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J. Biol. Chem., Vol. 277, Issue 20, 17657-17662, May 17, 2002

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Control of Ser²⁴⁴⁸ Phosphorylation in the Mammalian Target of Rapamycin by Insulin and Skeletal Muscle Load^*

Thomas H. Reynolds IV, Sue C. Bodine^§, and John C. Lawrence Jr.^¶

From the Departments of  Pharmacology and ^¶ Medicine, University of Virginia Health System, Charlottesville, Virginia 22908-0735 and ^§ Regeneron Pharmaceuticals, Inc., Tarrytown, New York 10591-6707

Received for publication, February 4, 2002, and in revised form, March 4, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have investigated the effects of insulin, amino acids, and the degree of muscle loading on the phosphorylation of Ser²⁴⁴⁸, a site in the mammalian target of rapamycin (mTOR) phosphorylated by protein kinase B (PKB) in vitro. Phosphorylation was assessed by immunoblotting with a phosphospecific antibody (anti-Ser(P)²⁴⁴⁸) and with mTAb1, an activating antibody whose binding is inhibited by phosphorylation in the region of mTOR that contains Ser²⁴⁴⁸. Incubating rat diaphragm muscles with insulin increased Ser²⁴⁴⁸ phosphorylation but did not change the total amount of mTOR. Insulin, but not amino acids, activated PKB, as evidenced by increased phosphorylation of both Ser³⁰⁸ and Thr⁴⁷³ in the kinase. Ser²⁴⁴⁸ phosphorylation was also modulated by muscle-loading. Overloading the rat plantaris muscle by synergist muscle ablation, which promotes hypertrophy of the plantaris muscle, increased Ser²⁴⁴⁸ phosphorylation. In contrast, unloading the gastrocnemius muscle by hindlimb suspension, which promotes atrophy of the muscle, decreased Ser²⁴⁴⁸ phosphorylation, an effect that was fully reversible. Neither overloading nor hindlimb suspension significantly changed the total amount of mTOR. In summary, our results demonstrate that atrophy and hypertrophy of skeletal muscle are associated with decreases and increases in Ser²⁴⁴⁸ phosphorylation, suggesting that modulation of this site may have an important role in the control of protein synthesis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Skeletal muscle mass is controlled by several factors including insulin, amino acids, and the degree of muscular activity (1-4). Muscle wasting is a hallmark of untreated Type 1 diabetes mellitus in humans (5), as well as in experimentally induced diabetes in animals (6). Restoring insulin to diabetics blocks the loss of muscle protein in humans (5) and increases muscle protein synthesis in animal models of diabetes (7-9). Although a net accumulation of protein cannot occur without an adequate supply of the precursor amino acids, certain amino acids exert a control on the rate of protein synthesis through a more specific mechanism. Branched chain amino acids, leucine in particular, stimulate protein synthesis by activating a nutrient-sensing pathway (4). Increasing the load on a muscle promotes hypertrophy (10), and unloading causes atrophy (11).

The effects of insulin, amino acids, and loading involve changes in the rates of muscle protein synthesis (4, 12, 13), a process that involves mRNA translation. The stimulatory effects on mRNA translation are mediated in part by the phosphorylation of the mRNA translational regulators, PHAS-I and p70^S6K (4). Nonphosphorylated PHAS-I binds to eIF4E,¹ the mRNA cap-binding protein, and prevents eIF4E from interacting with eIF4G. When phosphorylated in the appropriate sites, PHAS-I dissociates from eIF4E, allowing eIF4E to bind eIF4G to form an initiation complex needed for efficient binding and/or scanning by the 40 S ribosomal subunit. p70^S6K is a mitogen-activated protein kinase whose activation has been proposed to promote increased translation of messages, which have a polypyrimidine motif just downstream of the 5' cap (4).

