E3 Ubiquitin Ligase Cbl-b Research Articles

The E3 ubiquitin ligase Cbl-b to improve the prognosis of RANK positive breast cancer patients by inhibiting RANKL-induced cell migration and metastasis.

e22196 Background: The receptor activator of nuclear factor κ-B ligand (RANKL)/RANK pathway plays an important role in breast cancer progression. However, the prognostic value of RANK expression in...

Temporal proteomics of NGF-TrkA signaling identifies an inhibitory role for the E3 ligase Cbl-b in neuroblastoma cell differentiation.

SH-SY5Y neuroblastoma cells respond to nerve growth factor (NGF)-mediated activation of the tropomyosin-related kinase A (TrkA) with neurite outgrowth, thereby providing a model to study neuronal differentiation. We performed a time-resolved analysis of NGF-TrkA signaling in neuroblastoma cells using mass spectrometry-based quantitative proteomics. The combination of interactome, phosphoproteome, and proteome data provided temporal insights into the molecular events downstream of NGF binding to TrkA. We showed that upon NGF stimulation, TrkA recruits the E3 ubiquitin ligase Cbl-b, which then becomes phosphorylated and ubiquitylated and decreases in abundance. We also found that recruitment of Cbl-b promotes TrkA ubiquitylation and degradation. Furthermore, the amount of phosphorylation of the kinase ERK and neurite outgrowth increased upon Cbl-b depletion in several neuroblastoma cell lines. Our findings suggest that Cbl-b limits NGF-TrkA signaling to control the length of neurites.

Cbl-b and c-Cbl negatively regulate osteoblast differentiation by enhancing ubiquitination and degradation of Osterix

E3 ubiquitin ligase Cbl-b and c-Cbl play important roles in bone formation and maintenance. Cbl-b and c-Cbl regulate the activity of various receptor tyrosine kinases and intracellular protein tyrosine kinases mainly by regulating the degradation of target proteins. However, the precise mechanisms of how Cbl-b and c-Cbl regulate osteoblast differentiation are not well known. In this study, we investigated potential targets of Cbl-b and c-Cbl. We found that Cbl-b and c-Cbl inhibit BMP2-induced osteoblast differentiation in mesenchymal cells. Among various osteogenic transcription factors, we identified that Cbl-b and c-Cbl suppress the protein stability and transcriptional activity of Osterix. Our results suggest that Cbl-b and c-Cbl inhibit the function of Osterix by enhancing the ubiquitin–proteasome-mediated degradation of Osterix. Taken together, we propose novel regulatory roles of Cbl-b and c-Cbl during osteoblast differentiation in which Cbl-b and c-Cbl regulate the degradation of Osterix through the ubiquitin–proteasome pathway.

E3 Ubiquitin Ligase Cbl-b Regulates Thymic-Derived CD4+CD25+ Regulatory T Cell Development by Targeting Foxp3 for Ubiquitination.

CD28 costimulation is essential for the development of thymic-derived CD4(+)CD25(+)Foxp3(+) regulatory T cells ("tTregs"). E3 ubiquitin ligase Cbl-b has been shown to regulate CD28 dependence of T cell activation. In this paper, we report that the loss of Cbl-b partially but significantly rescues the defective development of tTregs in Cd28(-/-) mice. This partial rescue is independent of IL-2. Mechanistically, Cbl-b binds to Foxp3 upon TCR stimulation and, together with Stub1, targets Foxp3 for ubiquitination and subsequently degradation in the proteasome. As Cbl-b self-ubiquitination and proteasomal degradation is impaired in Cd28(-/-) T cells, the defective development of tTregs in Cd28(-/-) mice may in part be due to increased Foxp3 ubiquitination and degradation targeted by Stub1 and Cbl-b. Treating Cd28(-/-) mice with a proteasome inhibitor completely rescues defective tTreg development in these mice. Therefore, Cbl-b, together with Stub1, ubiquitinate Foxp3, and regulate tTreg development.

Development of a personalized cellular ex vivo CBL-b silencing cancer immune therapy.

3070 Background: The E3 ubiquitin ligase cbl-b has been identified as an important gatekeeper limiting T cell activation. Concordantly, the immune system of cbl-b deficient mice can effectively fig...

