TAK1 is a serine/threonine protein kinase featuring an N-terminal domain, a hinge region, and a C-terminal domain [17]. The N-terminal domain of TAK1 contains a conserved kinase domain, which is responsible for its enzymatic activity, phosphorylating specific serine and/or threonine residues on substrate proteins. ATP binds and transfers a phosphate group to the substrate in this kinase domain. The serine-rich and glycine-rich regions within this domain may regulate its kinase activity and play roles in protein–protein interactions (Fig. 1) [12, 16].

Fig. 1.

Schematic illustration of the domain structures of human transforming growth factor-$\beta$ (TGF-$\beta$) activated kinase 1 (TAK1), TAK1-binding proteins (TAB)1, TAB2, and TAB3. TAK1 contains a kinase domain at the N-terminus, followed by a TAB1 binding domain (TAB1 BD) and a TAB2/3 binding domain (TAB2/3 BD). TAB1 is characterized by a pseudo-phosphatase domain and a TAK1 binding domain (TAK1 BD). TAB2 and TAB3 are homologous proteins, and both have a CUE domain at the N-terminus, a TAK1 BD, and an Npl4 zinc-finger (NZF) domain at the C-terminus. CUE, Coupling of Ubiquitin to ER degradation.

Research on TAK1 in mice has cast uncertainty on its viability as a therapeutic target. While no TAK1 loss-of-function mutations have been identified in humans, a gain-of-function mutation resulting from a truncated kinase form leads to developmental phenotypes, including craniofacial abnormalities [18]. This highlights the complex role of TAK1 in embryogenesis and development [19]. The discovery underscores the challenges in targeting kinases therapeutically, given the potential for genetic contradictions and the complexity of kinase-mediated signaling pathways, which may involve redundant or compensatory mechanisms [20]. Moreover, the scaffolding roles played by TAK1 further complicate the interpretation of its genetic studies, emphasizing that a nuanced approach is needed in pharmacologically manipulating TAK1 for therapeutic benefits.

The biological actions of TAK1 span various signaling pathways, and its activity is regulated by diverse stimuli, including growth factors, cell adhesion, and inflammatory factors [21]. Upon activation, TAK1 regulates downstream proteins, including adaptor proteins in the NF-$\kappa$B and MAPK pathways, thereby influencing cell survival, proliferation, differentiation, and death [21]. Studies reveal that embryos lacking TAK1 exhibit developmental abnormalities and embryonic death, while conditional TAK1 knockout mice manifest a range of physiological defects associated with impaired TAK1 signaling, including reduced inflammation, abnormal cell differentiation, and compromised survival [11, 12, 22].

Genome-wide association studies (GWAS) have placed TAK1 (MAP3K7) in the spotlight, underscoring its critical role in health and disease, thus marking it as a potential therapeutic target [23, 24, 25, 26, 27, 28]. Such studies have connected TAK1 with various diseases, including Crohn’s disease, ulcerative colitis, and inflammatory bowel disorder. In particular, GWAS on Japanese rheumatoid arthritis (RA) patients highlighted a significant association of TAK1 with susceptibility to diseases and response to anti-TNF treatments, suggesting its pivotal role in inflammatory disease management [25, 29]. The association of TAK1 with diverse conditions, from immune responses to genetic traits affecting biological functions, demonstrates its broad biological impact, emphasizing the need for further research into its mechanisms and therapeutic applications.

Given these diverse roles, TAK1 is considered a key molecular node in signaling networks that determine cell fate and function. Understanding the regulation of TAK1 and its pathways offers potential therapeutic targets for various diseases where its signaling is aberrant.

TAK1 binding proteins (TABs) serve as specific binding partners for TAK1 and include TAB1, TAB2, and TAB3. The activation of TAK1 relies on the assembly of these proteins, forming a complex that crucially regulates the kinase activity of TAK1 [5].