Both PHAS-I and p70^S6K are controlled by mTOR, the 2459-amino acid mammalian counterpart of the tor1p and tor2p proteins that control protein synthesis and cellular growth in Saccharomyces cerevesiae (14). mTOR functions as a growth factor and nutrient-sensing signaling molecule in mammalian cells. Defining the role of mTOR has been greatly facilitated by a highly specific inhibitor, rapamycin. To inhibit mTOR rapamycin requires as a partner the FK506-binding protein, FKBP12, because it is only when complexed with FKBP12 that rapamycin is able to bind mTOR with high affinity (15). This high affinity interaction has been exploited to purify mTOR from mammalian tissues and cell lines (see for examples Refs. 16 and 17). The effects of both insulin and amino acids on increasing the phosphorylation of PHAS-I and p70^S6K in skeletal muscle are attenuated by rapamycin (18). Recently, rapamycin was shown to block both the compensatory hypertrophy of the plantaris muscle resulting from synergist ablation in vivo and the increase in muscle mass associated with recovery from disuse atrophy (2). These findings with rapamycin have implicated mTOR in the control of skeletal muscle mass by muscle loading.

The mechanisms through which mTOR signals and how mTOR activity is controlled are unclear. mTOR is able to phosphorylate both PHAS-I and p70^S6K in vitro (16, 19), although it is still not certain whether mTOR phosphorylates these proteins in cells. There is evidence that mTOR is controlled by PKB, which is activated in response to phospholipid products of the phosphatidylinositol 3-kinase reaction. PKB is able to phosphorylate Ser²⁴⁴⁸ in mTOR in vitro (20, 21), and this site becomes phosphorylated in response to PKB activation in cells (20-22). Phosphorylation of this region of mTOR was first detected using the antibody, mTAb1, whose reactivity with mTOR was decreased in response to phosphorylation (22). Subsequently, phosphorylation of Ser²⁴⁴⁸ was confirmed by using phosphospecific antibodies to this site (20, 21).

Interestingly, there is other evidence that the region surrounding Ser²⁴⁴⁸ is an important regulatory domain in mTOR. Binding of the antibody, mTAb1, to this region markedly increases the protein kinase activity of mTOR in vitro (21, 22). Also, recombinant mTOR in which the region containing the mTAb1 epitope has been deleted exhibits increased activity, both in vivo and in vitro (21). These findings suggest that phosphorylation of Ser²⁴⁴⁸ may be important in controlling mTOR. Therefore, we conducted experiments to determine whether treatments known to modulate skeletal muscle mass affect the phosphorylation of Ser²⁴⁴⁸.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Isolation and Incubation of Rat Diaphragm Muscles-- All incubations were performed at 37 °C in medium continuously bubbled with a 19:1 mixture of O₂/CO₂ essentially as described previously (18). Briefly, male Sprague-Dawley rats (~150 g) were sacrificed by decapitation, and diaphragm muscles were excised and incubated in Dulbecco's modified Eagle medium (30 ml/muscle) for 45 min to remove endogenous hormones. The muscles were then incubated with or without insulin (250 milliunits/ml) and/or a 2.5× mixture of minimal essential medium amino acids in Krebs-Henseleit buffer (118 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM potassium phosphate, and 25 mM NaHCO₃, pH 7.4) containing 5 mM glucose. After 20 min the muscles were blotted on filter paper and immediately frozen in liquid N₂.

Skeletal Muscle Loading and Unloading-- The procedures for loading plantaris muscles by synergist ablation and unloading gastrocnemius muscles by hindlimb suspension were conducted exactly as described previously (2). Briefly, rats (Sprague-Dawley, ~250 g) were anesthetized with ketamine/xylazine (50:10 mg/kg, intraperitoneal). For synergist ablation, the soleus, medial gastrocnemius, and lateral gastrocnemius muscles were removed bilaterally using aseptic surgical techniques (2). This produces a functional overload on the plantaris muscles (10). For hindlimb suspension studies, anesthetized animals were fitted with a tail-traction bandage. Following recovery from the anesthetic, a swivel hook was placed through the bandage and raised so that the hindlimbs were suspended just off the cage floor (11). At the appropriate time after surgery, the rats were sacrificed, and the plantaris muscles or gastrocnemius muscles were removed, frozen in liquid N₂, and stored at 80 °C. Muscles from weight-matched surgically untreated rats served as controls.

