Identification of Ser⁸⁶³ as a site of raptor phosphorylation in intact cells and generation of phosphospecific Ser⁸⁶³ antibodies. A, localization of cluster 1 (Ser⁶⁹⁶/Thr⁷⁰⁶) and cluster 2 (Ser⁸⁵⁵/Ser⁸⁵⁹/Ser⁸⁶³/Ser⁸⁷⁷) P-sites within the domain structure of raptor. The six P-sites identified map to a central, poorly conserved region of raptor that localizes between the N-terminal RNC (raptor N-terminal conserved) domains and HEAT repeats and the C-terminal WD40 repeats. B, low energy, collision-induced dissociation spectrum of the doubly charged raptor Ser(P)⁸⁶³ phosphopeptide. HEK293 cells were transfected with Myc-raptor, cultured in DMEM/FBS, and WCL was immunoprecipitated (IP) with Myc antibodies. A Coomassie-stained band of Myc-raptor (upper panel) was digested with trypsin after SDS-PAGE. Liquid chromatography-MS/MS and data analysis were conducted as described under “Experimental Procedures.” Note that the y₄–y₇ ion series clearly places the site of phosphorylation on Ser⁸⁶³. C, Ser(P)⁸⁶³ antibodies are site-specific. HEK293 cells were transfected with vector control (−) or with WT or S863A Myc-raptor alleles, as indicated. WCL was immunoprecipitated (IP) with Myc antibodies and immunoblotted (IB) with the indicated antibodies. D, Ser(P)⁸⁶³ antibodies are phosphospecific. HEK293 lysate was immunoprecipitated with preimmune (PI) antibodies (lane 1) or with anti-raptor antibodies (lanes 2–4). The immunoprecipitates were resuspended immediately in sample buffer (lanes 1 and 2) or washed in phosphatase (PPase) buffer and incubated in the absence (lane 3) or presence (lane 4) of λ-phosphatase. The immunoprecipitates were immunoblotted as indicated.

TSC/Rheb signaling modulates raptor Ser⁸⁶³ phosphorylation. A, TSC suppresses raptor Ser(P)⁸⁶³. Littermate-matched, 3T3 immortalized mouse embryonic fibroblasts derived from TSC1^+/+ or TSC^−/− animals were serum deprived. Triplicate lysates were immunoprecipitated (IP) with anti-raptor antibodies and immunoblotted (IB) as indicated. WCL was also immunoblotted directly to confirm the absence of TSC1 and the expected activation of mTORC1 signaling. Note: we find that TSC1^−/− fibroblasts express higher levels of total raptor protein when normalized for total protein content; thus, two-thirds of the immunoprecipitate from TSC1^−/− cells was loaded relative to TSC1^+/+ cells to normalize the amount of raptor between the two cell lines. WCL was loaded similarly. B, Rheb overexpression increases Myc-raptor Ser(P)⁸⁶³ and retards the mobility of Myc-raptor on SDS-PAGE. HEK293 cells were co-transfected with Myc-raptor (0.5 μg) and AU1-mTOR (2.5 μg) with or without FLAG-Rheb (2.5 μg), as indicated. The cells were serum deprived (lanes 1–5) and stimulated with insulin (lanes 6–8). WCL was immunoprecipitated with Myc antibodies and immunoblotted as indicated. WCL was also immunoblotted directly to confirm the expression of the various plasmids and the expected activation of mTORC1 signaling. C, rapamycin reduces Rheb-stimulated raptor Ser(P)⁸⁶³, performed as in B except that rapamycin was included in the culture medium during the entire ∼20 h serum deprivation period (lanes 6 and 7). D, Rheb-stimulated raptor Ser(P)⁸⁶³ requires active mTORC1. Performed as in B except that RR (lane 6) and RR/KD (lane 7) AU1-TOR alleles were also analyzed. At the time of serum deprivation, rapamycin was added to the culture medium (+ Rapa; lanes 5–7). ∼20 h later, cells were incubated in the absence or presence of insulin and lysed in Buffer B/CHAPS. WCL was immunoprecipitated with Myc antibodies and immunoblotted as indicated. WCL was also immunoblotted directly to confirm the expression of the various plasmids and the expected activation and/or inhibition of mTORC1 signaling by the various treatments.

