The Mammalian Target of Rapamycin Phosphorylates Sites Having a (Ser/Thr)-Pro Motif and Is Activated by Antibodies to a Region near Its COOH Terminus

The eukaryotic initiation factor 4E (eIF4E)-binding protein, PHAS-I, was phosphorylated rapidly and stoichiometrically when incubated with [γ-32P]ATP and the mammalian target of rapamycin (mTOR) that had been immunoprecipitated with an antibody, mTAb1, directed against a region near the COOH terminus of mTOR. PHAS-I was phosphorylated more slowly by mTOR obtained either by immunoprecipitation with other antibodies or by affinity purification using a rapamycin/FKBP12 resin. Adding mTAb1 to either of these preparations of mTOR increased PHAS-I phosphorylation severalfold, indicating that mTAb1 activates the mTOR protein kinase. mTAb1-activated mTOR phosphorylated Thr36, Thr45, Ser64, Thr69, and Ser82 in PHAS-I. All five of these sites fit a (Ser/Thr)-Pro motif and are dephosphorylated in response to rapamycin in rat adipocytes. Thus, our findings indicate that Pro is a determinant of the mTOR protein kinase specificity and that mTOR contributes to the phosphorylation of PHAS-I in cells.

The mammalian target of rapamycin, mTOR 1 (1) (also known as FRAP (2) or RAFT1 (3)), is a homolog of the Tor1p and Tor2p proteins that are required for cell cycle progression in Saccharomyces cerevisiae (4). Like the yeast proteins, mTOR has a COOH-terminal catalytic domain that is homologous to those found in yeast and mammalian phosphatidylinositol 3-OH kinases, although none of the TOR proteins have been shown to possess intrinsic lipid kinase activity (5). The function of mTOR is inhibited by incubating cells with rapamycin, a potent immunosuppressant and antiproliferative agent (5). FKBP12, an M r ϭ 12,000 member of a family of FK506-binding proteins, is the intracellular receptor for rapamycin and is required for the inhibitory effect of rapamycin on mTOR function. The rapamycin⅐FKBP12 complex binds with very high affinity to mTOR, a property that has proven useful for purifying the protein. The FK506⅐FKBP12 complex does not bind mTOR, and FK506 competitively inhibits the effects of rapa-mycin that are mediated by mTOR.
PHAS-I (6), also known as 4E-BP1 (7), is an important target of the mTOR-signaling pathway (8 -11). Nonphosphorylated PHAS-I binds tightly to eIF4E (7,12), the mRNA cap-binding protein, and prevents binding of eIF4E to eIF4G (13)(14)(15), a key step in translation initiation (16). When phosphorylated in response to insulin, growth factors, or other stimuli, PHAS-I dissociates from eIF4E, removing the impediment to initiation (17,18). In adipocytes, insulin promotes the phosphorylation of PHAS-I at five Ser/Thr residues, each of which is located on the NH 2 -terminal side of an adjacent Pro residue (19). Rapamycin promotes dephosphorylation of these sites and attenuates the effects of insulin (19).
Immunoprecipitates containing mTOR from rat brain were recently found to phosphorylate PHAS-I in vitro (9). The kinase reaction proceeded much more rapidly with Mn:ATP than with Mg:ATP, occurred on both Ser and Thr residues, and markedly decreased the eIF4E binding activity of PHAS-I. In this report, we describe an antibody that activates mTOR and identify the sites in PHAS-I that are phosphorylated by mTOR. These results provide important insight into both the substrate specificity of mTOR and mechanisms by which the kinase might be regulated.

EXPERIMENTAL PROCEDURES
Antibodies-Peptides having NH 2 -terminal C residues followed by sequences identical to positions 2433-2450 (DTNAKGNKRSRTRTD-SYS) and 1272-1290 (ARRVSKDDWLEWLRRLSLE) in mTOR (1) were coupled to keyhole limpet hemocyanin, and the peptide-hemocyanin conjugates were used to immunize rabbits by using the procedures described previously (20). Antibodies, designated mTAb1 and mTAb2, respectively, were purified by using columns containing affinity resins prepared by coupling peptides to Sulfolink beads (Pierce).