Affinity Purification of mTOR-- Crude extracts of skeletal muscle contain large amounts of myosin heavy chain, which comigrates with mTOR and prevents detection of mTOR by immunoblotting. Therefore, mTOR was affinity-purified prior to electrophoresis. The frozen muscles were manually ground with a porcelain mortar and pestle chilled in liquid N₂. Powdered tissue was homogenized on ice using a motor-driven tissue grinder (Teflon-glass) in 3 ml of homogenization buffer, which was composed of Buffer A (50 mM NaCl, 10 mM NaF, 0.25% Tween 20, 10% glycerol, 0.1 mM dithiothreitol, 500 nM microcystin-LR, 50 mM Tris/HCl, pH 7.4) supplemented with 0.1 mM phenylmethylsulfonyl fluoride and 10 µg/ml each of aprotinin, leupeptin, and pepstatin-A. The homogenates were rotated at 4 °C for 1 h and then centrifuged at 8,900 × g for 30 min at 4 °C. The protein concentrations of the supernatants were determined by the BCA method (Pierce).

To purify mTOR, 100 µg of a recombinant glutathione S-transferase (GST) FKBP12 fusion protein (GST-FKBP12), prepared as previously described (16, 17), were incubated with 25 µl of glutathione-Sepharose beads in Buffer B (145 mM NaCl and 10 mM sodium phosphate, pH 7.4) containing 10% bovine serum albumin for 1 h at 21 °C. The beads were then washed three times with homogenization buffer (1 ml/wash) and incubated with 1 ml of muscle extract (1 mg of protein) plus 10 µM rapamycin (Calbiochem) or where indicated, 10 µM FK506 (Fujisawa Pharmaceuticals). After 90 min at 4 °C, the beads were washed twice with Buffer A, twice with Buffer A plus 500 mM NaCl, and then twice in Buffer A.

Antibodies-- The mTOR antibodies mTAb1 and mTAb2, are the same as used in previous studies (16) and were generated by immunizing rabbits with peptides having sequences corresponding to residues 2433-2450 and 1272-1290, respectively, in mTOR. Antibodies to the -isoform of PKB were from Upstate Biotechnology. Phosphospecific antibodies to the Ser³⁰⁸ and Thr⁴⁷³ sites in PKB were from New England Biolabs.

To generate phosphospecific antibody to the Ser²⁴⁴⁸ site in mTOR, essentially the same procedures were used as described previously for the preparation of phosphospecific antibodies to sites in PHAS-I (23) except that different peptides were used. Briefly, rabbits were immunized with a phosphopeptide (CTRTDS*YSAGQS, where S* is phosphoserine) coupled to keyhole limpet hemocyanin. After the second booster injection, serum was collected and incubated with an affinity resin prepared by coupling a nonphosphorylated peptide to Sulfo-Link beads (Pierce). The unbound fraction was then incubated with a phosphopeptide resin. After exhaustively washing the resin, the anti-Ser(P)²⁴⁴⁸ antibodies were eluted with 0.3 M glycine/HCl (pH 2.7), neutralized, and further purified with protein A-Sepharose.

Electrophoretic Analyses and Immunoblotting of mTOR and PKB-- Samples of affinity-purified mTOR were subjected to SDS-PAGE (7.5% polyacrylamide gel) (24). The proteins were then electrophoretically transferred to Immobilon membranes and immunoblotted with mTOR and PKB antibodies as described previously (16, 22). After washing the membranes, the light generated by the alkaline phosphatase-conjugated secondary antibody and Tropix reagent was detected using x-ray film (Kodak XAR-5). Relative signal intensities of the mTOR bands were determined using a scanning laser densitometer (Molecular Dynamics). To assess mTOR phosphorylation relative to total mTOR, immunoblots prepared with anti-Ser(P)²⁴⁴⁸ or mTAb1 were stripped and reprobed with mTAb2, whose binding is not affected by phosphorylation of Ser²⁴⁴⁸. After correcting for the total amount of mTOR present, the statistical significance of differences among groups was assessed by analysis of variance and the Fisher least significant difference post-hoc test (25). An  level of p < 0.05 was accepted for statistical significance.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Identification of mTOR in Skeletal Muscle-- mTOR was affinity-purified from skeletal muscle extracts by using GST-FKBP12/rapamycin resin (16, 17) before immunoblots were prepared. mTOR appeared as a single band when immunoblotted with mTAb2 (Fig. 1, lane 1). The FKBP12/FK506 complex does not bind to mTOR (15). Therefore, as a control, the affinity purification procedure was conducted in an identical manner except that FK506 was substituted for rapamycin. As expected, no mTOR was detected in the sample generated with FK506 (Fig. 1, lane 2).