TAB1: Characterized by a pseudo-phosphatase domain and a TAK1 binding domain (TAK1 BD) (Fig. 1). The pseudo-phosphatase domain in TAB1 has a similar structure to protein phosphatase 2C (PP2C), despite lacking phosphatase activity. It interacts directly with the N-terminal kinase domain of TAK1 [30]. Disrupting this interaction can result in late-stage pregnancy edema and hemorrhage. In vitro experiments have found that TAB1 is indispensable for TAK1 activation through autophosphorylation [5, 31].

TAB2 and TAB3: Homologous proteins that share 48% of their amino acid sequences, indicating a significant evolutionary relationship. They feature an N-terminal coupling of ubiquitin to ER degradation (CUE) domain, a C-terminal TAK1 binding domain, and a Npl4 zinc-finger (NZF) domain [14, 16] (Fig. 1). The TAK1 binding domain in TAB2 and TAB3 is primarily responsible for binding with TAK1, while the CUE domain binds directly to ubiquitin, linking ubiquitinated proteins to endoplasmic reticulum degradation [32]. The NZF domain is responsible for binding polyubiquitin chains, specifically recognizing the polyubiquitin chains linked by lysine (K)63, crucial for TAK1 activation [33]. Although structurally similar, TAB2 and TAB3 can complement each other in certain functions. TAB2 gene knockout results in embryonic death in mice, while TAB3 gene knockout mice can be born normally, suggesting specific roles in regulating TAK1 activity [14, 16].

Through their binding domains, TAK1 and TAB1 are associated in unstimulated cells [31]. Upon stimulation, TAB2 and TAB3 bind to the C-terminal region of TAK1 through their respective binding domains, forming the TAK1–TAB1–TAB2/TAB3 complex. The functional assembly of TAK1 with TAB proteins in response to cellular stimuli underscores the complex’s versatility in regulating various biological processes. This interaction activates critical signaling pathways and reflects the complex’s adaptability to diverse cellular contexts, from immune responses to stress responses [12]. The detailed study of the TAK–TAB complex reveals its fundamental role in cellular signaling and disease mechanisms, presenting opportunities for therapeutic intervention.

TNF serves as a central cytokine in inflammatory responses and a common ligand in the NF-$\kappa$B and MAPK pathways [35, 36]. Upon TNF binding to TNF receptor 1 (TNF-R1), a trimeric complex form, which recruits the TNF receptor-associated death domain (TRADD) [37] through the death domain (DD). Subsequently, TRADD recruits TNF receptor-associated factors (TRAF) 2/5 and cellular inhibitors of apoptosis proteins 1/2 (c-IAP1/2), forming the TNF–R1 signaling complex. This complex recruits RIP1, leading to K63-linked polyubiquitination of RIP1. The polyubiquitin chains on RIP1 bind to the C-terminal NZF domain of TAB2/TAB3, promoting the formation of the TAK1/TAB1/TAB2/TAB3 complex and triggering conformational changes for TAK1’s autophosphorylation-dependent activation [38]. This activation further activates TAK1, initiating the IKK complex and ultimately activating NF-$\kappa$B. TRAF2-mediated K63-linked polyubiquitination plays a critical role in TNFR1-induced TAK1 activation.

Recently, GWAS studies have underscored the pivotal role of TNF–TAK1 complex signaling in the pathogenesis of diverse conditions, illuminating the critical function of TAK1 as a therapeutic target. For example, GWAS findings in Asian populations with rheumatoid arthritis (RA) underscore the involvement of MAP3K7 (TAK1) and its activator, TAB1, suggesting a nuanced genetic predisposition [23]. Furthermore, single-nucleotide polymorphisms (SNPs) within the TNF–TAK1 axis have been linked to a heightened risk of inflammatory diseases, including Crohn’s disease and ulcerative colitis, implicating genes such as MAP3K7 and TNFSF18, among others [24, 26, 27]. Despite the advancement of biological therapies targeting TNF, the emergence of resistance highlights the necessity of alternative strategies [39, 40]. The advent of selective TAK1 inhibitors represents a promising avenue, with preclinical studies validating the efficacy of blocking TAK1 in RA [41]. This development not only broadens our therapeutic arsenal but also emphasizes that it is imperative for further research to elucidate the intricate role of TAK1 within inflammatory pathways.

