TBK1 directly engages Akt/PKB survival signaling to support oncogenic transformation

The innate immune-signaling kinase, TBK1, couples pathogen surveillance to induction of host defense mechanisms. Pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here, we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low-nanomolar TBK1 inhibitor, indicates that this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling.     

环状RNA调控TBK1表达影响肿瘤发生发展

TANK结合酶1(TANK-bindingkinase 1,TBK1)是一种丝氨酸/苏氨酸激酶,在机体先天免疫调节、细胞选择性自噬、细胞凋亡、细胞再生中发挥重要作用,与肿瘤的发生密切相关.环状RNA(circle RNA,circRNA)是新型的闭合环状非编码RNA,其生物功能及与肿瘤的联系受到国内外学者广泛关注.近年...     

TXLNA enhances TBK1 phosphorylation by suppressing PPM1B recruitment

In recent years, there has been a notable increase in cancer incidence and mortality, and immune abnormalities have been closely linked to malignancy development. TANK-binding kinase 1 (TBK1) is a non-classical IκB kinase that regulates interferon and NF-κB signaling pathways and plays a crucial role in innate immunity. Recent studies have shown high expression levels of TBK1 and increased activity in various tumor cells, suggesting its involvement in the development and progression of multiple cancers. Targeting TBK1 for tumor therapy may be a possibility. However, little is known about the abnormal activation and dynamic regulation of TBK1 in cancer. First, we utilized the BioID biotinylation technique combined with TMT-based quantitative proteomics to analyze the TBK1 interacting proteins. Our results revealed that TXLNA interacts with TBK1 and binds to the α-helical scaffold of TBK1. The expression of TXLNA could affect the S172 phosphorylation of TBK1. PPM1B is a phosphatase that can dephosphorylate TBK1 S172, so we used the APEX2 proximity labeling technique combined with TMT-based quantitative proteomics to explore the interacting proteins of PPM1B and search for the regulatory pathway of TXLNA on TBK1 phosphorylation. We found that PPM1B interacts with TXLNA. Based on these results, we further found that TXLNA impairs the binding of PPM1B to TBK1, inhibiting the dephosphorylation of TBK1 and contributing to the abnormal enhancement of TBK1 activity in cancer cells. This study sheds light on the potential mechanism of aberrant activation and dynamic regulation of TBK1 in tumors and provides a potential target for tumor therapy.     

RKIP and TBK1 form a positive feedback loop to promote type I interferon production in innate immunity

TANK‐binding kinase 1 (TBK1) activation is a central event in type I interferon production in anti‐virus innate immunity. However, the regulatory mechanism underlying TBK1 activation remains unclear. Here we report that Raf kinase inhibitory protein (RKIP) is essential for TBK1 activation and type I interferon production triggered by viral infection. Upon viral infection, RKIP is phosphorylated at serine 109 (S109) by TBK1. Phosphorylation of RKIP enhances its interaction with TBK1 and in turn promotes TBK1 autophosphorylation. Mutation of RKIP S109 to alanine abrogates the interaction between RKIP and TBK1, and the anti‐viral function of RKIP. RKIP deficiency inhibits intracellular double‐stranded RNA‐ or DNA‐induced type I interferon production. Consistently, RKIP deficiency renders the mice more susceptible to vesicular stomatitis virus (VSV) and herpes simplex virus (HSV) infections. This study reveals a previously unrecognized positive feedback loop between RKIP and TBK1 that is essential for type I interferon production in anti‐viral innate immunity.     

Histone deacetylase 3 promotes innate antiviral immunity through deacetylation of TBK1

TANK-binding kinase 1 (TBK1),a core kinase of antiviral pathways,activates the production of interferons (IFNs).It has been reported that deacetylation activates TBK1;however,the precise mechanism still remains to be uncovered.We show here that during the early stage of viral infection,the acetylatlon of TBK1 was increased,and the acetylation of TBK1 at Lys241 enhanced the recruitment of IRF3 to TBK1.HDAC3 directly deacety-lated TBK1 at Lys241 and Lys692,which resulted in the activation of TBK1.Deacetylation at Lys241 and Lys692 was critical for the kinase activity and dimerizatlon of TBK1 respectively.Using knockout cell lines and transgenic mice,we confirmed that a HDAC3 null mutant exhibited enhanced susceptibility to viral challenge via impaired productlon of type I IFNs.Furthermore,activated TBK1 phosphorylated HDAC3,which promoted the deacetylation activity of HDAC3 and formed a feedback loop.In this study,we illustrated the roles the acetylated and deacetylated forms of TBK1 play in antivlral innate responses and clarified the post-translational modulations involved in the interaction between TBK1 and HDAC3.     