View larger version (43K): [in this window] [in a new window]   Fig. 1.   Affinity purification of mTOR by GST-FKBP12/rapamycin in rat gastrocnemius muscle. Rat muscle extracts were incubated with GST-FKBP12 resin and either rapamycin (RAP) (lane 1) or FK506 (lane 2). After washing the resins, proteins were eluted and subjected to SDS-PAGE. An immunoblot prepared with mTAb2 is shown.

Effects of Insulin and/or Amino Acids on Ser²⁴⁴⁸ Phosphorylation-- Because insulin and amino acids were known to modulate downstream effectors in the mTOR pathway, we conducted experiments to determine whether incubating muscles with these agents affected the phosphorylation of Ser²⁴⁴⁸ in mTOR. We chose to use hemidiaphragms for these experiments because these muscles may be maintained in a viable, insulin-responsive state during short term incubations in vitro. Phosphorylation of Ser²⁴⁴⁸ was assessed by immunoblotting with the phosphospecific antibody, anti-Ser(P)²⁴⁴⁸. mTOR that had been affinity-purified from control cells was readily detected with the anti-Ser(P)²⁴⁴⁸ antibody, indicating that this site contained some phosphate even in the basal state (Fig. 2A). Incubating muscles with either a mixture of amino acids² or insulin increased Ser²⁴⁴⁸ phosphorylation by ~1.5- or 2-fold, respectively (Fig. 2B). The combination of insulin plus amino acids was no more effective in increasing Ser²⁴⁴⁸ phosphorylation than insulin alone.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Effects of insulin and/or amino acids on phosphorylation of the COOH-terminal regulatory domain of mTOR in isolated diaphragm muscles. Muscles were incubated for 20 min in Krebs Henseleit buffer containing 5 mM glucose and the following: no additions (NA), a 2.5× mixture of minimum essential medium amino acids (AA), 250 milliunits/ml insulin (I), or the combination of insulin and/or amino acids (I+AA). mTOR was affinity-purified using rapamycin and GST-FKBP12. Samples were subjected to SDS-PAGE, and immunoblots were prepared with the anti-Ser(P)2448 (pSer2448) and mTAb1 antibodies (A). The pSer2448 blot was then stripped and reprobed with mTAb2. Band intensities from the pSer2448 immunoblots and the mTAb 1 immunoblots were determined by optical density scanning. These values were corrected for differences in the total amount of mTOR recovered, which was assessed by the mTAb2 signals. Results from pSer2448 (B), mTAb1 (C), and mTAb2 (D) are expressed as percentages of the respective control values and are means ± S.E. from four experiments. *, p < 0.05 versus NA; **, p < 0.01 versus NA.

Immunoblots were also prepared with mTAb1, whose binding is decreased by phosphorylation of one or more sites in the regulatory region surrounding Ser²⁴⁴⁸. Insulin plus amino acids decreased the mTAb1 signal by ~40% (Fig. 2C). Although the results were somewhat more variable than that observed with the anti-Ser(P)²⁴⁴⁸ antibody, the findings with mTAb1 provide independent confirmation that the phosphorylation of mTOR was increased by insulin. To determine whether the treatments affected the total amount of mTOR present, the blots were stripped and reprobed with mTAb2, whose binding is not affected by phosphorylation of Ser²⁴⁴⁸. The reactivity of mTOR with mTAb2 was not significantly changed by insulin or amino acids (Fig. 2D).

