Identification of a potent regulatory T cell epitope in factor V that modulates CD4+ and CD8+ memory T cell responses.

Identification of T cell epitopes that are recognized by Tregs may elucidate the relative contributions of thymic Tregs and induced Tregs to control of autoimmune diseases and allergy. One such T regulatory cell epitope or 'Tregitope', derived from blood Factor V, is described here. Tregs responding to Tregitope FV621 are potent suppressors of CD4+ T effector responses to Tetanus Toxoid in an in vitro bystander suppression assay, strongly inhibit proliferation of effector CD8+ T cells, down-modulate CD86 and HLA DR on antigen-presenting cells, and enhance expression of granzyme B in Tregs. Tregitope FV621 also suppresses anti-OVA immune responses in vivo. The immunomodulatory effect of Tregitope FV621 is enhanced when conjugated to albumin, suggesting that the short half-life of Tregitope peptides can be prolonged. The in silico tools used to prospectively identify the FV Tregitope described here, when combined with in vitro /in vivo validating assays, may facilitate future Tregitope discoveries.

Mouse natural killer cell development and maturation are differentially regulated by SHIP-1.

The SH2-containing inositol phosphatase-1 (SHIP-1) is a 5' inositol phosphatase known to negatively regulate the product of phosphoinositide-3 kinase (PI3K), phosphatidylinositol-3.4,5-trisphosphate. SHIP-1 can be recruited to a large number of inhibitory receptors expressed on natural killer (NK) cells. However, its role in NK cell development, maturation, and functions is not well defined. In this study, we found that the absence of SHIP-1 results in a loss of peripheral NK cells. However, using chimeric mice we demonstrated that SHIP-1 expression is not required intrinsically for NK cell lineage development. In contrast, SHIP-1 is required cell autonomously for NK cell terminal differentiation. These findings reveal both a direct and indirect role for SHIP-1 at different NK cell development checkpoints. Notably, SHIP-1-deficient NK cells display an impaired ability to secrete IFN-γ during cytokine receptor-mediated responses, whereas immunoreceptor tyrosine-based activation motif containing receptor-mediated responses is not affected. Taken together, our results provide novel insights on how SHIP-1 participates in the development, maturation, and effector functions of NK cells.

Ubiquitylation of an internalized killer cell Ig-like receptor by Triad3A disrupts sustained NF-κB signaling.

Killer cell Ig-like receptor (KIR) with two Ig-like domains and a long cytoplasmic domain 4 (2DL4; CD158d) is a unique KIR expressed on human NK cells, which stimulates cytokine production, but mechanisms regulating its expression and function are poorly understood. By yeast two-hybrid screening, we identified the E3 ubiquitin ligase, Triad3A, as an interaction partner for the 2DL4 cytoplasmic domain. The protein interaction was confirmed in vivo, and Triad3A expression induced polyubiquitylation and degradation of 2DL4. Overexpression of Triad3A selectively abrogated the cytokine-producing function of 2DL4, whereas Triad3A short hairpin RNA reversed ubiquitylation and restored cytokine production. Expression of Triad3A in an NK cell line did not affect receptor surface expression, internalization, or early signaling, but significantly reduced receptor turnover and suppressed sustained NF-κB activation. 2DL4 endocytosis was found to be vital to stimulate cytokine production, and Triad3A expression diminished localization of internalized receptor in early endosomes. Our results reveal a critical role for endocytosed 2DL4 receptor to generate sustained NF-κB signaling and drive cytokine production. We conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptor from early endosomes.

A SARS-CoV-2 NSP7 homolog of a Treg epitope suppresses CD4+ and CD8+ T cell memory responses.

Pathogens escape host defenses by T-cell epitope mutation or deletion (immune escape) and by simulating the appearance of human T cell epitopes (immune camouflage). We identified a highly conserved, human-like T cell epitope in non-structural protein 7 (NSP7) of SARS-CoV-2, RNA-dependent RNA polymerase (RdRp) hetero-tetramer complex. Remarkably, this T cell epitope has significant homology to a T regulatory cell epitope (Tregitope) previously identified in the Fc region of human immunoglobulin G (IgG) (Tregitope 289). We hypothesized that the SARS-CoV-2 NSP7 epitope (NSP7-289) may induce suppressive responses by engaging and activating pre-existing regulatory T cells. We therefore compared NSP7-289 and IgG Tregitopes (289 and 289z, a shorter version of 289 that isolates the shared NSP7 epitope) in vitro. Tregitope peptides 289, 289z and NSP7-289 bound to multiple HLA-DRB1 alleles in vitro and suppressed CD4+ and CD8+ T cell memory responses. Identification and in vitro validation of SARS-CoV-2 NSP7-289 provides further evidence of immune camouflage and suggests that pathogens can use human-like epitopes to evade immune response and potentially enhance host tolerance. Further exploration of the role of cross-conserved Tregs in human immune responses to pathogens such as SARS-CoV-2 is warranted.