Regulation of E3 ubiquitin ligase Cbl-b expression by protein tyrosine phosphatase SHP-1 (IRM10P.753)

Abstract Casitas B cell lymphoma protein-b (Cbl-b) is a RING finger family E3 ubiquitin ligase. It has been well documented that Cbl-b plays a crucial role in T cell activation and tolerance induction. Our recent study also indicates that Cbl-b regulates proallergic T cell development and allergic airway inflammation. Therefore, the regulation of Cbl-b expression in T cells is important for its biological functions. Our previous studies demonstrate that CD28 costimulation potentiates Cbl-b sel-ubiquitination, CTLA-4-B7 interaction induces Cbl-b expression. However, the molecular mechanism(s) of this regulation remains to be elucidated. In this study, we found that Cbl-b tyrosine phosphorylation in primary mouse T cells requires CD28 costimulation. The failure to detect Cbl-b tyrosine phosphorylation upon CD3 stimulation is not due to defective PTK activation, but rather interestingly SHP-1 is recruited to Cbl-b upon CD3 stimulation, while CD28 costimulation abrogates the interaction between Cbl-b and SHP-1. Furthermore, we identified Cbl-b N-terminal tyrosine residues within TKB domain and linker region (Y106, Y133, and Y363) are required for its E3 ubiquitin ligase activity. The aberrant Th2 phenotype observed in T cell-specific SHP-1-/- (tSHP-1-/-) mice is reminiscent of our recent observation that Th2 responses are heightened in Cbl-b-/- mice. In support of this notion, Cbl-b expression is down-regulated in naïve T cells of tSHP-1-/- mice.

The E3 ligase Cbl-b and TAM receptors regulate cancer metastasis via natural killer cells

Tumour metastasis is the primary cause of mortality in cancer patients and remains the key challenge for cancer therapy. New therapeutic approaches to block inhibitory pathways of the immune system have renewed hopes for the utility of such therapies. Here we show that genetic deletion of the E3 ubiquitin ligase Cbl-b (casitas B-lineage lymphoma-b) or targeted inactivation of its E3 ligase activity licenses natural killer (NK) cells to spontaneously reject metastatic tumours. The TAM tyrosine kinase receptors Tyro3, Axl and Mer (also known as Mertk) were identified as ubiquitylation substrates for Cbl-b. Treatment of wild-type NK cells with a newly developed small molecule TAM kinase inhibitor conferred therapeutic potential, efficiently enhancing anti-metastatic NK cell activity in vivo. Oral or intraperitoneal administration using this TAM inhibitor markedly reduced murine mammary cancer and melanoma metastases dependent on NK cells. We further report that the anticoagulant warfarin exerts anti-metastatic activity in mice via Cbl-b/TAM receptors in NK cells, providing a molecular explanation for a 50-year-old puzzle in cancer biology. This novel TAM/Cbl-b inhibitory pathway shows that it might be possible to develop a 'pill' that awakens the innate immune system to kill cancer metastases.

Development of a personalized cellular ex-vivo cbl-b silencing cancer immunotherapy

Meeting abstracts The E3 ubiquitin ligase cbl-b has been identified as an important gatekeeper limiting T cell activation. Concordantly, the immune system of cbl-b deficient mice can effectively fight tumors, thereby validating cbl-b as an excellent target to enhance anti-tumor immune activity. We

T Cell Activation Threshold Regulated by E3 Ubiquitin Ligase Cbl-b Determines Fate of Inducible Regulatory T Cells

E3 ubiquitin ligase Casitas-B-lineage lymphoma protein-b (Cbl-b) is critical for establishing the threshold for T cell activation and is essential for induction of T cell anergy. Recent studies suggest that Cbl-b is involved in the development of CD4(+)CD25(+) inducible regulatory T cells (iTregs). In this study, we report that the optimal induction of Foxp3 by naive CD4(+)CD25(-) T cells requires suboptimal TCR triggering. In the absence of Cbl-b, the TCR strength for optimal Foxp3 induction is downregulated in vitro. Using TCR-transgenic Rag(-/-) mice in combination with Cbl-b deficiency, we show that in vivo iTreg development is also controlled by Cbl-b via tuning the TCR strength. Furthermore, we show that Akt-2 but not Akt-1 regulates Foxp3 expression downstream of Cbl-b. Therefore, we demonstrate that Cbl-b regulates the fate of iTregs via controlling the threshold for T cell activation.