Because PKB has been shown to phosphorylate mTOR on Ser²⁴⁴⁸ in vitro (20, 21), we investigated the abilities of insulin and amino acids to activate this kinase. This was accomplished by using phosphospecific antibodies to assess changes in the phosphorylation of Ser³⁰⁸ and Thr⁴⁷³. These two sites become phosphorylated when PKB binds phospholipid products of the phosphatidylinositol 3-kinase reaction, and phosphorylation of both is required for full activation of the kinase (26). Insulin markedly increased the phosphorylation of both Ser³⁰⁸ and Thr⁴⁷³ (Fig. 3). In contrast, amino acids, either alone or in combination with insulin, had little if any effect on PKB phosphorylation (Fig. 3).

View larger version (38K): [in this window] [in a new window]   Fig. 3.   Effects of insulin and/or amino acids on PKB phosphorylation in isolated diaphragm muscles. Muscles were incubated as described in the legend to Fig. 2. Extracts were immunoblotted with PKB- or phosphospecific PKB antibodies.

Effects of Skeletal Muscle Loading on Ser²⁴⁴⁸ Phosphorylation-- Recent findings have implicated mTOR in the control of muscle fiber size in response to changes in load (2). Therefore, we conducted experiments to investigate the effect of loading on the phosphorylation of mTOR. In the first series of experiments, we investigated the phosphorylation of Ser²⁴⁴⁸ in a synergist ablation model of compensatory hypertrophy. In this model, the gastrocnemius and soleus muscles are surgically removed. This results in overloading of the plantaris muscle, which undergoes compensatory hypertrophy (10, 13). Under the conditions of the present experiments, the weight of the plantaris muscle was increased by 40% (2). In Fig. 4A, samples of plantaris muscles from control animals and samples obtained 2 weeks following synergist ablation were immunoblotted with mTOR antibodies. Immunoreactivity with the phosphospecific antibody to Ser(P)²⁴⁴⁸ was increased more than 2-fold by overloading the muscle (Fig. 4B). A reciprocal change was observed in the mTAb1 signal, although the effect was somewhat smaller than that observed with the anti-Ser(P)²⁴⁴⁸ antibody (Fig. 4C). Immunoblotting with mTAb2 indicated that the extract samples contained approximately equal amounts of mTOR (Fig. 4D). Thus, the changes in anti-Ser(P)²⁴⁴⁸ and mTAb1 binding indicate that synergist ablation increased the phosphorylation of mTOR.

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Effects of overloading rat plantaris muscles on mTOR phosphorylation. Plantaris muscles were isolated from rats 14 days following synergist muscle ablation (SA). mTOR was affinity-purified from muscle extracts and subjected to SDS-PAGE before immunoblots were prepared (A). Band intensities were measured by optical density scanning, and the values for anti-Ser(P)2448 (pSer2448) and mTAb1 were corrected for differences in the total amount of mTOR recovered, which was assessed by the mTAb2 signals. Results from pSer2448 (B), mTAb1 (C), and mTAb2 (D) are expressed as percentages of the respective control (CON) values and are means ± S.E. from four animals. *, p < 0.05 versus CON.

Because increasing muscle load increased phosphorylation of mTOR, we were interested in determining whether decreasing load would decrease phosphorylation of the protein. To investigate this possibility, rats were suspended by their tails to unload their hindlimbs. Fourteen days of this treatment decreased the weight of the gastrocnemius muscle by 35% (2). Unloading the gastrocnemius muscles in this manner decreased the phosphorylation of Ser²⁴⁴⁸ by ~60% (Fig. 5, A and B). Restoring load on the muscles by removing the suspension apparatus fully reversed the effect of unloading. Hindlimb suspension was without effect on binding of mTAb1 (Fig. 5C) or on the total amount of mTOR (Fig. 5D)

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effects of hindlimb suspension on mTOR phosphorylation in gastrocnemius muscle. Muscles were isolated from rats after 14 days of hindlimb suspension (HLS) or after 14 days of hindlimb suspension followed by 7 days of recovery (REC). mTOR was affinity-purified and immunoblots with the three mTOR antibodies were prepared (A). After optical density scanning to estimate band intensities, the values for mTAb1 and anti-Ser(P)2448 (pSer2448) were adjusted to correct for the total amount of mTOR recovered. The results from pSer2448 (B), mTAb1 (C), and mTAb2 (D) are expressed as percentages of the respective control (CON) values and are means ± S.E. from four animals. *, p < 0.05 versus CON; **, p < 0.01 versus HLS.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Insulin and muscle loading were found to increase the phosphorylation of Ser²⁴⁴⁸. These findings provide the first direct evidence of modification of mTOR in skeletal muscle and have potentially important implications in the control of muscle protein synthesis.

