Characterization of Rictor phosphorylation sites reveals direct regulation of mTOR complex 2 by S6K1

Abstract

The mammalian target of rapamycin (mTOR) functions within two distinct complexes (mTORC1 and mTORC2) to control cell growth, proliferation, survival, and metabolism. While there has been great progress in our understanding of mTORC1 regulation, the signaling mechanisms that regulate mTORC2 have not been defined. In this study, we use liquid chromatography-tandem mass spectrometry analyses to identify 21 phosphorylation sites on the core mTORC2 component Rictor. We find that one site, T1135, undergoes growth factor-responsive phosphorylation that is acutely sensitive to rapamycin and is phosphorylated downstream of mTORC1. We find that Rictor-T1135 is directly phosphorylated by the mTORC1-dependent kinase S6K1. Although this phosphorylation event does not affect mTORC2 integrity or in vitro kinase activity, expression of a phosphorylation site mutant of Rictor (T1135A) in either wild-type or Rictor null cells causes an increase in the mTORC2-dependent phosphorylation of Akt on S473. However, Rictor-T1135 phosphorylation does not appear to regulate mTORC2-mediated effects on SGK1 or PKC alpha. While the precise molecular mechanism affecting Akt is unknown, phosphorylation of T1135 stimulates binding of Rictor to 14-3-3 proteins. We provide evidence that Rictor-T1135 phosphorylation acts in parallel with other mTORC1-dependent feedback mechanisms, such as those affecting IRS-1 signaling to PI3K, to regulate the response of Akt to insulin.

FIG. 1.

The phosphorylation sites on Rictor identified by LC/MS/MS are clustered in a region conserved only in vertebrate orthologs. Rictor orthologs from the indicated species are shown schematically. Regions of similarity were determined by individually aligning full-length Rictor orthologs to human Rictor using BLAST. Two major regions of homology exist, the highly conserved region A (amino acids 8 to 1050 in humans) and the less-well-conserved region B (amino acids 1519 to 1694 in humans). The sizes of regions A and B represented in human Rictor correspond to the longest stretch of conservation in any other ortholog. Phosphorylation sites identified in this study are numbered based on full-length human Rictor and are summarized in Table 1. Accession numbers for orthologs are listed in Materials and Methods. H.s., H. sapiens; D.m., D. melanogaster; C.e., C. elegans; S.c., S. cerevisiae.

FIG. 2.

