ERK1/2 phosphorylate Raptor to promote Ras-dependent activation of mTOR complex 1 (mTORC1)

Abstract

The Ras/mitogen-activated protein kinase (MAPK) pathway regulates a variety of cellular processes by activating specific transcriptional and translational programs. Ras/MAPK signaling promotes mRNA translation and protein synthesis, but the exact molecular mechanisms underlying this regulation remain poorly understood. Increasing evidence suggests that the mammalian target of rapamycin (mTOR) plays an essential role in this process. Here, we show that Raptor, an essential scaffolding protein of the mTOR complex 1 (mTORC1), becomes phosphorylated on proline-directed sites following activation of the Ras/MAPK pathway. We found that ERK1 and ERK2 interact with Raptor in cells and mediate its phosphorylation in vivo and in vitro. Using mass spectrometry and phosphospecific antibodies, we found three proline-directed residues within Raptor, Ser(8), Ser(696), and Ser(863), which are directly phosphorylated by ERK1/2. Expression of phosphorylation-deficient alleles of Raptor revealed that phosphorylation of these sites by ERK1/2 normally promotes mTORC1 activity and signaling to downstream substrates, such as 4E-BP1. Our data provide a novel regulatory mechanism by which mitogenic and oncogenic activation of the Ras/MAPK pathway promotes mTOR signaling.

FIGURE 1.

Activation of the Ras/MAPK pathway induces ERK1/2-dependent phosphorylation of Raptor on proline-directed sites. A, HEK293 cells were transfected with empty vector or myc-tagged Raptor, serum-starved, and stimulated for 10 or 20 min with agonists of the Ras/MAPK pathway. Immunoprecipitated (IP) Raptor was then assayed for phosphorylation using a phospho motif antibody that recognizes the pS/T-P consensus motif. B, HEK293 cells were transfected with myc-tagged Raptor, serum-starved, and stimulated with 100 ng/ml PMA. Immunoprecipitated Raptor was then treated with 400 units of λ-phosphatase for 60 min at 30 °C and immunoblotted with the pS/T-P phospho motif antibody. C, model depicting the Ras/MAPK pathway and inhibitors and activators used in this study is shown. D, as in A, but cells were pretreated with 10 μm U0126, 10 μm PD184352, or 10 μm BI-D1870 for 30 min prior to 50 ng/ml PMA stimulation. E, HEK293 cells were cotransfected with control vector or myc-tagged Raptor, and siRNA duplexes were targeted against a scrambled sequence (Scr), ERK1 or ERK2, serum-starved, and stimulated with 50 ng/ml PMA. Immunoprecipitated Raptor was then assayed as in A. F, cells were cotransfected with myc-tagged Raptor and constitutively activated (G12V) or dominant negative (S17N) Ras, serum-starved overnight. Immunoprecipitated Raptor was then assayed as in A.

FIGURE 2.

ERK1/2 phosphorylate Raptor in vitro. A, immunoprecipitated HA-tagged WT ERK1 or HA₆-tagged ERK2 from serum-starved or PMA-stimulated cells lysed in a Nonidet P-40 containing buffer was incubated with immunopurified Raptor in a kinase reaction with [γ-³²P]ATP. The resulting samples were subjected to SDS-PAGE, and the dried Coomassie Blue-stained gel was autoradiographed. B, samples were treated as in A, but kinase reactions were performed without radioactivity. The resulting samples were immunoblotted for Raptor phosphorylation on pS/T-P consensus sites. C, as in A, but ERK2 was immunoprecipitated from cells treated with 50 ng/ml PMA in the presence or absence of 10 μm U0126. The levels of ³²P incorporation into Raptor were quantified and are shown below the autoradiogram. D, recombinant activated ERK1 was incubated with immunopurified WT or S3A Raptor in a kinase reaction, and the resulting samples were immunoblotted for Raptor phosphorylation on pS/T-P consensus sites.

FIGURE 3.

ERK2 interacts with Raptor in a mTOR-independent manner. A, HEK293 cells were transfected with myc-tagged Raptor and HA-tagged ERK2, stimulated with PMA, and lysed in either CHAPS or Nonidet P-40 buffer (NP-40). The presence of ERK2 was assayed within Raptor immunoprecipitates. B, cells were transfected with myc-tagged Raptor and lysed in Nonidet P-40 buffer. Cell extracts were incubated or not with 0.5 mg/ml DTSSP cross-linker for 45 min at room temperature. Cross-linking reactions were quenched by adding Tris, pH 7.4, and the presence of Raptor was assayed within endogenous ERK1/2 immunoprecipitates.

FIGURE 4.