The IKK‐related kinase TBK1 activates mTORC1 directly in response to growth factors and innate immune agonists

The innate immune kinase TBK1 initiates inflammatory responses to combat infectious pathogens by driving production of type I interferons. TBK1 also controls metabolic processes and promotes oncogene‐induced cell proliferation and survival. Here, we demonstrate that TBK1 activates mTOR complex 1 (mTORC1) directly. In cultured cells, TBK1 associates with and activates mTORC1 through site‐specific mTOR phosphorylation (on S2159) in response to certain growth factor receptors (i.e., EGF‐receptor but not insulin receptor) and pathogen recognition receptors (PRRs) (i.e., TLR3; TLR4), revealing a stimulus‐selective role for TBK1 in mTORC1 regulation. By studying cultured macrophages and those isolated from genome edited mTOR S2159A knock‐in mice, we show that mTOR S2159 phosphorylation promotes mTORC1 signaling, IRF3 nuclear translocation, and IFN‐β production. These data demonstrate a direct mechanistic link between TBK1 and mTORC1 function as well as physiologic significance of the TBK1‐mTORC1 axis in control of innate immune function. These data unveil TBK1 as a direct mTORC1 activator and suggest unanticipated roles for mTORC1 downstream of TBK1 in control of innate immunity, tumorigenesis, and disorders linked to chronic inflammation.     

RalB GTPase-mediated activation of the IkappaB family kinase TBK1 couples innate immune signaling to tumor cell survival

The monomeric RalGTPases, RalA and RalB are recognized as components of a regulatory framework supporting tumorigenic transformation. Specifically, RalB is required to suppress apoptotic checkpoint activation, the mechanistic basis of which is unknown. Reported effector proteins of RalB include the Sec5 component of the exocyst, an octameric protein complex implicated in tethering of vesicles to membranes. Surprisingly, we find that the RalB/Sec5 effector complex directly recruits and activates the atypical IkappaB kinase family member TBK1. In cancer cells, constitutive engagement of this pathway, via chronic RalB activation, restricts initiation of apoptotic programs typically engaged in the context of oncogenic stress. Although dispensable for survival in a nontumorigenic context, this pathway helps mount an innate immune response to virus exposure. These observations define the mechanistic contribution of RalGTPases to cancer cell survival and reveal the RalB/Sec5 effector complex as a component of TBK1-dependent innate immune signaling.     

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

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 (Mtor^A/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.     

TBK1 has a new Akt

TANK-binding kinase 1 (TBK1) is a noncanonical IκB kinase that plays an essential role in the innate immune response to foreign pathogens. Recent studies have highlighted additional roles for TBK1 in the regulation of metabolism, although the mechanisms of this regulation have not been well characterized. In a recent issue, Tooley et al. demonstrated that TBK1-dependent activation of downstream kinase Akt is mediated via mechanistic target of rapamycin complex 2. This novel action of TBK1 reveals a key role for this kinase in the regulation of cellular metabolism and growth by diverse environmental inputs.