Rictor is phosphorylated on T1135 in an insulin-responsive and rapamycin-sensitive manner. (A) Insulin and EGF stimulate phosphorylation of Rictor on a motif recognized by the phospho-Akt substrate antibody (p-Akt-sub). HEK-293E cells were transfected with empty vector (V) or cotransfected with myc-tagged mTOR, Rictor, mSIN1.1, and mLST8. After 16 h of serum starvation, cells were either left unstimulated (lanes −) or were stimulated for 30 min with insulin (250 nM) (lanes I) or EGF (25 ng/ml) (lanes E). Anti-myc immunoprecipitates (myc IP) were immunoblotted with either the phospho-Akt substrate antibody or the myc epitope antibody (Blot Ab). (B) The phosphorylation of endogenous Rictor on a motif recognized by the phospho-Akt substrate antibody is wortmannin and rapamycin sensitive. HEK-293E cells were serum starved for 16 h; pretreated with DMSO (lanes −), wortmannin (100 nM) (lanes W), or rapamycin (20 nM) (lanes R) for 5 min or U0126 (10 μM) (lanes U) for 1 h; and stimulated with either insulin (100 nM) or EGF (20 ng/ml) for 30 min, where indicated with I and E, respectively. Whole-cell lysates or immunoprecipitates of endogenous Rictor were immunoblotted with the indicated antibodies. (C) Rictor within mTORC2 is phosphorylated on a motif recognized by the phospho-Akt substrate antibody. HEK-293E cells were serum starved for 16 h and treated with insulin and/or rapamycin, where indicated, as described for panel B. Immunoprecipitates of endogenous Rictor, mSIN1, or mTOR were immunoblotted with the indicated antibodies. Note that the faster-migrating band detected in the mSIN1 immunoprecipitations that is not in those of Rictor or mTOR is either an isoform of mSIN1 that does not associate with mTORC2 in these cells or another protein with which the antibody cross-reacts. (D) A T1135A mutant eliminates insulin-stimulated phosphorylation of Rictor detected by the phospho-Akt substrate antibody. HEK-293E cells were transfected with empty vector (V), wild-type myc-Rictor (WT), or the indicated myc-Rictor phosphorylation site mutants. Cells were serum starved for 16 h before 30 min of stimulation with insulin (100 nM), where indicated. Whole-cell lysates and anti-myc immunoprecipitates were immunoblotted with either the phospho-Akt substrate antibody or the myc-epitope antibody. (E) Residual recognition of Rictor-T1135A by the phospho-Akt substrate antibody is not rapamycin sensitive. HEK-293E cells were transfected and treated as described for panel D but, where indicated, were pretreated for 5 min with rapamycin (20 nM). (F) Quantification of Rictor phosphorylation at four residues in response to insulin and rapamycin using an LC/MS/MS total ion current. HEK-293E cells were transfected with wild-type myc-Rictor, serum starved for 16 h, pretreated with rapamycin (20 nM) for 5 min, and stimulated with insulin (200 nM) for 30 min, as indicated. myc-Rictor was immunoprecipitated and separated on SDS-PAGE gels. Coomassie blue-stained bands were excised, proteolyzed, and subjected to targeted LC/MS/MS analyses. The relative phosphorylation levels, determined by quantification of the LC/MS/MS total ion current of the corresponding tryptic (S21, T1135, and S1219) or chymotryptic (S1113) peptides, are shown normalized to levels in the unstimulated sample. Although a significant number of S1113-containing peptides were detected, none were found to be phosphorylated. (G) The sequence surrounding human Rictor-T1135 is highly conserved among vertebrates. Sequences from the corresponding Rictor orthologs are aligned. See Materials and Methods for GenBank accession numbers. Numbers at the right of blots are molecular masses (in kilodaltons).

FIG. 3.

mTORC1-dependent phosphorylation of Rictor on T1135. (A) Confirmation of endogenous Rictor-T1135 phosphorylation [p-Rictor (T1135)] with a phospho-specific antibody. Littermate-derived Rictor^+/+ and Rictor^−/− MEFs were pretreated with rapamycin (20 nM) for 5 min followed by insulin (100 nM) for 30 min. Immunoblots of the indicated proteins are shown. *, nonspecific band. (B) Demonstration of the specificity of the Rictor phospho-T1135 antibody. HEK-293E cells were transfected as described for Fig. 2D, and anti-myc immunoprecipitates (myc IP) were immunoblotted with the indicated antibodies. V, empty vector; WT, wild type. (C) Phosphatase treatment of Rictor eliminates recognition by the phospho-T1135 antibody. Endogenous Rictor was immunoprecipitated from HEK-293E cells growing in full serum, treated with calf intestinal phosphatase (CIP), and immunoblotted with the phospho-T1135 and total Rictor antibodies. *, nonspecific band. (D) Rictor-T1135 is phosphorylated in a variety of cell types. 3T3-L1 adipocytes, C2C12 myotubes, and HepG2 cells were serum starved for 16 h, pretreated with rapamycin (20 nM) for 5 min, and stimulated with insulin (100 nM) for 30 min where indicated. Whole-cell lysates were immunoblotted for phospho-Rictor-T1135 and total Rictor. *, nonspecific band. (E) Phosphorylation of Rictor-T1135 in response to insulin at sequential time points. HEK-293E cells were serum starved for 16 h and stimulated for the indicated lengths of time. Whole-cell lysates were immunoblotted with the indicated antibodies. *, nonspecific band. (F) Amino acid refeeding stimulates Rictor-T1135 phosphorylation. HEK-293E cells were serum starved for 16 h, incubated in Dulbecco's phosphate-buffered saline for an additional 2 h, and then stimulated with fresh DMEM or DMEM with rapamycin (20 nM) for 30 min. (G) Constitutive phosphorylation of Rictor on T1135 in TSC2-deficient cells. HeLa cells stably expressing shRNAs targeting firefly luciferase (shLUC) or TSC2 (shTSC2) were serum starved for 16 h and, where indicated, were stimulated with insulin (100 nM) for 30 min. Lysates were immunoblotted with the indicated antibodies. *, nonspecific band. Numbers to the right of blots are molecular masses (in kilodaltons).

