similar conditions for exposure. unlike parental and vector control clones, C2C12 myoblasts expressing ⌬10mTORrr differentiated normally in the presence of high concentrations of rapamycin. This result strongly suggests that inhibition of mTOR by rapamycin is the mechanism responsible for the suppression of differentiation, as reported previously (12,27). Furthermore, deletion of the N-terminal 10 amino acids does not compromise mTOR-dependent myogenesis.
mTOR controls initiation of translation through two downstream pathways: 4E-BP1/eIF4E and ribosomal S6K1. mTOR directly phosphorylates S6K1 at Thr 389 , a residue whose phosphorylation is rapamycin-sensitive in vivo and is necessary for S6 kinase activity (21). When we exposed myoblasts that stably expressed the constitutively active S6K1 mutant to rapamycin, none of five clones examined differentiated, although differentiation occurred in DM in the absence of rapamycin. The basal level of S6K1 activity was increased in these cells and was relatively resistant to inhibition by rapamycin. These findings suggest that inhibition of S6K1 activity is not required for inhibition of myogenesis.
We next assessed inhibition of the mTOR/4E-BP1 pathway by rapamycin. mTOR phosphorylates 4E-BP1 at Thr 37 and Thr 46 and blocks its association with the cap-binding protein eIF4E in vitro. Phosphorylation of Thr 46 appears to be the major regulator of the 4E-BP1-eIF4E interaction in vivo (21). Most studies of the mTOR-4E-BP1 pathway have focused on its role in cell growth and proliferation, although a single report concluded that 4E-BP1 is unlikely to be involved in the rapamycin-sensitive regulation of differentiation in C2C12 cells (27). In our present study, Thr 37 and Thr 46 of 4E-BP1 were hyperphosphorylated in C2C12 cells cultured in either GM or DM, and their phosphorylation was slightly decreased in rapamycin-treated cells. We observed previously (28) that relatively small changes in 4E-BP1 phosphorylation were associated with inhibition of tumor growth by the rapamycin analogue CCI-779. Consistent with this finding, 4E-BP1 associated with eIF4E in rapamycin-treated C2C12 myoblasts. These results differ from those of Erbay and Chen (27). Our study demonstrates potent inhibition of mTOR signaling to 4E-BP1 in rapamycin-treated C2C12 cells. Residues Thr 37 and Thr 46 remained hyperphosphorylated in C2C12 cells expressing ⌬10mTORrr, and 4E-BP1 was not associated with eIF-4E in the presence of rapamycin. Importantly, C2C12⌬10mTORrr cells continued to differentiate in the presence of rapamycin, whereas neither C2C12⌬91mTORrr nor kinase-dead mTORrr-SIDA prevented rapamycin-induced hypophosphorylation of 4E-BP1 and association of 4E-BP1 with eIF4E.
It has been reported that a kinase-inactive rapamycin-resistant mTOR mutant (D2357E) can support myogenic differentiation of C2C12 myoblasts in the presence of rapamycin (27). That report is the first to describe an mTOR function that is independent of mTOR kinase activity. It has been shown that mTOR signaling to 4E-BP1 is dependent on its kinase function (15). Signaling to S6K1 was also shown to require kinase activity and was abrogated by deletion of 70 residues from the N terminus of mTOR (35). We therefore examined the ability of two other rapamycin-resistant deletion mutants to support proliferation and myogenesis in rapamycin-treated C2C12 cells. Proliferation of C2C12⌬91mTORrr and kinase-dead mTORrrSIDA cells was as sensitive to inhibition by rapamycin as that of control or parental C2C12 cells. The ⌬91mTORrr and kinase-dead mTORrrSIDA cells differentiated normally in DM medium, but neither differentiated in the presence of rapamycin. These results, which indicate that the kinase function of mTOR is required for myogenic differentiation, are contradictory to those of Erbay and Chen (27).
In our study, C2C12⌬10mTORrr myoblasts were rapamycin resistant and differentiated in the presence of rapamycin, but C2C12⌬91mTORrr myoblasts were sensitive to rapamycin, and their differentiation was inhibited. This result indicates that the region of amino acids 11-91 in the N terminus of mTOR has a functional domain that is crucial for mTOR downstream signaling to both 4E-BP1 and S6K1 (35). The N-terminal 1200 amino acids of mTOR proteins comprise a HEAT domain (named for the first proteins found to possess such a motif: Huntingtin, elongation factor 3, the regulatory A subunit of PP2A, and Tor1p; Refs. 36 -38). HEAT domains form curved rods that consist of ␣-loop-␣ repeats and provide a large hydrophobic surface area for potential protein-protein interactions (39 -42). Because ⌬91mTORrr is truncated in this HEAT sequence, protein interactions may be disturbed.
Taken together, our study substantiates the finding that mTOR plays a crucial role in controlling myogenesis in C2C12 cells. mTOR-dependent activation of the S6K1 pathway does not appear to be essential for muscle cell differentiation, whereas signaling to 4E-BP1 appears to be important. The N-terminal amino acids 11-91 and the kinase domain of mTOR appear to be essential for regulating myogenesis.
Acknowledgments-We thank Franklin Harwood for technical assistance and Sharon Naron for preparation of the manuscript.

FIG. 5. Rapamycin inhibits the proliferation and differentiation of C2C12 cells that stably express ⌬91mTORrr and kinase-dead mTORrrSIDA mutants.
A, C2C12⌬91mTORrr (f), C2C12mTORrrSIDA (OE), or vector control (q) cells were cultured in GM in the presence or absence of increasing concentrations of rapamycin. After 5 days, cells were counted as described under "Experimental Procedures" (n ϭ 3; error bars Ͻ symbol size). B, C2C12⌬91mTORrr (top panels) and C2C12mTORrrSIDA (bottom panels) cells were cultured in DM for 3 days in the presence or absence of rapamycin (1, 10, or 100 ng/ml) and examined by immunofluorescence for expression of myosin heavy chain.