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

Aaron Seth Tooley,Dubek Kazyken,Cagri Bodur,Ian E Gonzalez,Diane C Fingar

doi:10.1016/j.jbc.2021.100942

Aaron Seth Tooley, Dubek Kazyken + Show 3 more

Open Access

https://doi.org/10.1016/j.jbc.2021.100942

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Abstract

TBK1 responds to microbes 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) 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. Although TBK1 has been linked to Akt phosphorylation, a direct relationship between TBK1 and mTORC2, an Akt kinase, has not been described. By studying MEFs lacking TBK1, as well as MEFs, macrophages, and mice bearing an Mtor S2159A knock-in allele (MtorA/A) using in vitro kinase assays and cell-based approaches, we demonstrate here that TBK1 activates mTOR complex 2 (mTORC2) directly to increase Akt phosphorylation. We find that TBK1 and mTOR S2159 phosphorylation promotes mTOR-dependent phosphorylation of Akt in response to several growth factors and poly(I:C). Mechanistically, TBK1 coimmunoprecipitates with mTORC2 and phosphorylates mTOR S2159 within mTORC2 in cells. Kinase assays demonstrate that TBK1 and mTOR S2159 phosphorylation increase mTORC2 intrinsic catalytic activity. Growth factors failed to activate TBK1 or increase mTOR S2159 phosphorylation in MEFs. Thus, basal TBK1 activity cooperates with growth factors in parallel to increase mTORC2 (and mTORC1) signaling. Collectively, these results reveal cross talk between TBK1 and mTOR, key regulatory nodes within two major signaling networks. As TBK1 and mTOR contribute to tumorigenesis and metabolic disorders, these kinases may work together in a direct manner in a variety of physiological and pathological settings.

Highlights

Activation loop site autophosphorylation (S172) [6,7,8,9]
These results indicate that the kinase activity of TANK-binding kinase 1 (TBK1) rather than its scaffolding function promotes Akt phosphorylation. Consistent with this conclusion, the TBK1/ Iκβ kinase ε (IKKε) inhibitor amlexanox significantly reduced Akt P-S473 in response to epidermal growth factor (EGF) in mouse embryonic fibroblasts (MEFs) (Fig. 1D) and HEK293 cells (Fig. 1E). These results indicate that TBK1 kinase activity promotes mechanistic target of rapamycin (mTOR)-dependent phosphorylation of Akt S473 and T308 during EGF stimulation
By studying bone marrow–derived macrophages (BMDMs) in culture isolated from wildtype (Mtor+/+) and S2159A knock-in mice (MtorA/A), we demonstrated that mTOR S2159 phosphorylation is required for mTOR complex 1 (mTORC1) signaling and IFNβ production in macrophages stimulated with innate immune agonists (i.e., poly(I:C); LPS) [13]

Summary

Introduction

Activation loop site autophosphorylation (S172) [6,7,8,9]. TBK1/ IKKε in turn phosphorylate the transcription factors IRF3 and 7 (interferon regulatory factors), resulting in their translocation into the nucleus where they induce expression of type I interferons (e.g., IFNα/β), multifunctional cytokines that initiate host defense responses while limiting tissue damage [10, 11]. Consistent with our prior work [13], TBK1−/− MEFs displayed reduced S6K1 T389 phosphorylation, confirming that TBK1 promotes mTORC1 signaling (Fig. 1A).

Results

Conclusion