IL-1, also known as lymphocyte-activating factor, is mainly produced by monocytes/macrophages and exists in two forms: IL-1$\alpha$ and IL-1$\beta$. IL-1R has two types, with IL-1RI being the most abundant. IL-1RI binds to both IL-1$\alpha$ and IL-1$\beta$. In the IL-1$\beta$ signaling pathway, IL-1$\beta$ induces conformational changes in the extracellular domain of IL-1RI, recruiting the IL-1 receptor accessory protein (IL-1RAcP) to form the IL-1$\beta$/IL-1RI/IL-1RAcP trimeric complex. This complex recruits MyD88, initiating a signaling cascade involving IRAK4 and IRAK1 (IL-1 receptor-associated kinases) [42, 43, 44]. Phosphorylation of IRAK1 by IRAK4 releases IRAK1 into the cytoplasm, forming a signaling complex with E3 ubiquitin ligase TRAF6, which undergoes oligomerization, leading to the synthesis of K63-linked polyubiquitin chains by E2 enzymes Ubc13 and Uevi1A [45]. These polyubiquitin chains, binding to the highly conserved zinc-finger-NZF domains of TAB2 and TAB3, cause low oligomerization and autophosphorylation of TAK1 at Thr184 and Thr187 [46, 47]. Subsequently, TAK1 phosphorylates IKKs and MAPKs, activating NF-$\kappa$B and AP-1 downstream pathways, contributing to natural immunity and inflammation regulation [48].

Members of the TLR family recognize antigen components of different types of microorganisms and are well-studied pattern recognition receptors (PRRs). Both IL-1R and TLR receptors share Toll/interleukin-1 receptor domains in their intracellular signaling-related structures and, when activated, can bind MyD88 through these Toll/interleukin-1 receptor domains [42, 43, 49]. MyD88 [44], containing a death domain, then recruits IRAK1/4, TRAF6 and initiates downstream NF-$\kappa$B pathway activations [48, 50].

The TGF-$\beta$ signaling pathway is one of the most crucial signaling pathways, involved in regulating cell growth, proliferation, differentiation and aging, cell death, cell adhesion, migration, extracellular matrix synthesis, and remodeling [22, 51]. The TGF-$\beta$ family signals through the I-type receptor (T$\beta$RI) and II-type receptor (T$\beta$RII) complex integrity, which is essential for TGF-$\beta$ signal transduction. TGF-$\beta$ family ligands form a dimer and bind to T$\beta$RII and T$\beta$RI on the membrane, inducing the phosphorylation of T$\beta$RII by T$\beta$RI and activating its kinase activity. Subsequently, T$\beta$RI recruits and activates downstream signaling molecules, initiating the canonical Smad and non-Smad signaling pathways [52, 53, 54, 55].

Previous research has established TAK1 as a key upstream signaling molecule in the non-Smad signaling pathway induced by TGF-$\beta$1. However, the impact of TAK1 on activating the classic Smad signaling pathway remains a subject of debate [56]. Our previous studies have revealed that TAK1 can simultaneously affect the activation of both Smad and non-Smad signaling pathways induced by TGF-$\beta$1, thereby influencing the pathological changes associated with hypertensive nephropathy [57].

