Identification of Rictor as a target of mTORC1 signaling. (A) Schematic representation of the agonists and pharmacological inhibitors used in this study. (B) HEK293 cells were transfected with empty vector or HA-tagged Rictor; serum starved overnight; and stimulated for 10 or 20 min with PMA (50 ng/ml), insulin (100 nM), EGF (50 ng/ml), or fetal bovine serum (10%). Immunoprecipitated (IP) Rictor was then assayed for phosphorylation with a phospho-motif antibody that recognizes the RXRXXpS/T consensus motif. (C and D) As for panel B, except cells were pretreated with U0126 (20 μM), rapamycin (100 nM), or wortmannin (100 nM) for 30 min prior to PMA or insulin stimulation. (E and F) Endogenous Rictor was immunoprecipitated from HEK293 (E) or HeLa (F) cells and assayed as for panel B.

S6K1 is required for Rictor phosphorylation in cells. (A) HEK293 cells were cotransfected with HA-tagged Rictor and siRNA duplexes targeted against a scrambled sequence (Scr) or human S6K1. Cells were serum starved overnight and stimulated with PMA (50 ng/ml) or insulin (100 nM) for 20 min. Immunoprecipitated Rictor was then assayed for phosphorylation with the anti-RXRXXpS/T antibody. (B) HEK293 cells stably expressing an shRNA targeting a scrambled sequence (Scr) or S6K1 were transfected with HA-tagged Rictor, serum starved overnight, and treated with rapamycin (100 nM) for 30 min prior to insulin (100 nM) stimulation for 20 min. Immunoprecipitated Rictor was assayed as for panel A.

S6K1 is sufficient to promote Rictor phosphorylation in cells. (A) Schematic representation of S6K1 constructs used in this study. Constitutively activated S6K1 (CA) consists of F5A, T389E, and R410/413/414A (R3A) mutations. The kinase-inactive allele of S6K1 (KD) consists of a K100R mutation in subdomain II of the kinase domain. (B) HEK293 cells were cotransfected with myc-tagged Rictor and constructs expressing HA-tagged wild-type (wt), constitutively activated (CA), or kinase-inactive (KD) S6K1. Cells were serum starved overnight and treated with insulin (100 nM) for 20 min prior to harvesting. The phosphorylation of Rictor was assayed in anti-myc immunoprecipitates. (C and D) As for panel B, except S6K1 CA- and Rheb-expressing cells were pretreated with rapamycin (100 nM) for 30 min prior to harvesting.

Identification of Thr1135 as a rapamycin-sensitive phosphorylation site in Rictor. (A) Phosphorylation sites in known S6K1 substrates fit the K/RXRXXpS/T consensus. Rictor contains four residues fitting this consensus, corresponding to Ser21, Ser1113, Thr1135, and Ser1219. (B) MS/MS analyses of immunoprecipitated endogenous and exogenous Rictor from HEK293 cells revealed 13 phosphorylation sites, three of which are located within the K/RXRXXpS/T consensus motif (Ser21, Thr1135, and Ser1219). The white area identifies region of highest conservation between Rictor orthologs. (C) HEK293 cells were transfected with wild-type Rictor or potential S6K1 phosphorylation site mutants S21A, S1113A, T1135A, and S1219A; serum starved overnight; and stimulated with insulin (100 nM) for 20 min. Immunoprecipitated Rictor was then immunoblotted for phosphorylation at RXRXXpS/T sites. (D) Tandem mass spectrum of the precursor at m/z 825.03+, corresponding to the phosphopeptide TL(pT)EPSVDLNHSEDFTSSSAQK from murine Rictor. (E) As for panel C, except cells were stimulated with PMA (50 ng/ml) and assayed for Rictor phosphorylation by immunoblotting. (F and G) HEK293 cells were transfected with wild-type Rictor and wild-type Rheb (F) or constitutively activated S6K1 (G). Cells were serum starved overnight, and immunoprecipitated Rictor was immunoblotted for phosphorylation at RXRXXpS/T sites.

Detection of Rictor phosphorylation at Thr1135 using a novel phosphospecific antibody. (A) Primary sequence alignment showing conservation of Thr1135 as well as −3 and −5 basic residues within Rictor from different vertebrate species. (B) A phosphospecific antibody against Thr1135 was generated and tested on immunoprecipitated Rictor. HEK293 cells were transfected with wild-type Rictor or a phosphorylation site mutant, serum starved overnight and stimulated with insulin (100 nM) for 20 min. Rictor immunoprecipitates were assayed using the phospho-Thr1135 antibody. (C) HEK293 cells were transfected with wild-type Rictor and Rheb, serum starved overnight, and treated with rapamycin (20 nM) for 30 min where indicated. Rictor phosphorylation was assayed using the phospho-T1135 antibody. (D) TSC2^+/+ or TCS2^−/− MEFs were grown in the absence of serum and treated with rapamycin (100 nM) for 30 min where indicated. Endogenous Rictor was assayed for phosphorylation using the phospho-Thr1135 antibody. (E) HEK293 cells stably expressing HA-tagged wild-type Rictor were transfected with either constitutively activated Akt (Myr-Akt) or constitutively activated S6K1 (S6K-CA). Cells were serum starved overnight, treated with rapamycin (100 nM), and stimulated with insulin (100 nM) where indicated. Rictor phosphorylation was assayed using the phospho-T1135 antibody.