Measurements of PHAS-I Kinase Activity After Affinity Purification and Immunoprecipitation of mTOR-Rat brains were rinsed and homogenized (1 g of tissue/ml of buffer) by using a Polytron tissue disrupter (setting of 4 for 10 s, then setting of 8 for 10 s). Homogenization buffer contained solution A (100 mM NaCl, 10% glycerol, and 50 mM Tris/HCl, pH 7.4) supplemented with 2 mM ␤-mercaptoethanol, 10 g/ml leupeptin, 10 g/ml aprotinin, 5 g/ml pepstatin, and 1 mM phenylmethylsulfonyl fluoride. The homogenate was centrifuged at 14,500 ϫ g for 30 min at 4°C, and the supernatant was retained for purification of mTOR.
[His 6 ]PHAS-I (21) and GST-FKBP12 (1) were expressed in bacteria and purified as described previously. To purify mTOR, GST-FKBP12 (100 g) was incubated with 25 l glutathione-agarose (Sigma) in Tris-buffered saline (100 mM NaCl and 50 mM Tris/HCl, pH 7.4). After 1 h at 21°C, the resin was washed 3 times (1 ml/wash) with Trisbuffered saline, then suspended in homogenization buffer, and incubated (1 ml final volume) at 4°C with extract (3 mg of protein), 200 nM microcystin-LR, and 10 M rapamycin (Calbiochem). Control incubations were conducted with FK506 (Fujisawa Pharmaceuticals). After 1 h the beads were washed twice (1 ml/wash) with solution A plus 1 mM dithiothreitol, then washed twice with solution B (50 mM NaCl, 1 mM dithiothreitol, and 10 mM Na-HEPES, pH 7.4). For immunoprecipitations, antibodies (5 g) were incubated at 21°C with protein A-agarose * This work was supported by National Institutes of Health Grants DK52753, DK28312, and AR41180. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Electrophoretic Analyses-Samples were subjected to SDS-PAGE by using the method of Laemmli (22). 32 P-Labeled [His 6 ]PHAS-I was detected by autoradiography after drying the gels, and the amount of 32 P was determined by scintillation counting of gel slices containing the protein. mTOR (9) and PHAS-I (12) were detected by immunoblotting as described previously. 6 ]PHAS-I was phosphorylated with mTOR that had been immunoprecipitated using mTAb1 as described above. Samples of the phosphorylated protein were digested with lysyl endopeptidase C (WAKO Pure Chemical Industries) and chymotrypsin (Boehringer Mannheim), and phosphopeptides were resolved by reverse phase high performance liquid chromatography (HPLC) as described previously (19). The positions of the phosphorylated residues in the peptides were determined by the cycles in which 32 P was released when the peptides were subjected to sequential Edman degradation (23).

RESULTS AND DISCUSSION
Immunoprecipitation of mTOR with mTAb1 and mTAb2-mTOR was readily detected by immunoblotting after subjecting samples of rat brain extracts to SDS-PAGE (Fig. 1). The apparent M r of the mTOR estimated by SDS-PAGE was 240,000, which is similar to the M r of 289,000 predicted from the nucleotide sequence of the mTOR cDNA. The protein was recognized by either of the antibodies, mTAb1 and mTAb2, which were directed against two different regions of the mTOR molecule. As expected, the antibodies recognized mTOR that had been affinity-purified using rapamycin/FKBP12. No mTOR was detected if the affinity-purification procedures were conducted in an identical manner except using FK506⅐FKBP12, which does not bind mTOR. mTOR could be immunoprecipitated using either mTAb1 or mTAb2. In this case, the recoveries of mTOR were blocked by the respective antigenic peptides supporting the specificity of the antibodies.
Phosphorylation of PHAS-I by mTOR-When incubated in a reaction mixture containing Mn 2ϩ and [␥-32 P]ATP, [His 6 ]PHAS-I was phosphorylated by mTOR that had been immunoprecipitated with either mTAb1 or mTAb2. The rate of phosphorylation was much higher with mTAb1 than with mTAb2. mTAb1 was somewhat more efficient in immunoprecipitating mTOR than mTAb2 (Fig. 1); however, the higher rate of kinase activity could not be explained by the larger amount of mTOR present in the mTAb1 immunoprecipitates.
To investigate the possible stimulatory effect of mTAb1, mTOR was immunoprecipitated with mTAb2, then incubated with mTAb1 before assessing kinase activity (Fig. 2). Under these conditions, the rate of phosphorylation of PHAS-I was increased approximately 3-fold by mTAb1. In contrast, adding mTAb2 to mTOR that had been immunoprecipitated with mTAb1 decreased the rate of incorporation of 32 P into PHAS-I by approximately 50%. Previous studies demonstrated that mTOR autophosphorylation, although markedly diminished, still occurred when the protein was bound to rapamycin/ FKBP12, indicating that rapamycin/FKBP12 does not fully inhibit mTOR (24). When purified by rapamycin/FKBP12 affinity chromatography, mTOR phosphorylated PHAS-I at a relatively slow rate. Adding mTAb1 increased the PHAS-I kinase of the affinity-purified mTOR by approximately 10-fold (Fig. 2).