TANK-binding kinase 1 (TBK1), a serine/threonine kinase that belongs to the noncanonical IκB kinase family, plays an essential role in the innate immune response to viral and bacterial pathogens by regulating the type I interferon–mediated T cell response (1). Although TBK1 has been most widely studied in this context, more recent investigations using tissue-specific KO mice and drugs that inhibit kinase activity have revealed novel roles for this kinase in nonimmune cells, particularly at the intersection of immunity and metabolism. For example, TBK1 expression and activity are induced in adipose tissue in obesity by elevated expression of proinflammatory cytokines such as tumor necrosis factor α (2). TBK1 contributes to obesity by repressing energy expenditure and increasing anabolic functions as determined from analysis of mice with conditional adipose cell KO of TBK1 (3). TBK1 has also been reported to promote activation of Akt, a central kinase involved in metabolic regulation (4). However, the mechanism by which TBK1 regulates Akt has remained unclear.Akt is an essential regulator of glucose metabolism and plays an important role in controlling cellular glucose uptake and utilization through both positive and negative regulatory actions (4). Phosphorylation of Akt on T308 in its activation loop stimulates kinase activity, and phosphorylation on S473 further enhances activity and determines substrate specificity (4). 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. (6) contributed to the mechanistic understanding of TBK1 function in metabolic regulation by demonstrating a role for TBK1 in mechanistic target of rapamycin (mTOR) complex 2 (mTORC2) activation and subsequent phosphorylation of Akt.To investigate how TBK1 regulates Akt activation, mouse embryonic fibroblasts (MEFs) were stimulated with epidermal growth factor (EGF) and evaluated for Akt-S473 and Akt-T308 phosphorylation (6). The intensity and duration of Akt phosphorylation at both sites was diminished significantly, both in the absence of TBK1 and in the presence of the TBK1 inhibitor amlexanox. Restoration of endogenous levels of TBK1, but not kinase-dead TBK1, rescued EGF-stimulated Akt-S473 phosphorylation. The stimulation of Akt-S473 phosphorylation by EGF, as well as by other growth factors and the hormone insulin, was found to be dependent upon mTOR activity. Together, these results validate the ability of TBK1 to regulate Akt-S473 phosphorylation and show that in response to normal growth regulatory signaling, this regulation is mediated through mTOR kinase.The kinase mTOR is the core catalytic kinase of two multisubunit complexes, mTOR complex 1 (mTORC1) and mTORC2, which are distinguished by the scaffolding proteins Raptor and Rictor, respectively (7). mTORC1 is regulated by the combination of growth factor/hormone signaling and nutrient availability to drive anabolic metabolism. mTORC2, on the other hand, is regulated by growth factor/hormone signaling to activate Akt. Together, mTORC1 and mTORC2 are key signaling nodes in the regulation of cell growth and proliferation, and dysregulation of these signaling pathways contributes to metabolic disease and cancer. In previous investigations, the authors had demonstrated that phosphorylation of mTOR on S2159 by TBK1 enhanced mTORC1 activation and downstream signaling to promote cell growth and proliferation (8). To investigate if TBK1 acts upstream of mTORC2 to regulate Akt-S473 phosphorylation through a similar mechanism, MEFs derived from mice with an alanine knock-in at S2159 (Mtor^A/A) were stimulated with EGF. A marked reduction of Akt-S473 phosphorylation was observed in Mtor^A/A MEFs compared with WT MEFs (Mtor^+/+). Using immunoprecipitation of Rictor to isolate the mTORC2 complex, TBK1 was observed to interact with mTORC2 and directly phosphorylate mTOR-S2159 to activate mTORC2 intrinsic kinase activity toward Akt-S473. TBK1 activity is increased by phosphorylation of S172 in its activation loop in response to pathogens in the innate immunity pathway. In contrast, Tooley et al. (6) 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. However, when RAW264.7 macrophages and primary bone marrow–derived macrophages were stimulated with the dsRNA 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. Finally, the physiological regulation of mTORC2 activity by TBK1 was assessed by injection of Mtor^A/A and Mtor^+/+ mice with poly(I:C). 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 (Fig. 1) (6).Open in a separate windowFigure 1TBK1 promotes AKT activation through mTORC2. TBK1 interacts with and phosphorylates mTORC2 on S2159 of mTOR in response to either growth factor stimulation or innate immune agonists to promote AKT activation. Created using BioRender.com. mTORC2, mTOR complex 2; SGK, serum/glucocorticoid-regulated kinase; TBK1, TANK-binding kinase 1.TBK1 regulation of mTORC2-dependent phosphorylation of Akt shown in this study adds to the growing role of TBK1 as a signaling node in the regulation of cellular metabolism and growth by diverse environmental inputs. In response to foreign pathogens or inflammatory cytokines that stimulate TBK1 activation, or growth factor/hormone signaling that requires basal TBK1 activity, mTORC2 is activated to promote Akt-S473 phosphorylation and its downstream functions. Given that TBK1 expression and activity are enhanced in metabolic diseases and cancer, and the important role that Akt plays in these pathological conditions, identifying TBK1 as an upstream regulator of Akt reveals a potential novel approach to disrupt this signaling axis for therapeutic benefit (4, 9). 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. (6) only examined the TBK1-dependent phosphorylation of Akt-S473 by mTORC2; mTORC2 also has additional substrates, including serum/glucocorticoid-regulated kinase and members of the PKC family (Fig. 1) (4). These kinases regulate unique cellular functions, such as regulation of the actin cytoskeleton. It will be important to determine if TBK1 regulates the activation of these kinases through mTORC2 as well, to understand the full impact of inhibiting TBK1 function therapeutically.The mechanism by which TBK1 regulates mTORC2 function has not been established. Although the kinase activity of TBK1 is required for Akt-S473 phosphorylation, neither phosphorylation of S172 in the activation loop of TBK1 nor phosphorylation of mTOR-S2159 was increased by growth factor stimulation in this study. Phosphorylation of S172 stabilizes the active confirmation of TBK1 and it is possible that additional uncharacterized phosphorylation sites could serve a similar function. Alternatively, the interaction of TBK1 with mTORC2 could impact TBK1 conformation, or multimerization, to enhance activity. Intracellular localization of mTORC2 could also be determined by TBK1 interaction, which could affect substrate availability. As little is known about the upstream regulation of mTORC2, the next acts should be elucidating further the mechanism of its activation by TBK1 to reveal novel approaches for targeting the mTORC2-Akt signaling pathway.     

