PRAS40 and PRR5-like protein are new mTOR interactors that regulate apoptosis

Abstract

TOR (Target of Rapamycin) is a highly conserved protein kinase and a central controller of cell growth. TOR is found in two functionally and structurally distinct multiprotein complexes termed TOR complex 1 (TORC1) and TOR complex 2 (TORC2). In the present study, we developed a two-dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) based proteomic strategy to identify new mammalian TOR (mTOR) binding proteins. We report the identification of Proline-rich Akt substrate (PRAS40) and the hypothetical protein Q6MZQ0/FLJ14213/CAE45978 as new mTOR binding proteins. PRAS40 binds mTORC1 via Raptor, and is an mTOR phosphorylation substrate. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 kinase activity toward eIF-4E binding protein (4E-BP) and PRAS40 itself. HeLa cells in which PRAS40 was knocked down were protected against induction of apoptosis by TNFalpha and cycloheximide. Rapamycin failed to mimic the pro-apoptotic effect of PRAS40, suggesting that PRAS40 mediates apoptosis independently of its inhibitory effect on mTORC1. Q6MZQ0 is structurally similar to proline rich protein 5 (PRR5) and was therefore named PRR5-Like (PRR5L). PRR5L binds specifically to mTORC2, via Rictor and/or SIN1. Unlike other mTORC2 members, PRR5L is not required for mTORC2 integrity or kinase activity, but dissociates from mTORC2 upon knock down of tuberous sclerosis complex 1 (TSC1) and TSC2. Hyperactivation of mTOR by TSC1/2 knock down enhanced apoptosis whereas PRR5L knock down reduced apoptosis. PRR5L knock down reduced apoptosis also in mTORC2 deficient cells. The above suggests that mTORC2-dissociated PRR5L may promote apoptosis when mTOR is hyperactive. Thus, PRAS40 and PRR5L are novel mTOR-associated proteins that control the balance between cell growth and cell death.

Figure 1. Confirmation of PRAS40 and PRR5L binding to mTOR.

A. PRAS40 is associated specifically with mTORC1. mTOR, Rictor, Raptor and mock IPs were performed with HeLa and HEK293 extracts and analyzed by immunoblotting. PRAS40 was found specifically in mTOR and Raptor IPs. 1 h rapamycin treatment of cells dissociated a Raptor-PRAS40 subcomplex from mTOR. B. PRR5L is associated specifically with mTORC2. HeLa or HEK293 cells were transfected with GST-PRR5L or the empty plasmid. mTOR, Rictor, Raptor and mock IPs and GST pull downs were analyzed by immunoblotting. GST-PRR5L is detected specifically in mTOR and Rictor IPs. mTOR, Rictor and SIN1, but not Raptor, are detected specifically in GST-PRR5L pull downs.

Figure 2. PRAS40 interacts with mTORC1. A. PRAS40 binds mTOR via Raptor.

HEK293 cells were transfected with Raptor or control siRNA vectors and incubated for 4 days, followed by mTOR IP and immunoblotting. mTOR IPs and cell extracts were probed with antibodies directed against the indicated proteins. Since knock down of Raptor reduced the cellular amount of PRAS40, the PRAS40 signal in the IPs was quantified relative to PRAS40 levels in the corresponding extract. Quantitations were averaged over three independent experiments. Raptor knock down reduced the amount of PRAS40 associated with mTOR by 50%, as compared to control cells. B. mTOR kinase domain is involved in PRAS40 binding. HEK293 cells were transfected with a plasmid expressing wild type (WT) HA-mTOR or kinase dead (KD) HA-mTOR or an empty control plasmid, and incubated for 48 h followed by extract preparation and IP with an anti-HA antibody. PRAS40 levels in the extracts remained unaltered. PRAS40 association with mTORC1 containing HA-mTOR KD was moderately reduced as compared with mTORC1 containing HA-mTOR WT.

Figure 3. mTOR phosphorylates PRAS40 and PRR5L, and PRAS40 inhibits mTOR kinase activity. A. mTOR phosphorylates PRAS40 and PRR5L.