The effects of mTAb1 on both mTAb2-immunoprecipitated mTOR and the affinity-purified protein supports the conclusion that mTAb1 binding activates the mTOR kinase. A working hypothesis is that the COOH-terminal region of mTOR functions to inhibit the kinase domain. mTAb1 presumably acts by promoting a conformational change that releases the inhibition thereby activating the mTOR protein kinase. It is interesting to speculate that the activity of mTOR might be regulated by hormones and growth factors through modification of the COOH-terminal region.
PHAS-I could be stoichiometrically phosphorylated when incubated with mTAb1 activated by mTOR (Fig. 3). Based on the staining intensities of the mTOR band relative to that of a FIG. 1. Detection of mTOR by mTAb1 and mTAb2. 5 g each of nonimmune IgG (NI-IgG), mTAb1, and mTAb2 were bound to protein A-agarose, and GST-FKBP12 was bound to glutathione-agarose, as described under "Experimental Procedures." The samples were incubated for 20 min at 21°C in 0.5 ml of buffer containing no additions, and 1 mg/ml peptide 1 (CDTNAKGNKRSRTRTDSYS) or 1 mg/ml peptide 2 (ARRVSKDDWLEWLRRLSLE) before brain extract (0.5 ml, 3 mg of protein) either without or with 10 M rapamycin (Rap) or 10 M FK506 as indicated, was added to each. After incubating for 90 min at 4°C, the beads were washed before the proteins were eluted and subjected to SDS-PAGE. Immunoblots were then prepared using mTAb1 and mTAb2. known standard (bovine serum albumin) on silver stained polyacrylamide gels, we estimate that an mTOR immunoprecipitate from 3 mg of brain extract protein contained approximately 0.05 g of mTOR protein (data not shown). This amount of mTOR phosphorylated PHAS-I at a rate comparable to that of 0.1 g recombinant MAP kinase, which is by far the best of the kinases that have been reported to phosphorylate PHAS-I in vitro (21). The sensitivity of mTOR and MAP kinase to wortmannin and rapamycin/FKBP12 were distinctly different (Fig. 3). PHAS-I phosphorylation by mTOR was abolished by 1 M wortmannin, and the activity was markedly decreased by rapamycin/FKBP12. These agents had little if any effect on PHAS-I phosphorylation by MAP kinase.
Identification of Phosphorylation Sites-To investigate the specificity of mTOR, the sites in PHAS-I phosphorylated by mTOR were identified. [His 6 ]PHAS-I was phosphorylated using [␥-32 P]ATP and either mTAb1-activated mTOR or recombinant MAP kinase. The 32 P-labeled proteins were then digested with lysyl endopeptidase, and phosphopeptides were Samples of the 32 P-labeled proteins were digested with lysyl endopeptidase (A). The resulting peptides were applied to a reverse phase column and eluted at 1 ml/min with an increasing gradient of acetonitrile (19). The phosphorylation sites contained in the peaks generated from MAP kinase-phosphorylated samples have been identified previously (19). The positions of these peaks (LE-P1, LE-P2, LE-P3, and LE-P4) are indicated. LE-P4 fractions were pooled and incubated with chymotrypsin before the resulting phosphopeptides were resolved by reverse phase HPLC (B). The position of the previously characterized peaks, CT-P1 and CT-P2, are indicated.  His 6 ]PHAS-I was phosphorylated by mTAb1immunoprecipitated mTOR. After digesting the protein with lysyl endopeptidase, phosphopeptides were isolated by reverse phase HPLC as shown in Fig. 4. Samples LE-P4 were pooled and digested with chymotrypsin before the resulting phosphopeptides were resolved by HPLC. Samples of peak fractions from lysyl endopeptidase and chymotrypsin digestions were subjected to sequential Edman degradation. The amounts of 32 P retained on the filters after degradation were as follows: LE-P1, 55 cpm; LE-P2, 930 cpm; LE-P3, 1257 cpm; CT-P1, 473 cpm; and CT-P2, 405 cpm. The results represent the 32 P released in each cycle. The previously determined amino acid sequences of phosphopeptides in the peaks are shown. resolved by reverse phase HPLC (Fig. 4). mTOR phosphorylated sites that were recovered in phosphopeptides that eluted in peaks found in the same positions as LE-P1, LE-P2, LE-P3, and LE-P4, the four peaks previously characterized in analyses of PHAS-I phosphorylation by MAP kinase (19). LE-P1, LE-P2, and LE-P3 contain peptides in which Thr 69 , Ser 64 , and Ser 82 , respectively, are phosphorylated (19). LE-P4 contains both Thr 36 and Thr 45 . These sites may be resolved by digesting LE-P4 with chymotrypsin, which generates peptides that elute in two peaks, designated CT-P1 and CT-P2 (19), containing Thr 45 and Thr 36 , respectively. Digesting the pooled LE-P4 fractions from mTOR-phosphorylated [His 6 ]PHAS-I with chymotrypsin-generated 32 P-labeled peptides that eluted from the reverse phase column in precisely the same positions as CT-P1 and CT-P2 (Fig. 4).