Identification of nucleoporin 93 (Nup93) that mediates antiviral innate immune responses

RIG-I-like receptors (RLRs) are cytoplasmic sensors for viral RNA that elicit antiviral innate immune responses. RLR signaling culminates in the activation of the protein kinase TBK1, which mediates phosphorylation and nuclear translocation of IRF3 that regulates expression of type I interferon genes. Here, we found that Nucleoporin 93 (Nup93), components of nuclear pore complex (NPC), plays an important role in RLR-mediated antiviral responses. Nup93-deficient RAW264.7 macrophage cells exhibited decreased expression of Ifnb1 and Cxcl10 genes after treatment with a synthetic RLR agonist stimulation as well as Newcastle Disease Virus infection. Silencing Nup93 in murine primary macrophages and embryonic fibroblasts also resulted in reduced expression of these genes. IRF3 nuclear translocation during RLR signaling was impaired in Nup93-deficient RAW264.7 cells. Notably, the activation of TBK1 during RLR signaling was also decreased in Nup93-deficient cells. We found that Nup93 formed a complex with TBK1, and Nup93 overexpression enhanced TBK1-mediated IFNβ promoter activation. Taken together, our findings suggest that Nup93 regulates antiviral innate immunity by enhancing TBK1 activity and IRF3 nuclear translocation.     

抗病毒天然免疫通路中IRF-3调控新机制

干扰素调节因子-3(interferon regulatory factor-3,IRF-3)是IRF家族中重要 转录因子之一,在调控干扰素(interferon, IFN)基因表达和抗病毒天然免疫反应中具有重要作 用. 最新发现的MITA (mediator of IRF-3 activation, 又称STING/ERIS)蛋白是宿主抗病 毒天然免疫反应中的一种重要调节分子. 病毒侵染时,MITA与IRF-3相互作用,特异性激活 IRF-3,并募集TANK结合激酶1（TANK binding kinase 1, TBK1）与IFN通路中的线粒体抗 病毒信号蛋白MAVS(mitochondrial anti-viral signaling protein)形成复合物,且MITA可 被TBK1磷酸化,诱导Ⅰ型IFN及IFN刺激基因(interferon stimulate genes, ISG)的表达 ,诱发抗病毒天然免疫反应. 同时还发现,泛素连接酶RNF5（ring finger protein 5）可对MITA 发生泛素化修饰从而抑制其对IRF-3活化,实现对宿主抗病毒天然免疫反应负调节作用. 本 室研究发现,严重性急性呼吸系统综合症冠状病毒(severe acute respiratory syndrome co ronavirus, SARS-CoV）和人类新型冠状病毒（human coronavirus NL63, HCoV-NL63）的 木瓜样蛋白酶（papain-like protease, PLP）利用其特有的去泛素化酶（deubiquitinase, DUB）活性,通过宿主细胞泛素-蛋白酶体信号系统对IRF-3的泛素化等翻译后修饰进行调节 ,从而成为该种病毒逃逸机体抗病毒防御系统主要手段之一.     