Rheb promotes multisite raptor phosphorylation in Ser(P)⁸⁶³-dependent and -independent manners. A, raptor Ser(P)⁸⁶³ is required for Rheb-induced supershift of Myc-raptor on SDS-PAGE. HEK293 cells were co-transfected with WT or S863A Myc-raptor (0.5 μg) and AU1-mTOR (2 μg) with or without FLAG-Rheb (2 μg). Cells were serum deprived, WCLs were immunoprecipitated (IP) with Myc antibodies, and immunoprecipitates were resolved and immunoblotted (IB) as indicated. WCL was also immunoblotted directly to confirm expression of the transfected proteins as well as the expected activation of mTORC1 signaling by FLAG-Rheb. Note, immunoprecipitates were resolved on 8% SDS-PAGE and run at 35 mA for 8 (long run) or 4 h (short run). B, Rheb promotes raptor phosphorylation on multiple sites, with Ser⁸⁵⁵ and Ser⁸⁵⁹ phosphorylation occurring in a Ser(P)⁸⁶³-dependent manner. HEK293 cells were transfected and analyzed as in A, employing WT, S863A, and S863D Myc-raptor alleles. Myc-raptor immunoprecipitates from serum-deprived cells were immunoblotted with our panel of raptor phosphospecific antibodies (e.g. Ser(P)⁸⁶³, Ser(P)⁸⁵⁹, Ser(P)⁸⁵⁵, Ser(P)⁶⁹⁶, Thr(P)⁷⁰⁶, and Ser(P)⁸⁷⁷) as well as with a commercially available phosphospecific antibody (e.g. Ser(P)⁷⁹²). Note: immunoprecipitates were resolved on 6% SDS-PAGE and run at 35 mA for ∼2.5 h. C, phospho-specificity of P-raptor antibodies. HEK293 cells were co-transfected, treated, and immunoprecipitated as in A. In lanes 4, 7, and 10, Myc-raptor immunoprecipitates were incubated with λ-phosphatase in vitro and immunoblotted as indicated. WCL was also immunoblotted directly to confirm expression of the transfected proteins as well as the expected activation of mTORC1 signaling by FLAG-Rheb.

Model-Complex raptor phosphorylation promotes mTORC1 signaling. mTORC1 activation by diverse environmental cues (e.g. insulin, EGF, amino acids, energy) (step 1) enables mTOR-mediated phosphorylation of raptor on Ser⁸⁶³ and possibly on other sites (e.g. Ser⁶⁹⁶, Thr⁷⁰⁶, and Ser⁸⁷⁷) (step 2) as well as mTOR-mediated phosphorylation of PRAS40. Ser⁸⁶³ phosphorylation then primes raptor for subsequent phosphorylation events on Ser⁸⁵⁹/Ser⁸⁵⁵ and possibly on other unmapped sites (step 3) by unknown kinases. Cooperative, multisite raptor phosphorylation promotes substrate phosphorylation and biochemical signaling (step 4). mTOR Ser¹²⁶¹ phosphorylation in response to insulin and amino acid signaling cooperates to promote mTORC1 signaling (73). For simplicity, not shown are the AMPK- and RSK-mediated phosphorylation events on raptor, which reportedly down-regulate or augment mTORC1 activity, respectively (49, 51).