Previous results suggested that Ser²⁴⁴⁸ is important in the control of mTOR activity. Brunn et al. (16) demonstrated that mTAb1, which binds to the region of mTOR containing this site, markedly increased the PHAS-I kinase activity of mTOR. Scott et al. (22) later noted that insulin promoted the phosphorylation of mTOR in this region and proposed that phosphorylation of Ser²⁴⁴⁸ activated mTOR (22). Next, Sekulic et al. (21) generated a mutant recombinant mTOR lacking the 20-amino acid mTAb1 epitope and discovered that the mutant enzyme exhibited enhanced protein kinase activity. Based on these observations, it was suggested the region containing the epitope was an inhibitory regulatory domain and that phosphorylation of Ser²⁴⁴⁸ might activate mTOR by relieving the inhibition. Activation of mTOR would be expected to lead to increased phosphorylation of the downstream effectors, PHAS-I and p70^S6K, resulting in increased protein synthesis.

Results from mutagenesis studies have cast some doubt on the significance of Ser²⁴⁴⁸ phosphorylation. Sekulic et al. (21) investigated the effects of overexpressing an mTOR protein having an Ala²⁴⁴⁸ mutation on the phosphorylation of p70^S6K and PHAS-I in HEK293 cells. The effects of the Ala²⁴⁴⁸-mutated mTOR in supporting activation of p70^S6K in response to insulin were no different from those of mTOR with Ser²⁴⁴⁸. This finding would argue strongly against a role of Ser²⁴⁴⁸ phosphorylation as an important regulator of mTOR function were it not for the fact that the overexpression studies were conducted with forms of mTOR rendered rapamycin-resistant by mutating Ser²⁰³⁵ to Ile. We have recently discovered that this mutation markedly inhibits the ability of mTOR to phosphorylate PHAS-I in vitro.³ Thus, preventing phosphorylation of Ser²⁴⁴⁸ by the Ala²⁴⁴⁸ mutation in rapamycin-resistant mTOR (Ser²⁰³⁵ to Ile mutation) would not be expected to decrease activity toward PHAS-I, because this form of mTOR already exhibits reduced activity. Additional studies will be needed to address the functional effect of Ser²⁴⁴⁸ phosphorylation on mTOR activity.

The effects of insulin on increasing Ser²⁴⁴⁸ in hemidiaphragms were associated with activation of PKB (Fig. 3). This finding is consistent with other studies, which have generated three main lines of evidence supporting the conclusion that the phosphorylation of Ser²⁴⁴⁸ is controlled by PKB (20, 21). First, Ser²⁴⁴⁸ and the surrounding sequence is a good fit to the consensus motif (RXRXX(S/T)h), where X is any amino acid and h is a hydrophobic residue) for phosphorylation by PKB (27), and Ser²⁴⁴⁸ can be phosphorylated by purified PKB in vitro (20, 21). Second, increasing PKB in cells is associated with increased phosphorylation of Ser²⁴⁴⁸. Insulin activated endogenous PKB activity and increased the phosphorylation of Ser²⁴⁴⁸ in HEK293 cells, CHOIR800 cells, and 3T3L1 adipocytes (20, 22). Moreover, overexpressing a constitutively active PKB in HEK293 cells or activating a PKB-estrogen fusion protein with tamoxifen in MER-Akt cells decreased mTAb1 binding, suggestive of increased phosphorylation of Ser²⁴⁴⁸ (22). Third, inhibition of PKB is associated with decreases in Ser²⁴⁴⁸ phosphorylation. In 3T3L1 adipocytes, phosphorylation of Ser²⁴⁴⁸ was blocked by inhibitors of phosphatidylinositol 3-kinase, which also block activation of PKB (22). Also, overexpressing a kinase-dead PKB in HEK293 cells blocked Ser²⁴⁴⁸ phosphorylation (21).