Identification of Ras/MAPK-dependent proline-directed phosphorylation sites in Raptor. A, schematic representation of Raptor and phosphorylation sites identified by mass spectrometric analysis (proline-directed sites are indicated in boldface characters) is shown. B, cells transfected with wild-type (wt) Raptor, S8A, S696A, or S863A mutant, serum-starved, and stimulated with 50 ng/ml PMA for 20 min. Immunoprecipitated (IP) Raptor was assayed for phosphorylation using the pS/T-P phospho motif antibody. C, tandem MS spectrum of Glu-C peptide precursor ion m/z 727.3²⁺. Labeled ions correspond to b-type fragment ions. The location of Ser⁸ phosphorylation is confirmed by the observation of a y8 ion at m/z 865.4 and a b5 ion at m/z 589.1 D, cells transfected with wild-type or S3A mutant of Raptor (S8A/S696A/S863A) and treated as in B. E, primary sequence alignment showing conservation of the three proline-directed sites within different Raptor orthologs.

FIGURE 5.

ERK1/2 phosphorylate Raptor on Ser⁶⁹⁶ and Ser⁸⁶³in vivo. A, cells were transfected with myc-tagged Raptor and stimulated with 50 ng/ml PMA following pretreatment with 10 μm U0126 or vehicle for 30 min. Immunoprecipitated Raptor was then assayed for phosphorylation using phosphospecific antibodies. B, cells were transfected with myc-tagged Raptor and aui-tagged mTOR, serum-starved, and stimulated with 50 ng/ml PMA or 100 nm insulin following pretreatment with 10 μm PD184352 or 25 nm rapamycin and treated as in A. C, endogenous Raptor phosphorylation at Ser⁶⁹⁶ and Ser⁸⁶³ was determined in U2OS and HeLa cells. Both cell types were serum-starved overnight, and total cell lysates were assayed for Raptor phosphorylation using phosphospecific antibodies against Ser⁶⁹⁶ and Ser⁸⁶³. D, phosphorylation of endogenous Raptor in HEK293 cells stimulated with 50 ng/ml PMA or 25 μg/ml EGF for 20 and 10 min, respectively, is shown. Cells were either transfected with an siRNA with a scrambled sequence (Scr), or targeting both ERK1 and ERK2. Phosphorylation of endogenous Raptor in total cell lysates was performed as in A.

FIGURE 6.

Raptor phosphorylation by ERK1/2 does not regulate its ability to interact with mTOR or mTORC1 substrates. A, cells were cotransfected with FLAG-tagged mTOR and either wild-type (wt) Raptor or the S3A mutant, serum-starved, and stimulated with PMA. Associated mTOR was assayed within Raptor immunoprecipitates by immunoblotting. B, cells were cotransfected with HA-tagged S6K1 and either wild-type Raptor or the S3A mutant, serum-starved, and stimulated with PMA. Associated S6K1 was assayed within Raptor immunoprecipitates. C, cells were cotransfected with HA-tagged 4E-BP1 and either wild-type Raptor or the S3A mutant, serum-starved, and stimulated with PMA. Associated 4E-BP1 was assayed within Raptor immunoprecipitates.

FIGURE 7.

ERK1/2-mediated phosphorylation of Raptor is important for mTORC1 kinase activity. A, cells were cotransfected with wild-type (wt) mTOR and wild-type Raptor or the S3A mutant and serum-starved, and mTORC1 kinase activity was assayed within Raptor immunoprecipitates using GST-4E-BP1 as a substrate. The kinase reaction was performed in the presence of [γ-³²P]ATP, and the resulting samples were subjected to SDS-PAGE, and the dried Coomassie Blue-stained gel was autoradiographed. B, wild-type Raptor or the S3A mutant was transfected together with wild-type mTOR or a rapamycin-resistant mutant (S2035I) into HEK293 cells. After serum starvation, cells were treated with 20 nm rapamycin for 20 min to block endogenous mTORC1 activity and then stimulated with PMA for 20 min. Phosphorylation of 4E-BP1 on the mTORC1-dependent residues Thr³⁷/Thr⁴⁶ was detected by immunoblotting. The histogram shows quantifications of phosphorylated 4E-BP1 from three independent experiments. C, as in A, but cells were cotransfected with wild-type mTOR and Raptor and treated with 10 μm U0126, 10 μm PD184352, or vehicle for 30 min.

FIGURE 8.

Schematic representation of the molecular mechanisms employed by the Ras/MAPK pathway to regulate mTORC1 signaling. Activation of the Ras/MAPK pathway stimulates mTORC1 activity through the coordinated action of the kinases ERK1/2 and RSK on the TSC protein complex upstream mTORC1, and on Raptor, an important partner of mTOR within mTORC1.