mTORC1-activating stimuli promote the phosphorylation of raptor. A, insulin retards the electrophoretic mobility of raptor on SDS-PAGE in an amino acid-dependent manner. In lanes 1 and 2, HEK293 cells were serum deprived and stimulated with insulin (30 min). In lanes 3–6, HEK293 cells were similarly serum deprived, incubated in D-PBS/glucose (60 min) to effect amino acid deprivation, followed by stimulation with DMEM alone (as source of amino acids), insulin alone, or both DMEM and insulin (30 min). WCL was resolved on SDS-PAGE and immunoblotted (IB) with the indicated antibodies. Note: to observe this degree of raptor mobility shift, lysate was resolved on 6% SDS-PAGE and run at 35 mA for ∼5 h. B, phosphorylation underlies insulin-stimulated raptor electrophoretic mobility shift. HEK293 cells were serum deprived and stimulated with insulin (lanes 2–3). WCL was immunoprecipitated (IP) with anti-raptor antibodies, and the immunoprecipitates were washed in phosphatase buffer (lanes 1–3), incubated in the absence (lanes 1 and 2) or presence (lane 3) of λ-phosphatase, and immunoblotted as indicated. Note: immunoprecipitates were resolved on 6% SDS-PAGE and run at 35 mA for ∼5 h. C, phosphorylation underlies Rheb-induced raptor electrophoretic mobility shift. HEK293 cells were co-transfected with Myc-raptor (0.5 μg) and AU1-mTOR (2.5 μg) with or without FLAG-Rheb (2.5 μg), as indicated, and serum deprived. WCL was immunoprecipitated with Myc antibodies. Myc immunoprecipitates were washed in phosphatase buffer (lanes 4 and 5) and incubated in the absence (lane 4) or presence (lane 5) of λ-phosphatase. WCL was also immunoblotted directly to confirm the expression of the various plasmids and the expected activation of mTORC1 signaling. Note: immunoprecipitates were resolved on 8% SDS-PAGE and run at 35 mA for 8 (long run) or 4 h (short run).

Insulin/PI3K signaling promotes rapamycin-sensitive raptor Ser⁸⁶³ phosphorylation in 3T3-L1 adipocytes and HEK293 cells. A, regulation of raptor Ser(P)⁸⁶³ in mTORC1 in 3T3-L1 adipocytes. Differentiated adipocytes were serum deprived, pre-treated with wortmannin (lane 4, left panel) or with rapamycin (lane 8, right panel), and stimulated with insulin. WCL was immunoprecipitated (IP) with preimmune sera (PI) (lanes 1 and 5), anti-raptor antibodies (upper panels, lanes 2–4 and 6–8), or anti-mTOR antibodies (middle panels, lanes 2–4 and 6–8) and immunoblotted (IB) with the indicated antibodies. WCL was also immunoblotted directly (lower panels) with the indicated antibodies to confirm the expected activation and/or inhibition of mTORC1 signaling. B, regulation of raptor Ser(P)⁸⁶³ in HEK293 cells. HEK293 cells were serum deprived, pre-treated with wortmannin (W, lane 4) or rapamycin (R, lane 5), and stimulated with insulin, as indicated for the adipocyte experiment. WCL was immunoprecipitated with preimmune sera (PI) (lane 1) or anti-raptor antibodies (lanes 2–5), and immunoprecipitates were immunoblotted with the indicated antibodies. WCL was also immunoblotted directly with the indicated antibodies to confirm the expected activation and/or inhibition of mTORC1 signaling by the various treatments. C, time course for insulin-stimulated raptor Ser(P)⁸⁶³. HEK293 cells were serum deprived and stimulated with insulin for various amounts of time (5–60 min). WCL was immunoprecipitated with anti-raptor antibodies and immunoblotted with the indicated antibodies. WCL was also immunoblotted directly with the indicated antibodies to confirm the expected activation and/or inhibition of mTORC1 signaling by the various treatments. Note: the asterisk indicates the 70-kDa S6K1 αII species; double asterisks indicate the 85-kDa S6K1 αI species. If not indicated otherwise, all S6K1 immunoblots in this article represent the better studied S6K1 αII species. D, EGF and PMA promote raptor Ser(P)⁸⁶³ independently of PI3K. HEK293 cells were serum deprived and stimulated with 10% FBS (lane 2), insulin (lane 3), EGF (lane 5), or PMA (lane 6). WCL was immunoprecipitated with anti-raptor antibodies and immunoblotted with the indicated antibodies. WCL was also immunoblotted directly with the indicated antibodies to confirm the expected modulation of PI3K (P-Akt), MAPK (P-MAPK (Thr²⁰²/Tyr^Y204), and mTORC1 (P-S6) signaling by the various growth factors/mitogens. Note: p44^Mapk and p42^Mapk are also known as ERK1 and ERK2, respectively. E, MEK/MAPK signaling mediates EGF-stimulated raptor Ser(P)⁸⁶³. Myc-raptor-transfected HEK293 cells (0.5 μg) were serum deprived, pre-treated with UO126 (lane 5), and then stimulated with PMA (lane 3) or EGF (lanes 4–5). WCL was immunoprecipitated with Myc antibodies and immunoblotted with the indicated antibodies. WCL was also immunoblotted to confirm the expected activation/inhibition of MAPK and mTORC1 signaling.

