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

Figure 4. TBK1 promotes EGF‐stimulated mTORC1 signaling and catalytic activity in a manner dependent on site‐specific mTOR phosphorylation.

Figure 4

  1. mTOR S2159 phosphorylation is required for EGF‐stimulated mTORC1 signaling. HEK293 cells were co‐transfected with vector control, wild‐type, or rapamycin‐resistant (RR) AU1‐mTOR alleles (RR or RR/S2159A) together with HA‐S6K1. Cells were serum‐starved (20 h), treated −/+ rapamycin (30 min) to ablate endogenous mTORC1 function, and stimulated −/+ EGF [25 ng/ml] (30 min). HA‐S6K1 was immunoprecipitated, and immunoprecipitates (IP) and whole‐cell lysates (WCL) were immunoblotted (IB) as indicated. The arrow indicates AU1‐mTOR.
  2. TBK1 is required for EGF‐stimulated mTOR auto‐phosphorylation. TBK1+/+ and TBK1−/− MEFs were serum‐starved, EGF stimulated, and analyzed as in (A).
  3. EGF time course analysis of mTOR auto‐phosphorylation. TBK1+/+ and TBK1−/− MEFs were serum‐starved, EGF stimulated for 0‐60 min, and analyzed as in (A).
  4. TBK1 is required for EGF‐stimulated mTOR auto‐phosphorylation within mTORC1. TBK1+/+ and TBK1−/− MEFs were serum‐starved, pre‐treated with Ku‐0063794 [1 μM], and EGF stimulated as in (A). Raptor was immunoprecipitated, and IPs and WCL were analyzed.
  5. mTOR S2159 phosphorylation is required for EGF‐stimulated mTOR auto‐phosphorylation. HEK293 cells were transfected with Myc‐mTOR wild type (WT), S2159A, and kinase dead (KD). Cells were then serum‐starved, pre‐treated with Ku‐0063794 (30 min), and stimulated −/+ EGF as in (A).
  6. Model. EGF‐receptor signaling increases mTORC1 signaling through at least three pathways in MEFs: the PI3K/Akt, MAPK, and TBK1 pathways.