TORC-specific phosphorylation of mammalian target of rapamycin (mTOR): phospho-Ser2481 is a marker for intact mTOR signaling complex 2

Abstract

The mammalian target of rapamycin (mTOR) serine/threonine kinase is the catalytic component of two evolutionarily conserved signaling complexes. mTOR signaling complex 1 (mTORC1) is a key regulator of growth factor and nutrient signaling. S6 kinase is the best-characterized downstream effector of mTORC1. mTOR signaling complex 2 (mTORC2) has a role in regulating the actin cytoskeleton and activating Akt through S473 phosphorylation. Herein, we show that mTOR is phosphorylated differentially when associated with mTORC1 and mTORC2 and that intact complexes are required for these mTORC-specific mTOR phosphorylations. Specifically, we find that mTORC1 contains mTOR phosphorylated predominantly on S2448, whereas mTORC2 contains mTOR phosphorylated predominantly on S2481. Using S2481 phosphorylation as a marker for mTORC2 sensitivity to rapamycin, we find that mTORC2 formation is in fact rapamycin sensitive in several cancer cell lines in which it had been previously reported that mTORC2 assembly and function were rapamycin insensitive. Thus, phospho-S2481 on mTOR serves as a biomarker for intact mTORC2 and its sensitivity to rapamycin.

Fig. 1. mTORC1 and mTORC2 contain differentially phosphorylated mTOR

(A) Schematic of mTOR and multiple sequence alignment of the C-terminus of selected vertebrate and invertebrate TORs. Invertebrate species are Ciona intestinalis and C. savignyi (Cint and Csav); Drosophila melanogaster and D. virilis (Dmel and Dvir); Caenorhabditis elegans and C. briggsae (Cele and Cbrig); and Saccharomyces cerevisiae (TOR1 and TOR2). The region between the kinase domain (KD) and an N-terminal extension of the FATC domain (N-FATC) is conserved among vertebrates, including the marked phosphorylation sites S2448 and S2481. Asterisks indicate residues completely conserved in vertebrates. (B) HEK293 cells were serum-starved overnight. The indicated cells were stimulated with 200 nM insulin for 5 min at 37° C. Rictor and Raptor immunoprecipitates (IPs) from control and growth factor-stimulated cells were analyzed by immunoblotting with antibodies specific for mTOR phosphorylated on S2448 or S2481, or total mTOR. Whole cell lysates (WCL) were included as controls for total input. (C) Samples from actively growing U2OS cells were analyzed as in (B). Results are representative of multiple independent experiments.

Fig. 2. Intact mTORC1 and mTORC2 are necessary for mTOR phosphorylation

(A) HEK293 cells were infected with lentiviruses expressing a control shRNA or shRNAs targeting mTOR, Raptor or Rictor. Cells were selected with puromycin 24 hr. after infection and then serum-starved overnight 2 days post-selection. The indicated cells were stimulated with 200 nM insulin for 5 min. at 37°C. WCLs were normalized for total protein concentration and were analyzed by immunoblotting with antibodies specific for mTOR phosphorylated on S2448 or S2481. Blots for total mTOR, Rictor and Raptor were included as controls. (B) HEK293 cells were infected with lentiviruses expressing a control shRNA or shRNAs targeting Rictor or mSin1. Cells were treated as in (A) and analyzed by immunoblotting for mTOR phosphorylated on S2448 or S2481. Blots for total mTOR, Rictor and mSin1 were included as controls. (C) Wild type (WT) and Sin1^-/- mouse embryo fibroblasts (MEFs) were serum starved overnight. The indicated cells were stimulated with 200 nM insulin for 5 min. at 37°C. WCLs were normalized for total protein concentration and were analyzed by immunoblotting with antibodies specific for mTOR phosphorylated on S2481, total mTOR and Rictor. (D) HEK293 cells were infected with lentiviruses and were treated as in (A). Rictor and Raptor IPs from control and growth factor- stimulated cells were analyzed by immunoblotting for bound mTOR. WCLs were analyzed by immunoblotting with antibodies specific for mTOR phosphorylated on S2448 or S2481. Results are representative of multiple independent experiments.

Fig. 3. Prolonged treatment of cells with rapamycin inhibits mTOR phosphorylation on S2448 and S2481

(A) Serum-starved HEK293 cells were cultured in the presence or absence of 100 nM rapamycin for either 1 or 24 hr. The indicated cells were stimulated with 200 nM insulin for 5 min. at 37° C. WCLs were analyzed by immunoblotting with antibodies specific for mTOR phosphorylated on S2448 or S2481, or total mTOR. Rictor IPs were analyzed by immunoblotting with antibodies specific for Rictor, mTOR, and mTOR phosphorylated on S2481. (B) Actively growing U2OS cells were cultured in the presence or absence of 100 nM rapamycin for either 1 or 24 hr and analyzed as described as in (A). (C) Actively growing MDA 231, MDA 468, SKBR3 and A549 cells were treated as in (B). Rictor IPs were analyzed by immunoblotting with antibodies specific for Rictor and mTOR. WCLs were analyzed by immunoblotting for phospho-mTOR (S2481), phospho-Akt (S473), phospho-S6K (T389), total mTOR and total Akt. (D) Actively growing C2C12 myoblasts and HepG2 cells were treated as in (B) and WCLs were analyzed as in (C). Results are representative of multiple independent experiments.

Fig. 4. Depletion of mTORC2 renders S473 phosphorylation of Akt sensitive to rapamycin

MDA-MB-468 cells were infected with lentiviruses expressing a control shRNA or shRNAs targeting mTOR, Rictor or mSin1. Cells were selected with puromycin 24 hr. after infection and the indicated cells were treated with 100 nM rapamycin for an additional 24 hr. WCLs were normalized for total protein concentration and were analyzed by immunoblotting for phospho-mTOR (S2481), phospho-Akt (S473), phospho-S6K (T389), and total Akt, mTOR Rictor and mSin1. Results are representative of multiple independent experiments.