Tregitopes Improve Asthma by Promoting Highly Suppressive and Antigen-Specific Tregs.

Tregitopes (T regulatory epitopes) are IgG-derived peptides with high affinity to major histocompatibility complex class II (MHCII), that are known to promote tolerance by activating T regulatory cell (Treg) activity. Here we characterized the effect of IgG Tregitopes in a well-established murine model of allergic asthma, demonstrating in vivo antigen-specific tolerance via adoptive transfer of Tregitope-and-allergen-activated Tregs. Asthma is a heterogeneous chronic inflammatory condition affecting the airways and impacting over 300 million individuals worldwide. Treatment is suppressive, and no current therapy addresses immune regulation in severely affected asthmatics. Although high dose intra-venous immunoglobulin (IVIg) is not commonly used in the asthma clinic setting, it has been shown to improve severe asthma in children and in adults. In our laboratory, we previously demonstrated that IVIg abrogates airway hyperresponsiveness (AHR) in a murine model of asthma and induces suppressive antigen-specific T-regulatory cells. We hypothesized that IgG-derived Tregitopes would modulate allergic airway disease by inducing highly suppressive antigen-specific Tregs capable of diminishing T effector cell responses and establishing antigen-specific tolerance. Using ovalbumin (OVA-) and ragweed-driven murine models of allergic airway disease, we characterized the immunoregulatory properties of Tregitopes and performed Treg adoptive transfer to OVA- and ragweed-allergic mice to test for allergen specificity. Treatment with Tregitopes attenuated allergen-induced airway hyperresponsiveness and lung inflammation. We demonstrated that Tregitopes induce highly suppressive allergen-specific Tregs. The tolerogenic action of IgG Tregitopes in our model is very similar to that of IVIg, so we foresee that IgG Tregitopes could potentially replace steroid-based treatment and can offer a synthetic alternative to IVIg in a range of inflammatory and allergic conditions.

Immune Tolerance-Adjusted Personalized Immunogenicity Prediction for Pompe Disease.

Infantile-onset Pompe disease (IOPD) is a glycogen storage disease caused by a deficiency of acid alpha-glucosidase (GAA). Treatment with recombinant human GAA (rhGAA, alglucosidase alfa) enzyme replacement therapy (ERT) significantly improves clinical outcomes; however, many IOPD children treated with rhGAA develop anti-drug antibodies (ADA) that render the therapy ineffective. Antibodies to rhGAA are driven by T cell responses to sequences in rhGAA that differ from the individuals' native GAA (nGAA). The goal of this study was to develop a tool for personalized immunogenicity risk assessment (PIMA) that quantifies T cell epitopes that differ between nGAA and rhGAA using information about an individual's native GAA gene and their HLA DR haplotype, and to use this information to predict the risk of developing ADA. Four versions of PIMA have been developed. They use EpiMatrix, a computational tool for T cell epitope identification, combined with an HLA-restricted epitope-specific scoring feature (iTEM), to assess ADA risk. One version of PIMA also integrates JanusMatrix, a Treg epitope prediction tool to identify putative immunomodulatory (regulatory) T cell epitopes in self-proteins. Using the JanusMatrix-adjusted version of PIMA in a logistic regression model with data from 48 cross-reactive immunological material (CRIM)-positive IOPD subjects, those with scores greater than 10 were 4-fold more likely to develop ADA (p<0.03) than those that had scores less than 10. We also confirmed the hypothesis that some GAA epitopes are immunomodulatory. Twenty-one epitopes were tested, of which four were determined to have an immunomodulatory effect on T effector response in vitro. The implementation of PIMA V3J on a secure-access website would allow clinicians to input the individual HLA DR haplotype of their IOPD patient and the GAA pathogenic variants associated with each GAA allele to calculate the patient's relative risk of developing ADA, enhancing clinical decision-making prior to initiating treatment with ERT. A better understanding of immunogenicity risk will allow the implementation of targeted immunomodulatory approaches in ERT-naïve settings, especially in CRIM-positive patients, which may in turn improve the overall clinical outcomes by minimizing the development of ADA. The PIMA approach may also be useful for other types of enzyme or factor replacement therapies.