Regulation of naturally-occurring CD4+CD25+ regulatory T cell development by E3 ubiquitin ligase Cbl-b (P1041)

Abstract Naturally-occurring CD4+CD25+Foxp3+ regulatory T cells ("nTregs") are involved in shutting down immune responses after they have successfully eliminated invading organisms, and also in preventing autoimmunity. CD28 costimulation is essential for the development of nTregs in thymus. E3 ubiquitin ligase Cbl-b has been shown to regulate CD28 dependence of T cell activation. Here, we report that the loss of Cbl-b partially but significantly rescues the defective development of nTregs in CD28-/- mice. This partial rescue is likely to be independent of IL-2 because treating CD28-/- mice with IL-2/anti-IL-2 complex, which has been shown to selectively stimulate nTregs, fails to rescue defective development of CD4+CD8-CD25+Foxp3+ nTregs in thymuses of CD28-/- mice. Mechanistically, Cbl-b binds to Foxp3 upon TCR stimulation, and targets Foxp3 for ubiquitination and subsequently degradation in the proteasome. As Cbl-b self-ubiquitination and proteasomal degradation is impaired in CD28-/- T cells, the defective development of nTregs in CD28-/- mice may in part be due to increased Foxp3 ubiquitination and degradation targeted by Cbl-b. Therefore, we provide a previously-unappreciated function for Cbl-b in regulating Foxp3 expression via an ubiquitination-dependent mechanism.

T cell activation threshold regulated by E3 ubiquitin ligase Cbl-b determines fate of inducible regulatory T cells (P1025)

Abstract E3 ubiquitin ligase Cbl-b is critical for establishing the threshold for T cell activation, and is essential for induction of T cell anergy. Recent studies suggest that Cbl-b is involved in the development of inducible CD4+CD25+ regulatory T cells (iTregs). In this study, we report that the optimal induction of Foxp3 by CD4+CD25- T cells requires suboptimal TCR triggering. In the absence of Cbl-b, the TCR strength for optimal Foxp3 induction is down-regulated in vitro. Using TCR transgenic Rag-/- mice in combination with Cbl-b deficiency, we show that in vivo iTreg development is also controlled by Cbl-b via tuning the TCR strength. Furthermore, we show that Akt-2 but not Akt-1 regulates Foxp3 expression downstream of Cbl-b. Therefore, we demonstrate that Cbl-b regulates the fate of iTregs via controlling the threshold for T cell activation.

FOXP3, CBLBandITCHgene expression and cytotoxic T lymphocyte antigen 4 expression on CD4+CD25high T cells in multiple sclerosis

Expression of the forkhead box protein 3 (FoxP3) transcription factor is regulated by the E3 ubiquitin ligases Itch and Cbl-b and induces regulatory activity CD4(+) CD25(high) T cells. Treatment with interferon (IFN)-β enhances regulatory T cell activity in multiple sclerosis (MS). We studied the phenotype of CD4(+) CD25(high) T cells in MS by flow cytometry and its relationship with expression of the FOXP3, ITCH and CBLB genes. We found that untreated MS patients had lower cell surface expression of cytotoxic T lymphocyte antigen 4 (CTLA-4) on CD4(+) CD25(high) T cells and higher intracellular CTLA-4 expression than healthy controls. Cell surface expression of CTLA-4 on CD4(+) CD25(high) T cells correlated with expression of FOXP3 mRNA in untreated patients and increased significantly with time from most recent injection in patients treated with IFN-β. FOXP3 mRNA expression correlated with CBLB and ITCH and T helper type 2 cytokine mRNA expression in MS patients. These data link expression of FOXP3, CBLB and ITCH mRNA and CTLA-4 expression on the surface of CD4(+) CD25(high) T cell in MS. We hypothesize that this may reflect alterations in the inhibitory effect of CTLA-4 or in regulatory T cell function.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

E3 Ubiquitin Ligase Cbl-b Regulates Pten via Nedd4 in T Cells Independently of Its Ubiquitin Ligase Activity

E3 ubiquitin ligase Cbl-b plays a crucial role in T cell activation and tolerance induction. However, the molecular mechanism by which Cbl-b inhibits T cell activation remains unclear. Here, we report that Cbl-b does not inhibit PI3K but rather suppresses TCR/CD28-induced inactivation of Pten. The elevated Akt activity in Cbl-b(-/-) T cells is therefore due to heightened Pten inactivation. Suppression of Pten inactivation in T cells by Cbl-b is achieved by impeding the association of Pten with Nedd4, which targets Pten K13 for K63-linked polyubiquitination. Consistent with this finding, introducing Nedd4 deficiency into Cbl-b(-/-) mice abrogates hyper-T cell responses caused by the loss of Cbl-b. Hence, our data demonstrate that Cbl-b inhibits T cell activation by suppressing Pten inactivation independently of its ubiquitin ligase activity.

