The innate immune kinase TBK1 directly increases mTORC2 activity and downstream signaling to Akt

Abstract

TBK1 (TANK-binding kinase 1) responds to microbial pathogens to initiate cellular responses critical for host innate immune defense. We found previously that TBK1 phosphorylates mTOR (mechanistic target of rapamycin) (on S2159) to increase mTOR complex 1 (mTORC1) activity and signaling in response to the growth factor EGF and the viral dsRNA mimetic poly(I:C). mTORC1 and the less well studied mTORC2 respond to diverse cues to control cellular metabolism, proliferation, and survival. Here we demonstrate that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation at physiological levels of protein expression. We find that TBK1 phosphorylates mTOR S2159 within mTORC2 in vitro, phosphorylates mTOR S2159 in cells, and interacts with mTORC2 in cells. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A), we show that TBK1 and mTOR S2159 phosphorylation increase mTORC2 catalytic activity and promote mTOR-dependent downstream signaling to Akt in response to several growth factors and poly(I:C). While microbial-derived stimuli activate TBK1, we find that growth factors fail to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, we propose that basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal crosstalk between TBK1 and mTOR complexes (mTORCs), key nodes within two major signaling systems. As TBK1 and mTORCs have each been linked to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.

TBK1 has a new Akt

“…TBK1 activity is increased by phosphorylation of S172 in its activation J o u r n a l P r e -p r o o f loop in response to pathogens in the innate immunity pathway. In contrast, Tooley et al found that EGF stimulation did not enhance S172 phosphorylation, supporting that it is the basal activity of TBK1 that is important for mTORC2 signaling downstream of growth factors (6). However, when RAW264.7 macrophages and primary bone marrow-derived macrophages were stimulated with the double-stranded RNA mimetic poly(I:C), which induces TBK1-S172 phosphorylation, TBK1 and mTOR-S2159 were also found to be required for mTORC2-dependent phosphorylation of Akt-S473.…”

“…Spleen tissue isolated from Mtor A/A mice showed diminished Akt-S473 phosphorylation. Therefore, the authors conclude that under both basal and activated states, the activation of Akt by TBK1 is mediated through mTORC2 (Figure 1) (6).…”

“…Although it had been previously reported that TBK1 can directly phosphorylate Akt at S473 and T308 in in vitro kinase assays, the ability of TBK1 to mediate these phosphorylation events under physiological conditions was not known (5). In a recent study, Tooley et al, contribute to the mechanistic understanding of TBK1 function in metabolic regulation by demonstrating a role for TBK1 in mTORC2 activation and subsequent phosphorylation of Akt (6).…”

“…To investigate how TBK1 regulates Akt activation, mouse embryonic fibroblasts (MEFs) were stimulated with epidermal growth factor (EGF) and evaluated for Akt-S473 and -T308 phosphorylation (6).…”

“…In this regard, drugs such as amlexanox and other compounds are under investigation for their potential clinical use (10). Of note, the study by Tooley et al only examined the TBK1-dependent phosphorylation of Akt-S473 by mTORC2 (6); mTORC2 also has additional substrates, including serum/glucocoid regulated kinase (SGK) and members of the Protein kinase C (PKC) family (Figure 1) (4). These kinases regulate unique cellular functions, such as regulation of J o u r n a l P r e -p r o o f the actin cytoskeleton.…”