Inflammation-Induced Lactate Leads to Rapid Loss of Hepatic Tissue-Resident NK Cells.

The liver harbors two main innate lymphoid cell (ILC) populations: conventional NK (cNK) cells and tissue-resident NK (trNK) cells. Using the MCMV model of infection, we find that, in contrast to liver cNK cells, trNK cells initially undergo a contraction phase followed by a recovery phase to homeostatic levels. The contraction is MCMV independent because a similar phenotype is observed following poly(I:C)/CpG or α-GalCer injection. The rapid contraction phase is due to apoptosis, whereas the recovery phase occurs via proliferation in situ. Interestingly, trNK cell apoptosis is not mediated by fratricide and not induced by liver lymphocytes or inflammatory cytokines. Instead, we find that trNK cell apoptosis is the consequence of an increased sensitivity to lactic acid. Mechanistic analysis indicates that trNK cell sensitivity to lactate is linked to impaired mitochondrial function. These findings underscore the distinctive properties of the liver-resident NK cell compartment.

Shp-2 is critical for ERK and metabolic engagement downstream of IL-15 receptor in NK cells.

The phosphatase Shp-2 was implicated in NK cell development and functions due to its interaction with NK inhibitory receptors, but its exact role in NK cells is still unclear. Here we show, using mice conditionally deficient for Shp-2 in the NK lineage, that NK cell development and responsiveness are largely unaffected. Instead, we find that Shp-2 serves mainly to enforce NK cell responses to activation by IL-15 and IL-2. Shp-2-deficient NK cells have reduced proliferation and survival when treated with high dose IL-15 or IL-2. Mechanistically, Shp-2 deficiency hampers acute IL-15 stimulation-induced raise in glycolytic and respiration rates, and causes a dramatic defect in ERK activation. Moreover, inhibition of the ERK and mTOR cascades largely phenocopies the defect observed in the absence of Shp-2. Together, our data reveal a critical function of Shp-2 as a molecular nexus bridging acute IL-15 signaling with downstream metabolic burst and NK cell expansion.

Activation of Syk protein tyrosine kinase in response to osmotic stress requires interaction with p21-activated protein kinase Pak2/gamma-PAK.

The p21-activated serine/threonine protein kinase Pak2/gamma-PAK and the nonreceptor type of protein tyrosine kinase Syk are known to be activated when the cells are exposed to osmotic stress. The purpose of the present study was to examine whether Pak2 and Syk functionally cooperate in cellular signaling. Cotransfection studies revealed that Pak2 associates with Syk in COS cells. The constitutively active form of Cdc42 increases the association of Pak2 with Syk. Pak2 coexpressed with an inactive form of Cdc42 or kinase-inactive Pak2 interacts to a lesser extent with Syk, suggesting that Pak2-Syk association is enhanced by Pak2 activation. Interaction with Pak2 enhances the intrinsic kinase activity of Syk. This is supported by in vitro studies showing that Pak2 phosphorylates and activates Syk. Treatment of cells with sorbitol to induce hyperosmolarity results in the translocation of Pak2 and Syk to the region surrounding the nucleus and in dramatic enhancement of their association. Furthermore, cotransfection of Pak2 and Syk leads to the activation of c-Jun N-terminal kinase (JNK) under hyperosmotic conditions. Pak2 short interfering RNA suppresses sorbitol-mediated activation of endogenous Syk and JNK, thus identifying a novel pathway for JNK activation by Cdc42. These results demonstrate that Pak2 and Syk positively cooperate to regulate cellular responses to stress.

Ptpn11 Deletion in CD4+ Cells Does Not Affect T Cell Development and Functions but Causes Cartilage Tumors in a T Cell-Independent Manner.