FIG. 4.

S6K1 phosphorylates Rictor on T1135. (A) In vitro kinase assay demonstrating that T1135 is required for the phosphorylation of Rictor by S6K1 on the site(s) detected by the phospho-Akt substrate antibody (p-Akt-sub). In vitro kinase assays were performed with HA-S6K1 immunoprecipitated from insulin-stimulated HEK-293E cells. The Rictor substrate was immunoprecipitated from serum-starved, rapamycin-treated HEK-293E cells transfected with empty vector (V), wild-type myc-Rictor (WT), or myc-tagged versions of Rictor with the T1135A mutation alone or in combination with mutations at the indicated sites. The contents of the kinase reaction mixtures were immunoblotted with the indicated antibodies. (B) In vitro kinase assay demonstrating that S6K1 can directly phosphorylate Rictor-T1135. HA-S6K1 was immunoprecipitated from serum-starved HEK-293E cells that were left unstimulated, stimulated with insulin (100 nM) for 30 min, or pretreated with rapamycin (20 nM) for 5 min prior to insulin stimulation, as indicated. The Rictor substrate was obtained as described for panel A. (C) Rictor-T1135 phosphorylation becomes rapamycin resistant in cells expressing a rapamycin-resistant mutant of S6K1. HEK-293E cells were transfected with empty vector (V), wild-type S6K1 (WT), or a rapamycin-resistant mutant of S6K1 (RR). Cells were starved for 16 h and pretreated with rapamycin (20 nM) for 10 min prior to a 15-min stimulation with insulin (100 nM). Lysates were immunoblotted with the indicated antibodies. (D) S6K1 is required for the insulin-stimulated phosphorylation of T1135 in cells. HEK-293E cells were transfected with control (C) nontargeting siRNAs or siRNAs targeting either S6K1 or S6K2. Cells were serum starved for 16 h and either left unstimulated or stimulated with insulin (33 nM) for 30 min. Lysates were immunoblotted with the indicated antibodies. (E) S6K1 is required for phosphorylation of T1135 in cells growing in full serum. HEK-293E cells were transfected with siRNAs as described for panel D and grown in full serum, with a change of medium 16 h prior to lysis. Numbers to the right of blots are molecular masses (in kilodaltons).

FIG. 5.

The Rictor-T1135A mutant promotes increased phosphorylation of HA-Akt without altering the assembly or in vitro kinase activity of mTORC2. (A) Phosphorylation of T1135 does not affect mTORC2 assembly or in vitro kinase activity. mTORC2 was immunoprecipitated from HEK-293E cell lysates with myc-tagged Rictor (WT) or Rictor-T1135A following a 16-h serum starvation and, where indicated, stimulation for 30 min with insulin (100 nM). To demonstrate the specificity of the reaction, myc immunoprecipitates from vector (V)-transfected cells were used, and the mTOR kinase domain inhibitor LY294002 (15 μM; LY) was added to the indicated kinase reaction mixture prior to the addition of ATP. HA-tagged, kinase-dead Akt (HA-Akt^KD) was used as a substrate and was immunoprecipitated from serum-starved, wortmannin-treated (100 nM, 15 min) cells. The contents of the kinase reaction mixtures were immunoblotted with the indicated antibodies. (B) Expression of Rictor-T1135A increases phosphorylation of Akt at S473 [p-Akt (S473)] in HEK-293E cells. HA-Akt was coexpressed with empty vector (V), Rictor (WT), or Rictor-T1135A and was immunoprecipitated from cells that were serum starved for 16 h and then stimulated with insulin (100 nM) for the indicated times. HA immunoprecipitates (HA IP) and lysates were immunoblotted with the indicated antibodies.

FIG. 6.