In the context of disease, the dysregulation of TAK1/TAB ubiquitination has been implicated in the pathogenesis of autoimmune conditions, tumor growth, and neurodegenerative diseases [8, 21, 61]. The excessive or prolonged activation of NF-$\kappa$B signaling due to ubiquitination imbalance can exacerbate these conditions [62, 63]. Therefore, understanding and manipulating the ubiquitination process of the TAK1/TAB complex creates new therapeutic avenues. The potential to modulate this pathway offers promising strategies for treating related diseases and conditions. Moreover, alterations in the ubiquitination pattern of these proteins might serve as valuable biomarkers for the early detection, diagnosis, and prognosis of diseases [16]. This aspect of ubiquitination is critical for developing a holistic understanding of the role of the TAK1–TAB complex in health and disease [8, 45, 60].

Ubiquitination, a pivotal post-translational modification, intricately regulates cellular processes critical for maintaining homeostasis [8, 64], including the cell cycle, proliferation, and apoptosis [65]. The ubiquitin–proteasome system (UPS) is crucial for protein degradation, similar to the regulatory roles of kinases [66, 67]. Disruptions in ubiquitination pathways can lead to an imbalance in these processes, significantly contributing to many disorders, including cancer, neurodegenerative diseases, and issues related to adaptive and innate immunity [68, 69]. Recent studies have underscored the dual role of ubiquitination in either promoting or inhibiting tumor progression, depending on the context and the specific proteins being targeted, while the UPS was also demonstrated as a potent target for anticancer therapy [67, 68, 70]. Deubiquitinating enzymes (DUBs) have emerged as critical regulators within this context, capable of removing ubiquitin modifications and, thus, reversing the effects on target proteins. Many DUBs have demonstrated tumorigenic roles in multiple cancers, and many inhibitors have been investigated in clinical trials for diverse cancer treatments [70]. Given their central role in modulating the stability and activity of key proteins involved in tumorigenesis, DUBs represent promising targets for therapeutic intervention, offering the potential for developing novel anticancer strategies [70]. However, the FDA has currently only approved a few drugs that target the ubiquitin system [66].

The advancements in ubiquitin biology have led to innovative strategies for modulating the ubiquitination system, with significant implications for therapeutic development [67]. For the past decade, proteasome inhibitors have been utilized in treating multiple myeloma (MM), yet resistance to these inhibitors has emerged, impacting their effectiveness. Bortezomib (Velcade) and carfilzomib (Kyprolis) are FDA-approved proteasome inhibitors effective against multiple myeloma [66]. Techniques such as proteolysis-targeting chimera (PROTAC) molecules [71] and hydrophobicity tags (HyT) [72] have been developed to specifically target and manipulate the degradation of proteins, offering precise approaches to treat diseases caused by dysregulation of the ubiquitination process [68]. Furthermore, the advent of small molecule inhibitors targeting ubiquitination enzymes presents a promising direction in treating conditions such as cancer, highlighting the potential of these molecular interventions in precision medicine [66].

Ubiquitination and phosphorylation are among the most crucial and widely studied post-translational modifications. It is a dynamic and versatile mechanism for controlling various aspects of cellular function [8, 9]. Ubiquitin, a small yet pivotal protein in cellular regulation, consists of 76 amino acids and is notable for its seven lysine residues—K6, K11, K27, K29, K33, K48, and K63—plus an N-terminal methionine (M1) that can all serve as attachment points for forming polyubiquitin chains [64, 73]. The functions and implications of ubiquitination through K6, K27, K29, and K33 are less well-characterized and represent an area ripe for further research [74, 75]. This ubiquitination system is a key regulator of intracellular proteins, influencing as much as 80% of them [70]. The consequences of ubiquitination can vary greatly (Fig. 4).

Fig. 4.

Ubiquitin-mediated proteasomal degradation and signal transduction. K48-linked polyubiquitination is typically associated with proteasomal degradation, as illustrated by a polyubiquitinated protein directed to the 26S proteasome. In contrast, K63/M1-linked polyubiquitination is often involved in signal transduction processes. UB, ubiquitin.