The Syk-binding Ubiquitin Ligase c-Cbl Mediates Signaling-dependent B Cell Receptor Ubiquitination and B Cell Receptor-mediated Antigen Processing and Presentation

B cell receptor (BCR)-mediated antigen (Ag) processing and presentation lead to B cell-T cell interactions, which support affinity maturation and immunoglobulin class switching. These interactions are supported by generation of peptide-MHC class II complexes in multivesicular body-like MIIC compartments of B cells. Previous studies have shown that trafficking of Ag·BCR complexes to MVB-like MIIC occurs via an ubiquitin-dependent pathway and that ubiquitination of Ag·BCR complexes occurs by an Src family kinase signaling-dependent mechanism that is restricted to lipid raft-resident Ag·BCR complexes. This study establishes that downstream Syk-dependent BCR signaling is also required for BCR ubiquitination and BCR-mediated antigen processing and presentation. Knockdown studies reveal that of the two known Syk-binding E3 ubiquitin ligases c-Cbl and Cbl-b, only c-Cbl appears to have a central role in BCR ubiquitination, trafficking to MIIC, and ubiquitin-dependent BCR-mediated antigen processing and presentation. These results establish the novel role for Syk signaling and the Syk-binding ubiquitin ligase c-Cbl in the BCR-mediated processing and presentation of cognate antigen and define one mechanism by which antigen-induced BCR ubiquitination is modulated to impact the initiation and maturation of the humoral immune response.

Releasing the Brake: Targeting Cbl-b to Enhance Lymphocyte Effector Functions

The E3 ubiquitin ligase Cbl-b is an established nonredundant negative regulator of T-cell activation. Cbl-b fine-tunes the activation threshold of T cells and uncouples T cells from their vital need of a costimulatory signal to mount a productive immune response. Accordingly, mice deficient in cblb are prone to autoimmunity and reject tumors. The latter has been described to be mediated via CD8+ T cells, which are hyperactive and more abundant in shrinking tumors of cblb-deficient animals. This might at least also in part be mediated by resistance of cblb-deficient T cells to negative cues exerted by tumor-associated immuno-suppressive factors, such as TGF-β and regulatory T cells (Treg). Experiments using cblb-deficient T cells either alone or in combination with vaccines validate the therapeutic concept of enhancing the efficacy of adoptively transferred lymphocytes to treat malignant tumors. This paper summarizes the current knowledge about the negative regulatory role of Cbl-b in T-cell activation and its potential therapeutic implications for cancer immunotherapy.

E3 ubiquitin ligase Cbl-b negatively regulates T helper 2 cell responses by targeting Stat-6 for ubiquitination (152.19)

Abstract Casitas-B-lineage lymphoma protein-b (Cbl-b), an E3 ubiquitin ligase, belongs to the Cbl family. Recent studies indicate that Cbl-b emerges as a gatekeeper that controls the activation threshold of the antigen receptor and maintains the balance between tolerance and autoimmunity. However, the role of Cbl-b in T helper (Th) cell differentiation remains unknown. In this study, we show that Cbl-b deficiency selectively results in a biased Th2 differentiation in vitro. Furthermore, using a mouse model of asthma, we find that the absence of Cbl-b results in severe airway inflammation which associates with stronger Th2 responses including elevated IL-4, IL-5, and IL-13, and accumulation of eosinophils in bronchoalveolar lavage (BAL) fluid and higher titers of IgE in the serum. At the molecular level, Cbl-b selectively associates with Stat-6 upon IL-4 ligation, and targets Stat-6 for K48-linked polyubiquitination and subsequently degradation in the proteasome. These processes are heightened in the presence of TCR/CD28 costimulation. IL-4-induced Cbl-b-Stat6 association requires Cbl-b TKB domain, whereas TCR/CD28-induced Cbl-b-Stat6 interaction is mediated by tyrosine residues of Cbl-b and SH2 domain of Stat6. Therefore, we have discovered a previously uncharacterized function of Cbl-b in Th2 cell differentiation that may serve as an important therapeutic target for allergic diseases including asthma.

Reinforcement of cancer immunotherapy by adoptive transfer of cblb‐deficient CD8+ T cells combined with a DC vaccine