The ubiquitously expressed tyrosine phosphatase Src homology region 2 domain-containing phosphatase-2 (SHP-2, encoded by Ptpn11) is required for constitutive cellular processes including proliferation, differentiation, and the regulation of immune responses. During development and maturation, subsets of T cells express a variety of inhibitory receptors known to associate with phosphatases, which in turn, dephosphorylate key players of activating receptor signaling pathways. We hypothesized that SHP-2 deletion would have major effects on T cell development by altering the thresholds for activation, as well as positive and negative selection. Surprisingly, using mice conditionally deficient for SHP-2 in the T cell lineage, we show that the development of these lymphocytes is globally intact. In addition, our data demonstrate that SHP-2 absence does not compromise T cell effector functions, suggesting that SHP-2 is dispensable in these cells. Unexpectedly, in aging mice, Ptpn11 gene deletion driven by CD4 Cre recombinase leads to cartilage tumors in wrist bones in a T cell-independent manner. These tumors resemble miniature cartilaginous growth plates and contain CD4-lineage positive chondrocyte-like cells. Altogether these results indicate that SHP-2 is a cartilage tumor suppressor during aging.

Selective inhibition of Fcepsilon RI-mediated mast cell activation by a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus.

Ubiquitin-protein ligase Cbl-b negatively regulates high affinity IgE receptor (FcepsilonRI)-mediated degranulation and cytokine gene transcription in mast cells. In this study, we have examined the role of a truncated variant of Cbl-b related to the rat model of type 1 diabetes mellitus using the mast cell signaling model. Overexpression of the truncated Cbl-b that lacks the C-terminal region did not suppress the activation of proximal and distal signaling molecules leading to degranulation. FcepsilonRI-mediated tyrosine phosphorylation of Syk, Gab2, and phospholipase C-gamma1, and activation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAP kinase), and inhibitor of nuclear factor kappaB kinase (IKK), and generation of Rac1 are unaffected in cells overexpressing the truncated Cbl-b in the lipid raft. On the other hand, FcepsilonRI-mediated transcriptional activation of nuclear factor of activated T cells (NFAT), and transcription of interleukin-3 (IL-3) and IL-4 mRNA are inhibited by overexpression of the truncated variant of Cbl-b. This suppression parallels the re-compartmentalization of specific effector molecules in the lipid raft. These structural and functional analyses reveal the mechanism underlying the selective inhibition of cellular signaling by the truncated variant of Cbl-b related to insulin-dependent diabetes mellitus.

Role of type I interferon receptor signaling on NK cell development and functions.

Type I interferons (IFN) are unique cytokines transcribed from intronless genes. They have been extensively studied because of their anti-viral functions. The anti-viral effects of type I IFN are mediated in part by natural killer (NK) cells. However, the exact contribution of type I IFN on NK cell development, maturation and activation has been somewhat difficult to assess. In this study, we used a variety of approaches to define the consequences of the lack of type I interferon receptor (IFNAR) signaling on NK cells. Using IFNAR deficient mice, we found that type I IFN affect NK cell development at the pre-pro NK stage. We also found that systemic absence of IFNAR signaling impacts NK cell maturation with a significant increase in the CD27+CD11b+ double positive (DP) compartment in all organs. However, there is tissue specificity, and only in liver and bone marrow is the maturation defect strictly dependent on cell intrinsic IFNAR signaling. Finally, using adoptive transfer and mixed bone marrow approaches, we also show that cell intrinsic IFNAR signaling is not required for NK cell IFN-γ production in the context of MCMV infection. Taken together, our studies provide novel insights on how type I IFN receptor signaling regulates NK cell development and functions.

Expression of cDNAs in human Natural Killer cell lines by retroviral transduction.

Human NK-like cell lines are difficult to transfect using standard mammalian expression vectors and conventional transfection protocols, but they are susceptible to retroviral transduction as a means to introduce cDNAs. Our laboratory has exploited this technique to study a number of receptors in human NK cell lines. The method utilizes a bicistronic retroviral vector that co-expresses either drug resistance or enhanced green fluorescent protein (EGFP) in parallel with the gene of interest. After a single infection with recombinant retrovirus, transduced NK cells can be sorted for expression of EGFP or the transduced cell surface marker. Alternatively, cells expressing the transduced cDNAs can be selected for by treatment with neomycin, puromycin, or hygromycin. Using this method, the sorted/selected cells uniformly express the gene of interest and the expression is stable for many weeks of culture.

KIR2DL4 differentially signals downstream functions in human NK cells through distinct structural modules.

