Project Summary Cellular and organismal homeostasis requires integration of diverse local and systemic cues by cell signaling networks. The anabolic kinase mTOR (mechanistic target of rapamycin) integrates nutrient and growth factor availability, playing critical roles in cellular processes that modulate metabolism, tumorigenesis, and immune function. mTOR comprises the catalytic core of two functionally distinct, multi-subunit complexes, mTORC1 and mTORC2. Not surprisingly, aberrant mTOR function contributes to pathological states including obesity-linked type II diabetes, cancer, and immune disorders. Despite the physiological importance of mTOR, major gaps exist in our mechanistic understanding of mTOR regulation and function, particularly how mTOR communicates with other important regulatory systems to control cell and organismal physiology. Recent work from our lab provides the scientific premise for this proposal, which demonstrated that the non-canonical innate immune kinase TBK1 (TANK binding kinase 1) phosphorylates mTOR (on S2159) to increase mTORC1 and mTORC2 signaling upon cellular stimulation with innate immune agonists (e.g., the viral dsRNA mimetic poly(I:C); bacterial LPS), growth factors (EGF), or hormones (insulin) in cultured cells and in vivo (Bodur et al. 2018; Tooley et al. 2021). This work linked two physiologically important signaling systems not previously known to communicate. Classically, TBK1 responds to microbial-derived signals to initiate first-line host defense against viral and bacterial pathogens. These signals lead to phosphorylation and activation of TBK1, which drives ]production of type I interferons (e.g., IFNb) and IFN-stimulated genes (ISGs). More recent work implicates TBK1 in a broader range of functions including improved glycemic control during diet-induced obesity and tumorigenesis through poorly defined downstream mediators. We demonstrated recently that mice bearing a “TBK1 resistant”, phosphorylation deficient Mtor S2159A allele display systemic insulin resistance and hyperglycemia in the diet-induced obese but not the lean state (Bodur et al. 2022). Macrophages and adipose tissue from these mice display reduced expression of IFNb and the anti-inflammatory ISG IL-10 as well as elevated expression of pro-inflammatory cytokines (Bodur et al. 2022). The overarching goal of this application is to elucidate the regulation and function of TBK1-mTOR crosstalk in cells and in vivo. We will employ biochemical, cellular, and molecular approaches to better define how TBK1 promotes mTORC1 and mTORC2 signaling in the absence of microbial-derived signals (Aim 1). We will also investigate how TBK1- mTOR signaling protects against insulin resistance, hyperglycemia, and inflammation in the diet-induced obese but not lean state by studying mice in vivo and primary cells in culture (Aim 2). This project will not only elucidate basic mechanisms underlying control of TBK1-mTOR signaling but will also advance our knowl...