Rictor-T1135A promotes increased phosphorylation of endogenous Akt on S473 [p-Akt (S473)] in Rictor^−/− MEFs. (A) Introduction of Rictor-T1135A into Rictor^−/− cells leads to increased phosphorylation of endogenous Akt. Rictor^−/− MEFs were transfected with empty vector (V), Rictor (WT), or Rictor-T1135A, and confluent cells were serum starved for 16 h and stimulated with insulin (100 nM) for the indicated times. Lysates were immunoblotted with the indicated antibodies. Phosphorylation intensity in immunoblots was quantified using ImageJ software. Phospho-Akt (S473) [p-Akt (S473)] levels were divided by both total Akt and total Rictor levels and are graphed relative to levels at the zero time point for WT-Rictor-expressing cells. (B) Introducing Rictor-T1135A into Rictor^−/− cells does not affect total SGK1 levels, phosphorylation of the SGK1 substrate NDRG1, or total PKCα levels compared to those with the wild type. Cells were transfected and treated as described for panel A. Lysates were immunoblotted with the indicated antibodies. Phosphorylation intensity in immunoblots was quantified as described for panel A, was normalized to the total levels of phosphorylation of both the indicated protein and of Rictor, and is shown relative to the level of phosphorylation at the 10 min time point for WT-Rictor-expressing cells. (C) The increase in insulin-induced Akt-S473 phosphorylation in T1135A-expressing cells compares in magnitude to the increase in Akt-S473 phosphorylation resulting from a short rapamycin pretreatment. Rictor^−/− MEFs were transfected as described for panel A. Confluent cells were serum starved for 16 h, pretreated with rapamycin (20 nM) for 5 min, and stimulated with insulin (100 nM) for 10 or 30 min where indicated. Lysates were immunoblotted with the indicated antibodies. Phosphorylation in immunoblots was quantified as described for panel B and are shown relative to the quantity at the 10 min time point for untreated WT-Rictor-expressing cells.

FIG. 7.

Rictor-T1135 phosphorylation creates a 14-3-3 binding site. (A) Like Rictor-T1135A, Rictor-T1135D promotes increased phosphorylation of Akt-S473 [p-Akt (S473)]. Rictor^−/− MEFs were transfected with empty vector (V), wild-type myc-Rictor (WT), myc-Rictor-T1135A, or myc-Rictor-T1135D, and confluent cells were serum starved for 16 h and stimulated with insulin (100 nM) for the indicated times. Lysates were immunoblotted with the indicated antibodies. (B) Rictor-T1135 can mediate 14-3-3 binding in vitro. HEK-293E cells were transfected with empty vector (V), wild-type myc-Rictor (WT), myc-Rictor-T1135A, or double mutants of Rictor-T1135A in combination with second-candidate 14-3-3 binding sites. GST pulldown assays were performed on the corresponding lysates using recombinant GST or GST-14-3-3. Pulled-down proteins and lysates were immunoblotted with the indicated antibodies. (C) Rictor-T1135 can mediate 14-3-3 binding in cells. Rictor^−/− MEFs were cotransfected with Flag-14-3-3 and empty vector (V), myc-Rictor (WT), or myc-Rictor-T1135A. Anti-Flag immunoprecipitates (flag IP) and lysates from cells grown in full serum were immunoblotted with the indicated antibodies. Numbers at the right of blots are molecular masses (in kilodaltons).

FIG. 8.

Model of mTORC1-dependent feedback mechanisms affecting insulin-stimulated activation of Akt. Insulin and IGF1 stimulate IRS-1 binding to PI3K, thereby activating PI3K and increasing its production of phosphotidylinositol-3,4,5-trisphosphate (PIP3). PIP3 recruits PDK1 and Akt to the plasma membrane, where PDK1 phosphorylates Akt-T308. Through an unknown mechanism, insulin and IGF1 also activate mTORC2, which phosphorylates Akt-S473, leading to full activation of Akt. Activated Akt directly phosphorylates and inhibits TSC2, leading to Rheb activation and subsequent stimulation of mTORC1 activation. Both mTORC1 and its downstream target S6K1 can phosphorylate specific serine residues on IRS-1, and this feedback mechanism negatively regulates IRS-1 signaling to PI3K. As shown in the current study, S6K1 also directly phosphorylates the Rictor subunit of mTORC2 on T1135 and stimulates 14-3-3 binding to Rictor. S6K1-mediated phosphorylation of Rictor negatively regulates the ability of mTORC2 to phosphorylate Akt-S473 in cells. Therefore, mTORC1 activation triggers two parallel negative-feedback mechanisms affecting the levels of insulin/IGF1-stimulated Akt phosphorylation and activation.