Novel Upstream Activation of mTORC2 by the Innate Immune Kinase TBK1

Abstract

The mechanistic target of rapamycin (mTOR) senses diverse intracellular and extracellular cues to control key fundamental cellular processes, including cell growth and proliferation, cell survival, and metabolism. Consequently, aberrant mTOR activity contributes to various pathological diseases, including cancer, type 2 diabetes, and autoimmune diseases. mTOR nucleates the catalytic core of two multimeric complexes: mTOR complex 1 (mTORC1) and mTORC2. mTORC1 integrates nutrient and growth factor cues to promote cell growth, cell cycle progression, and anabolic processes, marked by increases in protein, lipid, and nucleotide biosynthesis. Growth factors and energetic stress activate mTORC2, which promotes cell survival and regulates metabolism. While upstream regulation of mTORC1, including the environmental stimuli and associated signaling intermediates, is far more comprehensively characterized, upstream regulation of mTORC2 remains woefully ill-defined. The noncanonical IKK-related kinases, Tank-binding kinase 1 (TBK1) and IKK epsilon (IKKe), initiate the innate immune response to circumvent pathogenic infection by integrating signals from various pattern recognition receptors’ detection of microbial-derived antigens. TBK1 and IKKe, in turn, phosphorylate and activate IRF-3/7 transcription factors to drive interferon beta (IFN-beta) production to promote eradication of infectious agents. Prior work from our lab identified TBK1 as a novel mTOR-S2159 kinase. In macrophages, TBK1-mediated phosphorylation of mTOR-S2159 within mTORC1 was found to promote IRF-3 nuclear translocation and IFN-beta production, and EGF-stimulated mTORC1 signaling was reduced in mouse embryonic fibroblasts (MEFs) lacking TBK1 or wild type MEFs treated with the TBK1 inhibitor amlexanox. While studying TBK1-mediated activation of mTORC1, we also observed reduced EGF-stimulated phosphorylation of Akt-S473, a well-established mTORC2 substrate. Here we demonstrate that TBK1-mediated phosphorylation of mTOR on S2159 increases mTORC2 intrinsic kinase activity and downstream signaling to Akt-S473 in response to EGF, and, more broadly, mTOR-S2159 phosphorylation promotes mTORC2 and mTORC1 signaling in response to a broader array of growth factors (EGF, FBS, PDGF, and insulin). Additionally, we demonstrate that mTOR-S2159 phosphorylation promotes TLR-3-mediated mTORC2 and mTORC1 signaling in response to the synthetic dsRNA mimetic, poly (I:C). Mechanistically, TBK1 interacts with mTORC2 in intact cells and phosphorylates mTOR-S2159 within mTORC2 in vitro. These results contribute important mechanistic insight toward our poor understanding of the upstream regulation of mTORC2. More importantly, since aberrant TBK1 and mTORC2 activity have both been implicated in pathological diseases, most notably obesity-driven type 2 diabetes and oncogenesis, the TBK1-mTORC2 axis may represent an attractive therapeutic target for such diseases.