Kinase assays were performed using mTORC1 or mTORC2 immunopurified from HEK293 cells, and purified PRAS40, GST-PRR5L (PRR5L) or 4E-BP as substrates. Rapamycin (100 nM) and purified FKBP12 were added directly to the reaction. Both PRR5L and PRAS40 are phosphorylated in vitro by both mTORCs. Phosphorylation by mTORC1 was rapamycin-sensitive. B. PRAS40 inhibits mTORC1 kinase activity toward 4E-BP and PRAS40 itself. Kinase assays were performed using mTORC1 immunopurified from HEK293 cells, purified 4E-BP as a substrate, and increasing concentrations of PRAS40. PRAS40 inhibits mTORC1 autophosphorylation and mTORC1 phosphorylation of 4E-BP and PRAS40, in a concentration-dependent manner.

Figure 4. PRAS40 and PRR5L are pro-apoptotic.

A. PRR5L and PRAS40 knock down cells are resistant to TNFα/cycloheximide induced apoptosis. HeLa cells were transfected with PRR5L, PRAS40 or control siRNA and incubated for 48 h, followed by 2 h induction of apoptosis with TNFα and cycloheximide. Cells were fixed and stained with DAPI and cleaved PARP antibody, and the percentage of apoptotic cells was quantified. B. PRAS40's effect on apoptosis is independent of mTORC1. HeLa cells were transfected with PRAS40 or control siRNA and incubated for 48 h, and treated with 100 nM rapamycin or carrier for 1 h before incubation with TNFα and cycloheximide for 2 h to induce apoptosis. Extracts were analyzed by immunoblotting with the indicated antibodies. C. PRR5L deficiency protects against apoptosis in SIN1 deficient cells. HeLa cells were transfected with diced PRR5L siRNA and/or synthetic siRNA against SIN1 as indicated, or the appropriate control siRNAs. Cells were incubated for 48 h, and apoptosis was induced with TNFα and cycloheximide for 2 h. Cells were fixed and stained with DAPI and cleaved PARP antibody, and the percentage of apoptotic cells was quantified. The efficiency of SIN1 knock down was assessed in parallel by immunoblotting (right panel).

Figure 5. PRR5L binds to mTORC2 via SIN1 and/or Rictor but does not affect mTORC2 integrity or kinase activity. A. PRR5L expression in HeLa and HEK293 cells, and PRR5L knock down efficiency.

Top panel: Total RNA was purified from HeLa or HEK293 cells, followed by reverse transcription and PCR with primers corresponding to PRR5L. As a negative control, reverse transcription without the transcriptase enzyme was performed. Endogenous PRR5L is expressed in both cell lines. Bottom panel: HEK293 cells were cotransfected with a GST-PRR5L vector and PRR5L siRNA or control siRNA, and incubated for 48 h. Immunoblots were performed on with antibody against GST or Actin. B. PRR5L binds mTOR via SIN1 and/or Rictor. HEK293 cells were cotransfected with a GST-PRR5L vector and a SIN1 siRNA vector or a control siRNA vectors, and incubated for 4 days. GST pull downs were immunoblotted with the indicated antibodies. GST-PRR5L was detected with an anti-GST antibody. mTOR binding to GST-PRR5L is weaker in the absence of SIN1 and Rictor. C. mTORC2 remains intact in PRR5L knock down cells. HEK293 cells were transfected with PRR5L siRNA or control siRNA and incubated for 48 h. Rictor IPs were immunoblotted with the indicated antibodies. D. mTORC2 readouts are unaltered in PRR5L knock down cells. HEK293 cells were transfected with PRR5L siRNA or control siRNA and incubated for 48 h. Immunoblots were performed on protein extracts with the indicated antibodies. The phosphorylation of Akt S473 and paxillin Y118 is unaltered by PRR5L knock down.

Figure 6. Analysis of TSC1/2 knock down cells. A. PRR5L is released from mTORC2 in TSC1/2 deficient cells.

TSC knock down was induced in TSCsi293 cells by tetracycline treatment for 4 days. Cells were cotransfected with GST-PRR5L vector and incubated for 2 days, followed by GST pull downs and immunoblots with the indicated antibodies. GST-PRR5L was detected with an anti-GST antibody. B. TSC1/2 knock down facilitates apoptosis. TSCsi293 and T-REx-293 (control) cells were treated with tetracycline for 4 days. Apoptosis was induced by 1.5 h treatment with TNFα and cycloheximide. Extracts were probed with the indicated antibodies.