E3 Ubiquitin Ligase CBL-B
Functionally, the N-terminus of Cbl-b encompasses the TKB domain, which comprises a four-helix bundle, a calcium-binding EF domain hand, and a variant Src homology2 (SH2) domain. The TKB domain recognizes target proteins for ubiquitin conjugation by binding to specific phosphotyrosine residues on target proteins (Meng et al. 1999). The RING finger domain of Cbl-b is highly conserved and has been shown to recruit ubiquitin-conjugating enzyme (E2)-ubiquitin complexes (Waterman et al. 1999). The proline-rich region of Cbl-b interacts with SH3 domain-containing proteins (Elly et al. 1999). Cbl-b also contains multiple tyrosine residues at its C-terminus, TKB domain, and also the linker region (Fig. 1). Several recent studies indicate that Cbl-b Y363, located in the helix-linker region between the TKB and the RING domains, is critical for the activation of its E3 ligase function (Kobashigawa et al. 2011; Dou et al. 2013).
Cbl-b is tyrosine phosphorylated by PTK(s), and Lck is the PTK that phosphorylates Cbl-b in T cells (Xiao et al. 2015). Interestingly, Cbl-b tyrosine phosphorylation is undetectable in naïve T cells upon TCR stimulation, and this is because TCR stimulation induces the association of protein tyrosine phosphatase SHP-1 with Cbl-b, which prevents Cbl-b phosphorylation. CD28 co-stimulation facilitates the dissociation of SHP-1 from Cbl-b, thus allowing Cbl-b to be phosphorylated by Lck (Xiao et al. 2015). In addition to Cbl-b Y363, Cbl-b Y106 and Y133 within the TKB domain are essential for its E3 ubiquitin ligase activity (Xiao et al. 2015). Cbl-b also has a ubiquitin-associated (UBA) domain at its C-terminus (Fig. 1). Although Cbl-b and c-Cbl shares high sequence and functional similarity, the UBA domains from Cbl-b and c-Cbl display remarkably different in vitro properties. Cbl-b UBA binds to ubiquitin, but c-Cbl UBA fails to do so (Peschard et al. 2007). In contrast to c-Cbl, Cbl-b does not have a PI3-K p85-binding motif, which also highlights potentially important divergence roles and modes of action of these two highly similar regulatory proteins (Beckwith et al. 1996).
Cbl-b in T Cell Activation and Tolerance Induction
Accumulated evidence indicated that ubiquitination mediated by Cbl-b is a novel and crucial mechanism for TCR signaling modification in order to negatively regulate T cell activation and immune tolerance (Liu 2004; Bhoj and Chen 2009). Cbl-b deficiency uncouples the requirement of T-cell proliferation and IL-2 production from CD28 co-stimulation (Bachmaier et al. 2000; Chiang et al. 2000), suggesting that Cbl-b is a negative regulator of CD28 co-stimulatory activity. Guanine nucleotides are activated upon TCR and CD28 co-stimulation and function as key regulators of cytoskeleton reorganization at the immune synapse (Krawczyk and Penninger 2001; Zeng et al. 2003). Cbl-b appears to be a negative regulator of GDP/GTP exchange factor (Vav1) phosphorylation (Krawczyk et al. 2000). In addition to Vav, Cbl-b inhibits TCR-induced activation of NF-κB via Akt and PKC-θ dependent mechanisms (Qiao et al. 2008). Cbl-b has been shown to regulate PI3-K/Akt activation by inactivating Pten via Nedd4 (Guo et al. 2012).
It has been demonstrated that reduction of the T cell activation threshold (caused by the loss of Cbl-b) correlates with an increased susceptibility to the development of autoimmunity (Bachmaier et al. 2000; Chiang et al. 2000), suggesting that Cbl-b is a key regulator of the susceptibility to autoimmunity. These studies also suggest that the regulation of Cbl-b expression is critical for T cell activation and tolerance induction. CD28 co-stimulation potentiates TCR-induced Cbl-b ubiquitination and degradation, whereas CTLA-4-B7 interaction induces Cbl-b expression (Zhang et al. 2002; Li et al. 2004). Therefore, CD28 and CTLA-4 control the threshold for T cell activation, at least in part, through regulating the negative regulator Cbl-b. Mechanistically, Cbl-b degradation may be mediated by two mechanisms. Firstly, Nedd4, which has been shown to target Cbl-b for ubiquitination and degradation (Yang et al. 2008), and PKC-θ which phosphorylates Cbl-b at Ser282 in the TKB domain, may facilitate Cbl-b ubiquitination and degradation (Gruber et al. 2009). Secondly, Cbl-b undergoes autoubiquitination, and this process is regulated by Cbl-b tyrosine phosphorylation at N-terminal TKB domain and linker region (Peschard et al. 2007; Dou et al. 2013; Xiao et al. 2015).