The finding that phosphopeptides derived from the mTORphosphorylated sample eluted identically from the reverse phase column as those from the MAP kinase-phosphorylated protein suggested that mTOR phosphorylated the same sites as MAP kinase. However, because the peptides contain multiple Ser/Thr residues, it was necessary to determine the position of the phosphorylated residue in each peptide to confirm the site assignment. Subjecting LE-P1, LE-P2, LE-P3, CT-P1, and CT-P2 from mTOR-phosphorylated samples to sequential Edman degradation resulted in release of 32 P in cycles 1, 8, 10, 3, and 3, indicative of phosphorylation of Thr 69 , Ser 64 , Ser 82 , Thr 45 , and Thr 36 , respectively (Fig. 5).
Although it facilitated the phosphorylation site analyses, the finding that mTOR phosphorylated the same sites in PHAS-I as MAP kinase was unexpected in view of the dissimilar catalytic domains of the two enzymes (25,26). An initial concern was that the PHAS-I phosphorylation observed with mTOR immunoprecipitates might be due to contamination with MAP kinase. Both biochemical and pharmacological evidence indicate that this was not the case. mTOR phosphorylated Thr 69 much more rapidly than this site was phosphorylated by MAP kinase (19). Phosphorylation by mTOR was markedly inhibited by rapamycin/FKBP12, which was without effect on the phosphorylation of PHAS-I by MAP kinase (Fig. 3); and, wortmannin, which binds and covalently modifies mTOR, abolished phosphorylation by mTOR but not phosphorylation by MAP kinase (Fig. 3). Moreover, the finding that the PHAS-I kinase associated with affinity-purified mTOR was increased by mTAb1 (Fig. 2) provides strong evidence that PHAS-I phosphorylation is mediated by mTOR itself.
The similarity of the kinetics and stoichiometry of phosphorylation of PHAS-I by mTOR and MAP kinase was also unexpected, although subtle but potentially important differences in the activities of these enzymes toward PHAS-I are apparent. Comparing the phosphate incorporated into a single phosphorylation site relative to the total phosphate in all of the sites, mTOR reproducibly phosphorylated Thr 69 more rapidly than did MAP kinase ( Fig. 4 and Ref. 19). Additionally, the stoichiometry of phosphorylation of PHAS-I by either enzyme appears to plateau at just over 1 mol of phosphate/mol of PHAS-I (Fig.  3) indicating that mono-phosphorylated PHAS-I may not be a preferred substrate for further phosphorylation. Taken together these findings indicate that relative site preferences of PHAS-I kinase(s) may play a role in regulating PHAS-I function. It will be interesting to determine the relative role each phosphorylation site or combination of sites plays in determining the affinity of PHAS-I for eIF4E.
All of the sites phosphorylated by mTOR conform to a (Ser/ Thr)-Pro motif. An implication is that Pro on the COOH-terminal side of a Ser/Thr residue might be an important determinant in the specificity of mTOR. The sites phosphorylated by mTOR in vitro are phosphorylated in response to insulin in adipocytes (19), and their phosphorylation markedly decreases the ability of PHAS-I to bind to eIF4E (9). Moreover, treating adipocytes with rapamycin has been shown to decrease phosphorylation of the sites. These findings support the conclusion that the sites in PHAS-I phosphorylated by mTOR are physiologically relevant and that mTOR contributes to the phosphorylation of PHAS-I in cells.