TBK1, IKBKE phosphorylate RIPK1 at T189

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

TNFα与TNF受体1（TNFR1）的结合通过连续形成多个信号复合物，即复合物-I、IIa和IIb（Micheau O和Tschopp J 2003；Walczak H 2011），从而诱导细胞存活或细胞死亡。这些复合物的动态组装受到RIPK1（受体相互作用丝氨酸/苏氨酸蛋白激酶1）及其它TNFα信号通路成分的蛋白水解、泛素化、去泛素化和磷酸化的严格调控。快速形成的复合物-I（TNFR1信号复合物）在受体的胞浆尾部组装，由TNFR1、TNFR1相关死亡结构域（TRADD）、TNF受体相关因子-2（TRAF2）和RIPK1组成（Micheau O和Tschopp J 2003）。活化的TNFR1信号复合物（复合物-I）招募多种E3泛素（Ub）连接酶，如细胞凋亡抑制因子cIAP1/2（BIRC2、BIRC3）、mind bomb 2（MIB2）或线性泛素链组装复合物（LUBAC）（Micheau O和Tschopp J 2003；Feltham R等. 2018；Yuan J等. 2019）。BIRC2、BIRC3和MIB2介导的K63链接泛素化和LUBAC介导的RIPK1及其它复合物成分的Met1链接线性泛素化稳定了膜结合的促存活TNFR1信号复合物，同时抑制了细胞质中死亡诱导复合物IIa（TRADD:TRAF2:RIPK1:FADD:CASP8）和IIb（RIPK1:RIPK3:MLKL）的形成。LUBAC的催化活性还使TRAF关联的NF-κB激活剂（TANK）结合激酶1（TBK1）和抑制κ-B激酶（IKK）ε（IKKε或IKBKE）招募到TNFR1信号复合物中（Lafont E等. 2018；Xu D等. 2018）。LUBAC由HOIP、HOIL-1L和SHARPIN亚基组成。HOIP缺乏显著降低了TBK1或IKBKE在人类宫颈癌细胞系HeLa细胞和肺癌细胞系A549细胞中复合物-I的招募（Lafont E等. 2018）。蛋白质印迹分析显示，TBK1和IKBKE被招募到并强烈磷酸化在多种TNFα刺激的人类细胞系（包括A549、角质形成细胞HaCaT、单核细胞U937和THP-1细胞）的TNFR1复合物中（Lafont E等. 2018）。类似地，TBK1被发现以LUBAC依赖的方式与小鼠细胞复合物-I的成分相关联（Xu D等. 2018）。在TBK1缺乏（由W619*功能丧失突变引起）的人类端粒酶逆转录酶（TERT）永生化真皮成纤维细胞中，检测到TNFR1复合物中的IKBKE，而不是TBK1（Taft J等. 2021）。TBK1/IKBKE的酶活性由位于TBK1和IKKε激酶结构域T环中的S172位点的磷酸化启动（Shimada T等. 1999；Kishore N等. 2002；Gu L等. 2013）。已知的激活TBK1和IKBKE可触发干扰素调节因子3（IRF3）和IRF7的磷酸化以及下游模式识别受体（如Toll样受体3和4（TLR3、TLR4）、cGAS/STING和RIG-I样受体）的I型干扰素（IFNs；IFN-α/β）的表达（Fitzgerald KA等. 2003；Fang R等. 2017）。尽管TBK1或IKBKE在人类A549和鼠胚胎成纤维细胞（MEF）中TNF诱导的基因表达中表现出有限的作用（Lafont E等. 2018），但两种激酶通过抑制人类和鼠细胞中的RIPK1激活来防止TNFα诱导的细胞死亡（Lafont E等. 2018；Xu D等. 2018；Taft J等. 2021）。机制上，TBK1和/或IKBKE直接在TNFR1信号复合物中对RIPK1进行磷酸化，以防止TNFR1下游的RIPK1激酶依赖性细胞死亡信号（Lafont E等. 2018；Xu D等. 2018）。质谱分析表明，人类RIPK1在T189位点被TBK1磷酸化（Xu D等. 2018）。使用磷酸化特异性抗体，在体外激酶分析和在标记蛋白共表达的人类胚胎肾HEK293T细胞中，检测到RIPK1的T189位点的磷酸化，并被TBK1/IKBKE抑制剂MRT67307抑制（Xu D等. 2018）。在TNF刺激的Jurkat细胞中检测到内源性RIPK1在T189位点的磷酸化（Xu D等. 2018）。其他研究者报告称TBK1或IKBKE在多个位点磷酸化RIPK1（Lafont E等. 2018）。此外，TBK1或IKBKE缺乏增强了TNF诱导的RIPK1自磷酸化、RIPK3与caspase-8:FADD的结合以及人类A549和HeLa细胞中磷酸化MLKL的水平（Lafont E等. 2018）。类似的结果在鼠细胞中也得到报道（Lafont E等. 2018；Xu D等. 2018）。体内研究表明，Tbk1−/−小鼠的胚胎致死性是由TNFα刺激的RIPK1激酶活性的过度激活引起的（Xu D等. 2018）。此外，携带TBK1（W619*、R440*和Y212D）同源功能丧失突变的患者的慢性系统性自身炎症与TNFα诱导的RIPK1依赖性细胞死亡的增强有关（Taft J等. 2021）。此外，TBK1介导的CYLD磷酸化也可能通过调节RIPK1的泛素化状态来控制TNFR1信号通路（Xu X等. 2020；Taft J等. 2021）。这些数据表明，TBK1和IKBKE通过降低复合物-I中RIPK1的自磷酸化来下调RIPK1，从而防止TNFα诱导的RIPK1激酶活性依赖性细胞死亡。此Reactome事件描述了TBK1/IKBKE介导的RIPK1在TNFR1信号复合物中的T189位点的磷酸化。