A variety of mechanisms are engaged in the induction of T cell tolerance, such as deletion of self-reactive T cells, involvement of regulatory T cells, and induction of T cell anergy (Kamradt and Mitchinson 2001; Walker and Abbas 2002). T cell anergy has been considered as a critical mechanism to maintain T cell tolerance, and genetic and biochemical evidences have indicated T cell anergy is partly a consequence of proteolytic activation mediated by E3 ubiquitin ligases (Heissmeyer et al. 2004; Mueller 2004; Paolino and Penninger 2009). Ablation of Cbl-b in mice results in resistance to T cell anergy induction both in vitro and in vivo, suggesting that Cbl-b is critical for establishing T-cell tolerance to specific antigens (Jeon et al. 2004). It has been shown that different anergy-inducing conditions selectively induce enhanced expression of Cbl-b, which targets the key molecules such as phospholipase C-γ1 (PLC-γ1) and protein kinase C-θ (PKC-θ) for degradation, resulting in the instability of mature immunological synapses (Heissmeyer et al. 2004; Jeon et al. 2004). Cbl-b is therefore considered to be an important negative regulator in initiating and maintaining T cell anergy both in vitro and in vivo.
Regulatory T cells (Tregs) play an important role in regulating peripheral T cell tolerance. CD28 co-stimulation is required for the Tregs development. It was reported that CD4+CD25− effector T cells (Teffs) are resistant to regulation by thymic-derived Tregs (tTregs) in vitro and that there is a defect in the conversion of CD4+CD25− T cells into CD4+Foxp3+ inducible Tregs (iTregs) in Cblb −/− mice in vitro and in vivo (Qiao et al. 2013). However, it is unknown why Cblb −/− Teffs are resistant to regulation by nTregs, and whether Cbl-b deficiency affects peripheral conversion of naïve CD4+CD25− T cells into CD4+Foxp3+ iTregs in vivo. Taken together, these studies strongly indicate that Cbl-b deficiency not only affects Teffs but also facilitates iTreg development. The impaired iTreg development in the absence of Cbl-b seems to be due to heightened activation of Akt-2, which phosphorylates Foxo1/Foxo3a (Harada et al. 2010). Interestingly, Cbl-b deficiency has been demonstrated to partially rescue the decrease of tTregs) in CD28 −/− mice. Together with Stub1, Cbl-b targets Foxp3 for ubiquitination and proteasome-mediated degradation (Zhao et al. 2015). Therefore, Cbl-b is crucial to maintain the expression of Foxp3 at the steady state. Further studies using specific deletion of Cbl-b in the Tregs are required to firmly confirm the role of Cbl-b in the Treg development and function.
Cbl-b in T Helper Cell (TH) Development
Naïve TH cells differentiate in response to antigen stimulation into TH1, TH2, TH9, TH17, or iTreg effector cells, which are characterized by the secretion of different sets of cytokines (Zhu et al. 2010; Christie and Zhu 2014). Since Cblb −/− mice are highly susceptible to experimental autoimmune encephalomyelitis (EAE) and collagen-induced arthritis (CIA), both of which are believed to be mediated at least in part by TH17, it was expected that Cbl-b might inhibit TH17 cell development. To our surprise, however, the loss of Cbl-b facilitates TH2 and TH9 cell differentiation in vitro. Consistent with this, in a mouse model of asthma, the absence of Cbl-b results in severe airway inflammation and stronger TH2 and TH9 responses. This is achieved by targeting Stat6, a critical transcription factor for TH2 and TH9 downstream of the IL-4 receptor, for ubiquitination (Qiao et al. 2014). Therefore, these data indicate that Cbl-b inhibits TH2 and TH9 cell development and allergic airway inflammation.
