Reactome | TBK1, IKBKE phosphorylate RIPK1 at T189

TBK1, IKBKE phosphorylate RIPK1 at T189

Stable Identifier

R-HSA-9817397

Type

Reaction [transition]

Species

Homo sapiens

Compartment

cytosol, plasma membrane

ReviewStatus

5/5

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Binding of TNFα to TNF receptor 1 (TNFR1) induces either cell survival or cell death through the sequential formation of several signaling complexes, namely complex-I, IIa and IIb (Micheau O and Tschopp J 2003; Walczak H 2011). The dynamic assembly of these complexes is tightly regulated by proteolysis, ubiquitination, deubiquitination and phosphorylation of receptor-interacting serine/threonine protein kinase 1 (RIPK1) and other components of the TNFα signaling pathway. The rapidly forming complex-I (the TNFR1 signaling complex) is assembled at the receptor’s cytoplasmic tail and consists of TNFR1, TNFR1-associated death domain (TRADD), TNF receptor associated factor-2 (TRAF2) and RIPK1 (Micheau O and Tschopp J 2003). The activated TNFR1 signaling complex (complex-I) recruits several E3 ubiquitin (Ub) ligases, such as cIAP1/2 cellular inhibitor of apoptosis (BIRC2, BIRC3), mind bomb 2 (MIB2) or linear ubiquitin chain assembly complex (LUBAC) (Micheau O and Tschopp J 2003; Feltham R et al. 2018; Yuan J et al. 2019). K63-linked ubiquitination by BIRC2, BIRC3 and MIB2 as well as LUBAC-mediated Met1-linked linear ubiquitination of RIPK1 and other complex components stabilize the membrane-bound pro-survival TNFR1 signaling complex, while suppressing the formation of the cytosolic death-inducing complex IIa (TRADD:TRAF2:RIPK1:FADD:CASP8) and IIb (RIPK1:RIPK3:MLKL). The catalytic activity of LUBAC also enables recruitment of TRAF-associated NF-κB activator (TANK) binding kinase 1 (TBK1) and inhibitor-kappa-B kinase (IKK) epsilon (IKKε or IKBKE) to the TNFR1 signaling complex (Lafont E et al. 2018; Xu D et al. 2018). LUBAC is composed of the HOIP, HOIL-1L, and SHARPIN subunits. HOIP deficiency strongly diminished recruitment of TBK1 or IKBKE to the complex-I in human cervical carcinoma epithelial HeLa cells and lung carcinoma epithelial A549 cells (Lafont E et al. 2018). Western blot analysis revealed that TBK1 and IKBKE are recruited to and strongly phosphorylated within the TNFR1 complex in various TNFα-stimulated human cell lines including A549, keratinocyte HaCaT, monocytic U937 and THP-1 cells (Lafont E et al. 2018). Similarly, TBK1 was found to associate with components of the complex-I in the LUBAC-dependent manner in mouse cells (Xu D et al. 2018). IKBKE, but not TBK1, was detected within the TNFR1 complex in human telomerase reverse transcriptase (TERT)-immortalized dermal fibroblasts derived from patients with TBK1 deficiency as a result of loss-of-function mutation at W619* (Taft J et al. 2021). The enzymatic activity of TBK1/IKBKE is initiated by phosphorylation at S172 located in the T loop of the TBK1 and IKKε kinase domains (Shimada T et al. 1999; Kishore N et al. 2002; Gu L et al. 2013). Activated TBK1 and IKBKE are known to trigger phosphorylation of interferon regulatory factor 3 (IRF3) and IRF7 and subsequent expression of type I interferons (IFNs; IFN-α/β) downstream of pattern recognition receptors, such as Toll-like receptors 3 and 4 (TLR3, TLR4), cGAS/STING and RIG-I-like receptors (Fitzgerald KA et al. 2003; Fang R et al. 2017). While TBK1 or IKBKE showed limited effect on TNF-induced gene expression in human A549 and mouse embryonic fibroblasts (MEF) (Lafont E et al. 2018), both kinases prevented TNFα-induced cell death by suppressing RIPK1 activation in human and mouse cells (Lafont E et al. 2018; Xu D et al. 2018; Taft J et al. 2021). Mechanistically, TBK1 and/or IKBKE directly phosphorylate RIPK1 within the TNFR1 signaling complex to prevent RIPK1 kinase-dependent cell death signaling downstream of TNFR1 (Lafont E et al. 2018; Xu D et al. 2018). Mass spectrometry analysis suggests that human RIPK1 is phosphorylated by TBK1 at T189 (Xu D et al. 2018). Using a phospho-specific antibody, T189 of RIPK1 was detected as a phosphorylation site upon incubation with TBK1 in in vitro kinase assay and upon co-expression of tagged proteins in human embryonic kidney HEK293T cells. This phosphorylation was inhibited by TBK1/IKBKE inhibitor MRT67307 (Xu D et al. 2018). Phosphorylation of endogenous RIPK1 at T189 was detected in TNF-stimulated Jurkat cells (Xu D et al. 2018). Others reported that TBK1 or IKBKE phosphorylate RIPK1 at multiple residues (Lafont E et al. 2018). Further, TBK1 or IKBKE deficiency enhanced TNF-induced autophosphorylation of RIPK1, association of RIPK3 with caspase-8:FADD and levels of phosphorylated MLKL in human A549 and HeLa cells (Lafont E et al. 2018). Similar findings were reported for mouse cells (Lafont E et al. 2018; Xu D et al. 2018). In vivo studies showed that the embryonic lethality of Tbk1−/− mice was caused by TNFα-stimulated hyperactivation of RIPK1 kinase activity (Xu D et al. 2018). Moreover, chronic systemic autoinflammation in patients carrying homozygous loss-of-function mutations in TBK1 (W619*, R440* and Y212D) is associated with enhanced TNFα-induced RIPK1-dependent cell death (Taft J et al. 2021). In addition, TBK1-mediated phosphorylation of CYLD may also control the TNFR1 signaling pathway by regulating the ubiquitination status of RIPK1 (Xu X et al. 2020; Taft J et al. 2021). These data suggest that TBK1 and IKBKE downregulate RIPK1 auto-phosphorylation within the complex-I, thereby preventing TNFα- induced RIPK1-kinase-activity-dependent cell death.

