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bioRxiv · 10.1101/2022.10.11.511671

Complex Intracellular Mechanisms of TBK1 Kinase Activation Revealed by a Specific Small Molecule Inhibitor

Ye, J.·Dephoure, N.·Maniatis, T.
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Abstract

TANK-binding kinase 1 (TBK1), a dimeric serine/threonine protein kinase, plays a critical role in multiple signaling pathways including innate immunity, autophagy and cell death. TBK1 is activated by the phosphorylation of an essential serine residue 172 (S172) within the activation loop of the kinase domain, and this phosphorylation can occur by trans-autophosphorylation: one TBK1 dimer phosphorylates a second dimer at S172. Here we show that phosphorylation of TBK1 S172 in cultured human cells in response to multiple inducers is reduced, but not abolished by the highly specific and potent TBK1 small molecule inhibitor GSK8612. Thus, upstream kinase(s) must phosphorylate TBK1 in response to inducers in cultured cells. We show that distinct upstream kinases are recruited for the activation of TBK1 in response to different inducers. We also identify extensive crosstalk among TBK1, IKK{beta} and IKK{varepsilon} kinases in the cellular response to various inducers. In addition, we show that dsDNA and dsRNA trigger dynamic intracellular translocation of TBK1, leading to its localization and activation on the Golgi apparatus or mitochondria, respectively. GSK8612 does not block the intracellular localization of TBK1. We conclude that TBK1 is activated by both upstream kinase phosphorylation and by trans-autophosphorylation, and the signal-dependent spatial engagement of TBK1 with other signaling molecules is a fundamental mechanism for its specific activation by multiple inducers.

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Ye, J., Dephoure, N., Maniatis, T.. 2022-10-12. Complex Intracellular Mechanisms of TBK1 Kinase Activation Revealed by a Specific Small Molecule Inhibitor. https://doi.org/10.1101/2022.10.11.511671

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