S6K1 phosphorylates Rictor on Thr1135 in vitro. (A) S6K1 was immunoprecipitated from HEK293 cells stimulated with insulin (100 nM) and incubated in a kinase reaction with [γ-³²P]ATP with GST-Rictor fusion proteins containing wild-type and T1135A sequences. (B) As for panel A, except cells were pretreated with rapamycin (100 nM) for 30 min and stimulated with insulin or PMA for 20 min prior to immunoprecipitation of S6K1. (C) Immunoprecipitated wild-type or kinase-inactive S6K1 from insulin-stimulated cells was incubated with immunopurified full-length wild-type or T1135A Rictor in a kinase reaction without radioactivity. The resulting samples were subjected to SDS-PAGE and immunoblotted for Rictor phosphorylation on RXRXXpS/T consensus sites.

A Rictor mutant that cannot be phosphorylated on Thr1135 promotes mTORC2-directed phosphorylation of Akt. (A) HEK293 cells were transfected with siRNA duplexes targeted against a scrambled sequence or human Raptor. Cells were grown in the presence of serum or serum starved overnight and stimulated with EGF (25 ng/ml) or insulin (100 nM). Akt phosphorylation was assayed by immunoblotting from cell lysates using Akt phospho-Ser473 antibodies. (B) HEK293 cells were serum starved overnight and treated with rapamycin for 30 min prior to stimulation with insulin (25 nM) (left panel) or EGF (25 ng/ml) (right panel). Akt phosphorylation was assayed as for panel A. DMSO, dimethyl sulfoxide. (C) HEK293 cells stably expressing an empty vector, Rictor wild-type Rictor, or the T1135A or T1135D mutant were seeded at similar densities and serum starved overnight. Cells were stimulated with EGF (25 ng/ml) for 5 or 10 min and assayed for Akt and NDRG1 phosphorylation using NDRG1 phospho-Thr346/Thr356/Thr366 antibodies. (D and E) Results from three independent experiments as for panel C were quantified, and the mean (± SD) fold stimulation in Akt (D) and NDRG1 (E) phosphorylation was calculated compared to an empty-vector control. (F) siRNA duplexes targeted against a scrambled sequence or human Rictor were transfected in HEK293 cells stably expressing an empty vector or siRNA-resistant wild-type Rictor or the T1135A or T1135D mutant. Cells were serum starved overnight, stimulated with EGF (25 ng/ml), and harvested for FACS analysis of Ser473 phosphorylation (see Materials and Methods for details). Levels of phosphorylated Akt were determined in HA-Rictor positive cells. Data are expressed as fold stimulation in Akt phosphorylation compared to unstimulated empty-vector-transfected cells.

Rictor phosphorylation at Thr1135 inhibits Akt signaling and cell proliferation. (A) HEK293 cells were transfected with Flag-tagged mTOR and wild-type HA-tagged Rictor or Thr1135 mutants. Associated mTOR was determined in anti-HA immunoprecipitates using anti-Flag antibodies. (B) mTORC2 kinase assays were optimized by immunoprecipitating endogenous Rictor from HEK293 cells stimulated with EGF (25 ng/ml) and treated with the dual PI3K/mTOR inhibitor PI-103 where indicated (left panel). Using these assay conditions, mTORC2 activity was measured in HA-tagged Rictor wild-type or mutant immunoprecipitates from unstimulated or EGF-stimulated cells (right panel). mTORC2 activity toward kinase-inactive GST-Akt purified from mammalian cells was assayed. (C) Subcellular localization of wild-type Rictor and the Rictor Thr1135 mutants. Confocal images of serum-growing HEK293 cells stably expressing HA-tagged wild-type Rictor or Thr1135 mutants are shown. (D) HEK293 cells stably expressing wild-type Rictor or Thr1135 mutants were serum starved overnight and stimulated with EGF (25 ng/ml). Phosphorylation of Akt substrates FoxO1/3a and GSK3α/β was assayed by immunoblotting. (E) HEK293 cells stably expressing wild-type Rictor or the Thr1135 mutants were grown in culture medium containing 5% FBS. The relative number of viable cells was measured during five consecutive days using an MTS assay. (F) Upon growth factor stimulation, PI3K is recruited to the plasma membrane using IRS-dependent and -independent mechanisms. PIP3-mediated recruitment of Akt to the plasma membrane promotes PDK1- and mTORC2-dependent phosphorylation of Akt at Thr308 and Ser473, respectively. Upon activation, Akt phosphorylates TSC2, which releases the inhibitory function of the TSC complex on Rheb. GTP-loaded Rheb then activates mTORC1, which phosphorylates and activates S6K1. Activated S6K1 phosphorylates several substrates, including IRS-1 and Rictor, which play roles in the negative regulation of mTORC2 signaling.