Insulin-stimulated raptor Ser⁸⁶³ phosphorylation requires sufficient levels of amino acids and cellular energy. A, effect of amino acid and insulin stimulation alone and in combination on raptor Ser(P)⁸⁶³. HEK293 cells were serum deprived followed by incubation in D-PBS/glucose (60 min) to effect amino acid deprivation (lanes 5–14). Amino acid-deprived cells were then pre-treated with rapamycin (R) (lanes 9 and 14), and stimulated with amino acids alone, insulin alone, or both amino acids and insulin, as indicated. In lanes 6, 8, and 9, DMEM was used as source of amino acids, whereas in lanes 11, 13, and 14, 5× minimal essential medium amino acids were used. In lanes 1–4, the cells were not amino acid deprived, they were simply serum deprived and stimulated with insulin (30 min). WCL was immunoprecipitated (IP) with preimmune (PI) or raptor antibodies and immunoblotted (IB) as indicated. WCL was also immunoblotted directly to confirm the expected activation and/or inhibition of mTORC1 signaling by the various treatments. B, amino acid (AA) withdrawal decreases, whereas amino acid re-addition increases raptor Ser(P)⁸⁶³ in a rapamycin-sensitive manner. HEK293 cells cultured in DMEM/FBS were incubated in D-PBS/glucose containing 10% dialyzed FBS (60 min) to effect amino acid deprivation in the continued presence of serum growth factors (lanes 3–8). Amino acid-deprived cells were pre-treated with rapamycin (lanes 7 and 8) and stimulated with DMEM/FBS (30 min) (lanes 5–8). WCL was immunoprecipitated with preimmune (PI) or raptor antibodies and immunoblotted as indicated. WCL was also immunoblotted directly to confirm the expected activation and/or inhibition of mTORC1 signaling. C, insulin-stimulated raptor Ser(P)⁸⁶³ requires amino acids in 3T3-L1 adipocytes. Differentiated adipocytes were serum deprived and then amino acid deprived (60 min). The cells were then pre-treated with rapamycin (R; lane 6) or wortmannin (W; lane 7) and stimulated with amino acids alone (using DMEM), insulin alone, or both amino acids and insulin (30 min) as indicated. WCL was immunoprecipitated with preimmune or raptor antibodies and immunoblotted as indicated. WCL was also immunoblotted directly to confirm the expected activation and/or inhibition of mTORC1 signaling by the various treatments. D, energy stress mediates raptor Ser⁸⁶³ de-phosphorylation. HEK293 cells were transfected with Myc-raptor (0.5 μg). Cycling cells were re-fed with DMEM/FBS that lacked (lanes 2 and 3) or contained (lanes 4 and 5) 2-deoxyglucose (2DG) (25 mm) and incubated 15 min. WCL was immunoprecipitated with Myc antibodies and immunoblotted as indicated. WCL was also immunoblotted directly to confirm the expected down-regulation of mTORC1 signaling and up-regulation of AMPK in response to energy stress.

mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward GST-4EBP1. HEK293 cells on 10-cm plates were co-transfected with Myc-raptor alleles (2 μg) (WT, S863A, or 6A) together with AU1-mTOR alleles (8 μg) (WT or KD), serum deprived, and stimulated with insulin for 20 min. Myc-raptor was immunoprecipitated (IP) with Myc antibodies, and the in vitro kinase activity of co-precipitated AU1-mTOR (and endogenous mTOR) was assayed using recombinant GST-4EBP1 (isolated from bacteria) as substrate in the presence of both cold and hot [³²P]ATP. In vitro kinase reactions were analyzed by Thr(P)³⁷/Thr(P)⁴⁶-4EBP1 Western blotting (IB) and by autoradiographic analysis of ³²P incorporation into GST-4EBP1. The graph shows the level of ³²P incorporated into GST-4EBP1 as determined using a phosphorimager.