Like insulin, the degree of muscle loading can alter mRNA translation, protein synthesis, and muscle mass (1, 2, 12, 13). Load-dependent changes in skeletal muscle size are partly explained by changes in mRNA translation (1-3). Muscle atrophy induced by hindlimb suspension (2, 3) is associated with a decrease in the phosphorylation of p70^S6K; whereas, muscle hypertrophy following resistance training is associated with an increase in p70^S6K phosphorylation (1). Furthermore, overload-induced hypertrophy of the rat plantaris muscle is associated with an increase in p70^S6k activity and a decrease in PHAS-1 bound to eIF4E, indicating an increase in mRNA translation initiation (2). Interestingly, the changes in Ser²⁴⁴⁸ phosphorylation observed with muscle loading and unloading correlate well with the changes in PKB phosphorylation noted by Bodine et al. (2). Thus, both muscle mass and PKB phosphorylation were decreased in gastrocnemius muscles following hindlimb suspension, and synergist ablation resulted in an ~40% increase in the mass of the plantaris muscle and promoted a severalfold increase in the phosphorylation of PKB (2). These findings are consistent with the hypothesis that changes in PKB activity are responsible for the changes in Ser²⁴⁴⁸ phosphorylation observed in muscles undergoing atrophy or hypertrophy in response to changes in muscle load.

Nave et al. (20) have presented evidence that insulin and amino acid signaling converge at mTOR in HEK293 cells. The present findings in skeletal muscle are consistent with such a view, as insulin promoted the phosphorylation of Ser²⁴⁴⁸. However, there are some differences between our results and those of Nave et al. (20). In HEK293 cells amino acid withdrawal decreased Ser²⁴⁴⁸ phosphorylation and abolished the ability of insulin to stimulate Ser²⁴⁴⁸ phosphorylation. In hemidiaphragms exogenous amino acids were not required for the stimulatory effect of insulin on Ser²⁴⁴⁸ phosphorylation. Thus, there may be differences among cell types in the relative requirements for insulin and amino acids for the phosphorylation of Ser²⁴⁴⁸. This would not be surprising as different cells exhibit differing requirements for exogenous amino acids for the stimulatory effects of insulin on the phosphorylation of PHAS-I and p70^S6K (28, 29). However, in muscles incubated in vitro there may be adequate endogenous amino acids to allow for an insulin effect in the absence of exogenous amino acids. Amino acids did not activate PKB in skeletal muscle (Fig. 3), which is in agreement with results obtained in numerous cell types (28, 29).

In summary, the present study indicates that insulin activates PKB and promotes the phosphorylation of Ser²⁴⁴⁸ in mTOR in skeletal muscle. Furthermore, the results demonstrate that increasing muscle load leads to increased phosphorylation of this site. The fact that these treatments increase mRNA translation (1, 2, 4), indicates that increased phosphorylation of Ser²⁴⁴⁸ might have an important functional role and is at the very least a marker for increased protein synthesis in skeletal muscle.

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants DK-52753 and AR-41180.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Pharmacology, University of Virginia Health System, P.O. Box 800735, 1300 Jefferson Park Ave., Charlottesville, VA 22908-0735. Tel.: 434-924-1584; Fax: 434-982-3575; E-mail: jcl3p@virginia.edu.

Published, JBC Papers in Press, March 7, 2002, DOI 10.1074/jbc.M201142200

² Although the amino acid and control values were judged to be not significantly different by an analysis of variance test, p was less than 0.05 by Student's t test.

³ L. McMahon and J. C. Lawrence, Jr., manuscript in preparation.

	ABBREVIATIONS

The abbreviations used are: eIF, eukaryotic initiation factor; mTOR, mammalian target of rapamycin; GST, glutathione S-transferase; HEK, human embryonic kidney cells; PKB, protein kinase B.

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