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. 2017 Nov 17;37(1):19–38. doi: 10.15252/embj.201696164

Figure 1. TBK1 interacts with and phosphorylates mTOR within mTORC1.

Figure 1

  1. The mTOR S2159 sequence fits TBK1 and IKKε consensus phosphorylation motifs.
  2. TBK1/IKKε phosphorylate mTOR S2159 directly in vitro. In vitro kinase (IVK) assays with recombinant (re) active TBK1 or IKKε [50 ng] (Invitrogen) and recombinant GST‐mTOR substrate [200 ng] for 30 min at 30°C. Reactions were pre‐incubated on ice 30 min with amlexanox [500, 250 or 50 μM], BX‐795 [10 μM] or MRT‐67307 [10 μM] and immunoblotted (IB) as indicated.
  3. TBK1/IKKε phosphorylate full‐length mTOR on S2159. Myc‐mTOR wild type (WT) and S2159A were immunoprecipitated (IP) from transfected HEK293 cells and incubated with re‐TBK1 or re‐IKKε. IVK assays were performed as above and immunoblotted (IB) as indicated.
  4. TBK1 overexpression increases mTOR P‐S2159, and poly(I:C) boosts this phosphorylation further. HEK293‐TLR3 cells were co‐transfected with Flag‐TBK1 and Myc‐mTOR. Cells were serum‐starved (20 h) and stimulated −/+ poly(I:C) [50 μg/ml] (2 h). Myc‐mTOR immunoprecipitates were immunoblotted (IB) as indicated.
  5. TBK1 and IKKε overexpression increases mTOR P‐S2159 within mTORC1. HEK293‐TLR3 cells were co‐transfected with Flag‐TBK1 or Flag‐IKKε, Myc‐mTOR, and HA‐raptor. HA‐raptor immunoprecipitates and whole‐cell lysates (WCL) were immunoblotted (IB) as indicated.
  6. Flag‐TBK1 and Flag‐IKKε co‐immunoprecipitate with endogenous mTORC1. HEK293T cells were transfected with Flag‐TBK1 or Flag‐IKKε wild type (+) or kinase dead (KD). Endogenous raptor immunoprecipitates and WCL were immunoblotted (IB) as indicated.
  7. mTOR is phosphorylated on S2159 in wild type but not TBK1 null MEFs. TBK1+/+ and TBK1−/− MEFs were serum‐starved (20 h) and stimulated ± EGF [25 ng/ml]. WCL was immunoblotted (IB) as indicated. The arrow indicates mTOR phosphorylated on S2159.
  8. The TBK1‐ and IKKε‐activating agonists poly(I:C) and LPS increase mTOR P‐S2159 in a BX‐795‐sensitive manner in cultured macrophages. RAW264.7 macrophages were pre‐treated with BX‐795 [10 μM] (2 h) and stimulated −/+ poly(I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).
  9. Poly(I:C) and LPS increase mTOR P‐S2159 in primary bone marrow‐derived macrophages (BMDMs). BMDMs were stimulated −/+ poly(I:C) [30 μg/ml] or LPS [100 ng/ml] (60 min).
  10. LPS increases mTOR P‐S2159 in vivo. Mice (C57BL6, 6 weeks old) were injected intraperitoneally with PBS or LPS [1 mg/kg BW] (2 h). mTOR was immunoprecipitated from spleen tissue, and IPs and WCL were immunoblotted as indicated. The graph depicts levels of mTOR P‐S2159 relative to total mTOR in spleen tissue −/+ LPS. n = 8 from 3 independent experiments ± SD. *P = 0.004 relative to PBS‐treated control mice by paired t‐test (two‐tailed).

Source data are available online for this figure.