The success of cancer immunotherapy is limited by potent endogenous immune-evasion mechanisms, which are at least in part mediated by transforming growth factor-β (TGF-β). The E3 ubiquitin ligase Cbl-b is a key regulator of T cell activation and is established to regulate TGF-β sensitivity. cblb-deficient animals reject tumors via CD8(+) T cells, which make Cbl-b an ideal target for improvement of adoptive T-cell transfer (ATC) therapy. In this study, we show that cblb-deficient CD8(+) T cells are hyper-responsive to T-cell receptor (TCR)/CD28-stimulation and are in part protected against the negative cues induced by TGF-β in vitro. Notably, adoptive transfer of polyclonal, non-TCR transgenic cblb-deficient CD8(+) T cells is not sufficient to reject B16-ova or EG7 tumors in vivo. Thus, cblb-deficient ATC requires proper in vivo re-activation by a dendritic cell (DC) vaccine. In strict contrast to ATC monotherapy, this approach delayed tumor outgrowth and significantly increased survival rates, which is paralleled by increased CD8(+) T-cells infiltration to the tumor site and enrichment of ova-specific and interferon-γ (IFN-γ)-secreting CD8(+) T cell in the draining lymph node (LN). Moreover, CD8(+) T cells from cblb-deficient mice vaccinated with the DC vaccine show increased cytolytic activity in vivo. In summary, our data using cblb-deficient polyclonal, non-TCR-transgenic adoptively transferred CD8(+) T cells into immuno-competent non-lymphodepleted recipients suggest that targeting Cbl-b might serve as a novel 'adjuvant approach', suitable to augment the effectiveness of established anti-cancer immunotherapies.

Visualizing the Role of Cbl-b in Control of Islet-Reactive CD4 T Cells and Susceptibility to Type 1 Diabetes

The E3 ubiquitin ligase Cbl-b regulates T cell activation thresholds and has been associated with protecting against type 1 diabetes, but its in vivo role in the process of self-tolerance has not been examined at the level of potentially autoaggressive CD4(+) T cells. In this study, we visualize the consequences of Cbl-b deficiency on self-tolerance to lysozyme Ag expressed in transgenic mice under control of the insulin promoter (insHEL). By tracing the fate of pancreatic islet-reactive CD4(+) T cells in prediabetic 3A9-TCR × insHEL double-transgenic mice, we find that Cbl-b deficiency contrasts with AIRE or IL-2 deficiency, because it does not affect thymic negative selection of islet-reactive CD4(+) cells or the numbers of islet-specific CD4(+) or CD4(+)Foxp3(+) T cells in the periphery, although it decreased differentiation of inducible regulatory T cells from TGF-β-treated 3A9-TCR cells in vitro. When removed from regulatory T cells and placed in culture, Cblb-deficient islet-reactive CD4(+) cells reveal a capacity to proliferate to HEL Ag that is repressed in wild-type cells. This latent failure of T cell anergy is, nevertheless, controlled in vivo in prediabetic mice so that islet-reactive CD4(+) cells in the spleen and the pancreatic lymph node of Cblb-deficient mice show no evidence of increased activation or proliferation in situ. Cblb deficiency subsequently precipitated diabetes in most TCR:insHEL animals by 15 wk of age. These results reveal a role for peripheral T cell anergy in organ-specific self-tolerance and illuminate the interplay between Cblb-dependent anergy and other mechanisms for preventing organ-specific autoimmunity.

E3 ubiquitin ligase Cbl-b potentiates the apoptotic action of arsenic trioxide by inhibiting the PI3K/Akt pathway

Arsenic trioxide (ATO) is a strong inducer of apoptosis in malignant hematological cells. Inducible phosphatidyl inositol 3 kinase (PI3K)-Akt activation promotes resistance to ATO. In the present study, we evaluated whether E3 ubiquitin ligase Cbl-b, a negative regulator of PI3K activation, is involved in the action of ATO. The effect of ATO on cell viability was measured by the Trypan blue exclusion assay or by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis was determined by flow cytometry and protein expression was assayed by Western blotting. ATO decreased the viability of HL60 cells and induced cellular apoptosis, which was accompanied by transient activation of Akt. The PI3K/Akt inhibitor, LY294002, significantly increased ATO-induced apoptosis (P < 0.05). In addition, ATO up-regulated the expression of Cbl-b proteins. Furthermore, ATO inhibited cell viability with an IC50 of 18.54 μM at 24 h in rat basophilic leukemia-2H3 cells. ATO induced cellular apoptosis with transient activation of Akt and Cbl-b was also up-regulated. Rat basophilic leukemia-2H3 cells transfected with a dominant negative (DN) Cbl-b mutation showed overexpression of Cbl-b (DN) and enhanced Akt activation. Compared with cells transfected with vector, ATO-induced apoptosis was decreased and G2/M phase cells were increased at the same concentration (P < 0.05). The PI3K/Akt inhibitor, LY294002, re-sensitized Cbl-b (DN) overexpressing cells to ATO and reversed G2/M arrest (P < 0.05). Taken together, these results suggest that Cbl-b potentiates the apoptotic action of ATO by inhibition of the PI3K/Akt pathway.