This Reactome event describes TBK1/IKBKE-mediated phosphorylation of RIPK1 at T189 within the TNFR1 signaling complex.

Literature References

PubMed ID	Title	Journal	Year
30420664	TBK1 and IKKε prevent TNF-induced cell death by RIPK1 phosphorylation de Miguel, D, Draberova, H, Lafont, E, Reichert, M, Bhamra, A, Rieser, E, Walczak, H, Draber, P, Kupka, S, Linkermann, A, Henderson, S, Wojdyla, K, Chalk, A, Surinova, S, von Mässenhausen, A	Nat. Cell Biol.	2018
30146158	TBK1 Suppresses RIPK1-Driven Apoptosis and Inflammation during Development and in Aging Xu, D, Levin, JZ, Aron, L, Adiconis, X, Jin, T, Mifflin, L, Meng, H, Ofengeim, D, Zhu, H, Amin, P, Naito, MG, Shan, B, Li, W, Yankner, BA, Yuan, J, Chen, H, Pan, H, Li, Y, Pan, L, Zou, C	Cell	2018
34363755	Human TBK1 deficiency leads to autoinflammation driven by TNF-induced cell death Sabli, IKD, Wokke, BHA, Schnappauf, O, van Laar, JAM, Taft, J, Gruber, C, van Pelt, P, Legarda, D, Chavan, PP, Mancini, GMS, Chan, M, Sancho-Shimizu, V, Markson, M, Richardson, A, Aksentijevich, I, Khubchandani, R, Cüceoğlu, MK, Malle, L, Bogunovic, D, Patel, R, Buta, S, Özen, S, Kastner, DL, Ting, AT, Hollink, IHIM, Martín-Fernández, M	Cell	2021