Proteasomal degradation: Ubiquitin chains, particularly those linked through the K48 residue, often signal the protein to be directed to the 26S proteasome for degradation. This is a key process for removing damaged, misfolded, or unneeded proteins, thereby regulating protein levels and quality control within the cell [73]. Altering substrate activity: Ubiquitination can also change the functional state of proteins, either activating or inhibiting their enzymatic activity, altering their cellular location, or affecting their ability to interact with other molecules [64]. Mediating protein–protein interactions: Ubiquitin chains can serve as a platform for assembling protein complexes, which is critical in various signaling pathways. For example, K63-linked ubiquitin chains are often involved in processes such as DNA repair, NF-$\kappa$B signaling, and the regulation of cellular trafficking [73]. Given its versatility, ubiquitination is a fundamental regulatory process essential for maintaining cellular homeostasis and responding to stress.

Ubiquitination encompasses non-proteolytic roles such as receptor internalization, multiprotein complex assembly, intracellular trafficking, and key signaling pathways, including those governing inflammation, autophagy, DNA repair, and enzymatic regulation [76, 77, 78]. Deregulation can trigger oncogenic pathways, disrupt cellular metabolism, and lead to inadequate protein complex formation critical for inflammation response or DNA repair, resulting in the accumulation of misfolded proteins, causing diseases such as neurodegeneration or misdirecting proteins away from their functional locations within the cell [8, 79].

Ubiquitination is a multistep process typically involving the coordinated action of three ubiquitination enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3) [64]. Initially, the E1 forms a high-energy thioester bond between the carboxyl group of the C-terminal lysine residue of ubiquitin and the thiol group of its own cysteine residue, utilizing ATP-provided energy. Subsequently, the activated ubiquitin is transferred onto the cysteine residue of the E2. Finally, a member of the highly conserved ubiquitin-protein ligase family, E3, recognizes specific target proteins to be ubiquitinated and catalyzes the transfer of ubiquitin molecules from E2 to the lysine residue of the target protein (Fig. 5) [64]. Really interesting new gene (RING) finger [RNF] proteins containing RING domains act as E3 ubiquitin ligases that mediate the covalent linkage of ubiquitin to target proteins [9]. Depending on the ubiquitin chain’s topology, ubiquitination can lead to proteasomal degradation or mediate protein–protein interactions, influencing various cellular processes [64]. Understanding these processes provides insights into the molecular mechanisms of diseases involving the TAK1–TAB complex and offers new opportunities for drug development.

Fig. 5.

The ubiquitin modification system. Ubiquitination begins with the activation of ubiquitin (Ub) by the E1 enzyme, which is ATP-dependent. Activated ubiquitin is then transferred to the E2 conjugating enzyme. In the final step, the E3 ligase enzyme facilitates the attachment of ubiquitin to the substrate protein, completing the ubiquitination process.

The activation of NF-$\kappa$B via the TAK1–TAB complex is intricately controlled through various regulatory mechanisms, including the critical process of deubiquitination, which counteracts ubiquitination [60, 116, 117, 118]. Deubiquitination involves the removal of ubiquitin moieties from proteins by specific deubiquitinating enzymes (DUBs) [66]. The interplay between ubiquitination and deubiquitination is a dynamic and finely balanced process central to maintaining cellular protein homeostasis. This selective deubiquitination can affect protein–protein interactions, influencing the formation and disassembly of protein complexes essential for accurate signal transduction and cellular responses [119]. The disruption of these processes is often implicated in cancer pathogenesis, underlining the importance of these mechanisms in controlling cell growth and proliferation [120].

Several DUBs, each with distinct roles, are instrumental in regulating the TAK1–TAB complex. By selectively removing ubiquitin chains from these proteins, these enzymes can alter the activity, stability, and interaction of TAK1 and TABs, thereby fine-tuning the signaling pathways they are involved in [118, 121, 122, 123]. This targeted deubiquitylation is crucial for precisely controlling signaling events that govern cellular processes such as inflammation, cell survival, and proliferation, with implications for both normal physiology and disease states, including cancer [63].