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The innate immune kinase TBK1 initiates inflammatory responses to combat infectious pathogens by driving production of type I interferons. TBK1 also controls metabolic processes and promotes oncogene-induced cell proliferation and survival. Here, we demonstrate that TBK1 activates mTOR complex 1 ( mTORC1) directly. In cultured cells, TBK1 associates with and activates mTORC1 through site-specific mTOR phosphorylation (on S2159) in response to certain growth factor receptors (i.e., EGF-receptor but not insulin receptor) and pathogen recognition receptors ( PRRs) (i.e., TLR3; TLR4), revealing a stimulus-selective role for TBK1 in mTORC1 regulation. By studying cultured macrophages and those isolated from genome edited mTOR S2159A knock-in mice, we show that mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFN-β production. These data demonstrate a direct mechanistic link between TBK1 and mTORC1 function as well as physiologic significance of the TBK1- mTORC1 axis in control of innate immune function. These data unveil TBK1 as a direct mTORC1 activator and suggest unanticipated roles for mTORC1 downstream of TBK1 in control of innate immunity, tumorigenesis, and disorders linked to chronic inflammation. [ABSTRACT FROM AUTHOR]

The mammalian target of rapamycin complex 1 (mTORC1) functions as an environmental sensor to promote critical cellular processes such as protein synthesis, cell growth, and cell proliferation in response to growth factors and nutrients. While diverse stimuli regulate mTORC1 signaling, the direct molecular mechanisms by which mTORC1 senses and responds to these signals remain poorly defined. Here we investigated the role of mTOR phosphorylation in mTORC1 function. By employing mass spectrometry and phospho-specific antibodies, we demonstrated novel phosphorylation on S2159 and T2164 within the mTOR kinase domain. Mutational analysis of these phosphorylation sites indicates that dual S2159/T2164 phosphorylation cooperatively promotes mTORC1 signaling to S6K1 and 4EBP1. Mechanistically, S2159/T2164 phosphorylation modulates the mTOR-raptor and raptor-PRAS40 interactions and augments mTORC1-associated mTOR S2481 autophosphorylation. Moreover, mTOR S2159/T2164 phosphorylation promotes cell growth and cell cycle progression. We propose a model whereby mTOR kinase domain phosphorylation modulates the interaction of mTOR with regulatory partner proteins and augments intrinsic mTORC1 kinase activity to promote biochemical signaling, cell growth, and cell cycle progression. [ABSTRACT FROM AUTHOR]

The rapamycin-sensitive mTOR complex 1 (mTORC1) promotes protein synthesis, cell growth, and cell proliferation in response to growth factors and nutritional cues. To elucidate the poorly defined mechanisms underlying mTORC1 regulation, we have studied the phosphorylation of raptor, an mTOR-interacting partner. We have identified six raptor phosphorylation sites that lie in two centrally localized clusters (cluster 1, Ser696/Thr706 and cluster 2, Ser855/Ser859/Ser863/Ser877) using tandem mass spectrometry and generated phosphospecific antibodies for each of these sites. Here we focus primarily although not exclusively on raptor Ser863 phosphorylation. We report that insulin promotes mTORC1-associated phosphorylation of raptor Ser863 via the canonical PI3K/TSC/Rheb pathway in a rapamycin-sensitive manner. mTORC1 activation by other stimuli (e.g. amino acids, epidermal growth factor/MAPK signaling, and cellular energy) also promote raptor Ser863 phosphorylation. Rheb overexpression increases phosphorylation on raptor Ser863 as well as on the five other identified sites (e.g. Ser859, Ser855, Ser877, Ser696, and Thr706). Strikingly, raptor Ser863 phosphorylation is absolutely required for raptor Ser859 and Ser855 phosphorylation. These data suggest that mTORC1 activation leads to raptor multisite phosphorylation and that raptor Ser863 phosphorylation functions as a master biochemical switch that modulates hierarchical raptor phosphorylation (e.g. on Ser859 and Ser855). Importantly, mTORC1 containing phosphorylation site-defective raptor exhibits reduced in vitro kinase activity toward the substrate 4EBP1, with a multisite raptor 6A mutant more strongly defective that single-site raptor S863A. Taken together, these data suggest that complex raptor phosphorylation functions as a biochemical rheostat that modulates mTORC1 signaling in accordance with environmental cues. [ABSTRACT FROM AUTHOR]