Hepatitis B Virus Polymerase Blocks Pattern Recognition Receptor Signaling via Interaction with DDX3: Implications for Immune Evasion

Viral infection leads to induction of pattern-recognition receptor signaling, which leads to interferon regulatory factor (IRF) activation and ultimately interferon (IFN) production. To establish infection, many viruses have strategies to evade the innate immunity. For the hepatitis B virus (HBV), which causes chronic infection in the liver, the evasion strategy remains uncertain. We now show that HBV polymerase (Pol) blocks IRF signaling, indicating that HBV Pol is the viral molecule that effectively counteracts host innate immune response. In particular, HBV Pol inhibits TANK-binding kinase 1 (TBK1)/IκB kinase-ε (IKKε), the effector kinases of IRF signaling. Intriguingly, HBV Pol inhibits TBK1/IKKε activity by disrupting the interaction between IKKε and DDX3 DEAD box RNA helicase, which was recently shown to augment TBK1/IKKε activity. This unexpected role of HBV Pol may explain how HBV evades innate immune response in the early phase of the infection. A therapeutic implication of this work is that a strategy to interfere with the HBV Pol-DDX3 interaction might lead to the resolution of life-long persistent infection.     

RalB signaling: a bridge between inflammation and cancer

A connection between the genetic events that lead to tumor formation and the signaling pathways of the innate immune response has been established. In this issue, Chien et al. (2006) show that the RalB GTPase regulates the IKK family member TBK1, providing an unexpected link between the signaling pathways that promote inflammation and cancer. In tumor cells the RalB/TBK1 pathway inhibits apoptosis and in nontumorigenic cells it stimulates an innate immune response.     

Select paramyxoviral V proteins inhibit IRF3 activation by acting as alternative substrates for inhibitor of kappaB kinase epsilon (IKKe)/TBK1

V accessory proteins from Paramyxoviruses are important in viral evasion of the innate immune response. Here, using a cell survival assay that identifies both inhibitors and activators of interferon regulatory factor 3 (IRF3)-mediated gene induction, we identified select paramyxoviral V proteins that inhibited double-stranded RNA-mediated signaling; these are encoded by mumps virus (MuV), human parainfluenza virus 2 (hPIV2), and parainfluenza virus 5 (PIV5), all members of the genus Rubulavirus. We showed that interaction between V and the IRF3/7 kinases, TRAF family member-associated NFkappaB activator (TANK)-binding kinase 1 (TBK1)/inhibitor of kappaB kinase epsilon (IKKe), was essential for this inhibition. Indeed, V proteins were phosphorylated directly by TBK1/IKKe, and this, intriguingly, resulted in lowering of the cellular level of V. Thus, it appears that V mimics IRF3 in both its phosphorylation by TBK1/IKKe and its subsequent degradation. Finally, a PIV5 mutant encoding a V protein that could not inhibit IKKe was much more susceptible to the antiviral effects of double-stranded RNA than the wild-type virus. Because many innate immune response signaling pathways, including those initiated by TLR3, TLR4, RIG-I, MDA5, and DNA-dependent activator of IRFs (DAI), use TBK1/IKKe as the terminal kinases to activate IRFs, rubulaviral V proteins have the potential to inhibit all of them.     

Regulation of TBK1 activity by Optineurin contributes to cell cycle-dependent expression of the interferon pathway

The innate immune system has evolved to detect and neutralize viral invasions. Triggering of this defense mechanism relies on the production and secretion of soluble factors that stimulate intracellular antiviral defense mechanisms. The Tank Binding Kinase 1 (TBK1) is a serine/threonine kinase in the innate immune signaling pathways including the antiviral response and the host defense against cytosolic infection by bacteries. Given the critical roles of TBK1, important regulatory mechanisms are required to regulate its activity. Among these, Optineurin (Optn) was shown to negatively regulate the interferon response, in addition to its important role in membrane trafficking, protein secretion, autophagy and cell division. As Optn does not carry any enzymatic activity, its functions depend on its precise subcellular localization and its interaction with other proteins, especially with components of the innate immune pathway. This review highlights advances in our understanding of Optn mechanisms of action with focus on the relationships between Optn and TBK1 and their implication in host defense against pathogens. Specifically, how the antiviral immune system is controlled during the cell cycle by the Optn/TBK1 axis and the physiological consequences of this regulatory mechanism are described. This review may serve to a better understanding of the relationships between the different functions of Optn, including those related to immune responses and its associated pathologies such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget’s disease of bone.