Cbl-b in Humoral Immunity
Cbl-b has also been shown to be a negative regulator of BCR signaling. Cblb −/− B cells display heightened phosphorylation of Igα, Syk, and PLC-γ2 in response to BCR stimulation which leads to prolonged Ca2+ mobilization and increased activation of ERK and JNK, and surface expression of the activation marker, CD69. Cbl-b exerts its inhibitory role on BCR signaling by targeting Syk and Igα for ubiquitination and proteasomal degradation (Yasuda et al. 2002; Niiro et al. 2012). Consistent with these data, B cell-specific ablation of both c-Cbl and Cbl-b (Cbl −/− Cblb −/− ) develop systemic lupus erythematosus (SLE)-like autoimmune disease possibly due to the breakdown of B cell anergy (Kitaura et al. 2007). B cells lacking both c-Cbl and Cbl-b display enhanced tyrosine phosphorylation of Syk, PLC-γ2, and Vav and Ca2+ mobilization and substantial attenuation of tyrosine phosphorylation of adaptor protein BLNK. Taken together, Cbl proteins regulate B cell-intrinsic checkpoints of immune tolerance, possibly via coordinating multiple BCR-proximal signaling pathways during anergy induction. In addition to BCR signaling, Cbl-b also inhibits CD40-mediated B cell activation. Cblb −/− mice display enhanced thymus-dependent antibody responses and germinal center formation, whereas introduction of CD40 deficiency abolishes these effects. Cbl-b selectively down-modulates CD40-induced activation of NF-κB and JNK, thereby suppressing B cell responses.
Cbl-b in Tumor Immunity
In recent years there has been growing awareness of the importance of antitumor immunotherapies. Cbl-b is a checkpoint regulator for T cell activation and is a potential drug target for antitumor immunity. Cblb −/− mice reject spontaneous tumors and transplanted tumors, and this is partially mediated by CD8+ T cells (Chiang et al. 2007; Loeser et al. 2007). Most recent studies indicate that Cbl-b also regulates antitumor activity by NK cells by targeting the TAM tyrosine kinase receptors Tyro3, Axl, and Mer for ubiquitination (Paolino et al. 2014). Therefore, CD8+ cytotoxic T cells and NK cells are considered as the main contributors during tumor rejection process. In support of a crucial role of Cbl-b in antitumor immunity, adoptive transfer of Cblb-silenced CD8+ T cells augments tumor vaccine efficacy in a B16 melanoma model (Hinterleitner et al. 2012). Furthermore, silencing Cblb gene has been shown to enhance the immune activation of T cells against RM-1 prostate cancer cells in vitro (Shi et al. 2014). Therefore, targeting Cbl-b represents a novel immunotherapeutic approach against tumors.
Cbl-b in Innate Immunity
Although Cbl-b is known to inhibit T cell activation, tolerance induction, and TH2/9 cell differentiation, its role in innate immune responses remains to be defined. An initial study showed that loss of Cbl-b augments lung inflammatory responses in Lipopolysaccharides (LPS)-induced septic shock and cecal ligation and puncture-induced sepsis. Cbl-b was believed to target TLR4 for ubiquitination, thus dampening TLR4-mediated NF-κB activation (Bachmaier et al. 2007). However, Cbl-b was also reported to target MyD88 and TRIF for ubiquitination in co-transfection experiments using HEK293 cells (Han et al. 2010). Recently, several studies illustrated that although Cbl-b may not regulate TLR signaling, it plays a crucial role in antifungal innate immune response against disseminated candidiasis. Genetic deficiency of Cbl-b increases survival rate of mice challenged with lethal fungal infection, which is in consistent with hyper-expression of TNF-α and IL-6, increased release of reactive oxygen species, and enhanced fungal killing capacity. Cbl-b exerts its anti-inflammatory response by targeting C-type lectin receptors (CLRs) dectin-1, dectin-2, and dectin-3 and SYK for K48-linked ubiquitination and subsequent degradation (Wirnsberger et al. 2016; Xiao et al. 2016; Zhu et al. 2016). Silencing Cblb gene or inhibition of Cbl-b protein by a peptide inhibitor protects mice from lethal Candida infection (Wirnsberger et al. 2016; Xiao et al. 2016). Thus, Cbl-b appears to be a promising therapeutic target in lethal disseminated candidiasis, and possibly other fungal infections. However, further translational studies remain to be performed before moving forward into clinical application.
The Cbl-b E3 ligase functions as a T cell gatekeeper in regulating the balance between T cell activation and tolerance induction. Cbl-b is crucial for maintaining T cell anergy status by targeting PLC-θ and PKC-θ for ubiquitination and degradation, leading to immunological synapse instability. Cbl-b also facilitates iTreg development, suggesting that Cbl-b regulates T cell tolerance via multiple mechanisms. Loss of Cbl-b elicits the spontaneous rejection of solid and hematopoietic tumors, as well as transplanted tumors, which is possibly mediated by CD8+ T cells and NK cells. Therefore, Cbl-b appears to serve as an attractive therapeutic strategy for cancer immunotherapy. Although Cbl-b may not be directly involved in TLR signaling, it does inhibit signaling derived from dectin-1, dectin-2, and dectin-3, thus negatively regulating the intensity of antifungal immune responses. Targeting Cbl-b may open a new avenue for fighting lethal fungal infections.
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