KIR2DL4 (2DL4) is a member of the killer cell Ig-like receptor (KIR) family in human NK cells. It can stimulate potent cytokine production and weak cytolytic activity in resting NK cells, but the mechanism for 2DL4-mediated signaling remains unclear. In this study we characterized the signaling pathways stimulated by 2DL4 engagement. In a human NK-like cell line, KHYG-1, cross-linking of 2DL4 activated MAPKs including JNK, ERK, and p38. Furthermore, 2DL4 cross-linking resulted in phosphorylation of IkappaB kinase beta (IKKbeta) and the phosphorylation and degradation of IkappaBalpha, which indicate activation of the classical NF-kappaB pathway. Engagement of 2DL4 was also shown to activate the transcription and translation of a variety of cytokine genes, including TNF-alpha, IFN-gamma, MIP1alpha, MIP1beta, and IL-8. Pharmacological inhibitors of JNK, MEK1/2 and p38, blocked IFN-gamma, IL-8, and MIP1alpha production, suggesting that MAPKs are regulating 2DL4-mediated cytokine production in a nonredundant manner. Activation of both p38 and ERK appear to be upstream of the stimulation of NF-kappaB. Mutation of a transmembrane arginine in 2DL4 to glycine (R/G mutant) abrogated FcepsilonRI-gamma association, as well as receptor-mediated cytolytic activity and calcium responses. Surprisingly, the R/G mutant still activated MAPKs and the NF-kappaB pathway and selectively stimulated the production of MIP1alpha, but not that of IFN-gamma or IL-8. In conclusion, we provide evidence that the activating functions of 2DL4 can be compartmentalized into two distinct structural modules: 1) through transmembrane association with FcepsilonRI-gamma; and 2) through another receptor domain independent of the transmembrane arginine.

Protein-tyrosine kinase Syk is required for pathogen engulfment in complement-mediated phagocytosis.

The protein tyrosine kinase Syk plays a central role in Fcgamma receptor-mediated phagocytosis in the adaptive immune system. We show here that Syk also plays an essential role in complement-mediated phagocytosis in innate immunity. Macrophage-like differentiated HL60 cells and C3bi-opsonized zymosan comprised the pathogen-phagocyte system. C3bi-opsonized zymosan particles promptly attached to the cells and were subsequently engulfed via complement receptor 3. During this process, Syk became tyrosine phosphorylated and accumulated around the nascent phagosomes. The transfer of Syk-siRNA or dominant-negative Syk (DN-Syk) into HL60 cells resulted in impaired phagocytosis. Quenching assays using fluorescent zymosan revealed that most of the attached zymosan particles were located inside parental HL60 cells, whereas few were ingested by the mutant cells. These data indicated that Syk is required for the engulfment of C3bi-opsonized zymosan. During C3bi-zymosan-induced phagocytosis, actin accumulation occurred around phagosomes and was followed by depolymerization, and further RhoA was activated together with tyrosine phosphorylation of Vav. These responses including the actin remodeling were suppressed in Syk-siRNA- or DN-Syk-expressing cells. Our results demonstrated that Syk plays an indispensable role in complement-mediated phagocytosis by regulating both actin dynamics and the RhoA activation pathway and that these functions of Syk lead to phagosome formation and pathogen engulfment.

Tyrosine phosphorylation of adaptor protein 3BP2 induces T cell receptor-mediated activation of transcription factor.

Molecular adaptors/scaffolds have indispensable roles in the activation of lymphocytes. In this report, we have demonstrated the role of tyrosine phosphorylation of an adaptor protein 3BP2 (c-Abl-SH3 domain binding protein-2, also known as SH3BP2) in T cell receptor (TCR)-mediated activation of transcription factor. Short interfering RNA for 3BP2 suppresses the expression level of endogenous 3BP2 and inhibits TCR-mediated activation of interleukin (IL)-2 promoter and nuclear factor of activated T cells (NFAT) element. Engagement of TCR induces tyrosine phosphorylation and lipid raft translocation of 3BP2. The overexpression studies reveal that substitution of 3BP2-Tyr(183), Tyr(446), or Arg(486) in the SH2 domain suppresses TCR-mediated activation of NFAT. Point mutations of 3BP2 cannot affect the translocation of 3BP2 into the lipid raft. Phosphorylation of Tyr(183) is required for the interaction with Vav1, the guanine nucleotide exchanging factor of Rac1. In fact, overexpression of dominant-negative form of Rac1 inhibits TCR-mediated activation of NFAT. Phosphorylation of Tyr(446) recruits the SH2 domain of Lck for the optimal activation of transcription factors. Furthermore, point mutation of Arg(486) in the 3BP2-SH2 domain that couples ZAP-70 to LAT dramatically reduces NFAT activation. These results suggest that the site-directed functions of 3BP2 induce the activation of transcription factors.

Point mutations of 3BP2 identified in human-inherited disease cherubism result in the loss of function.

Adaptor protein 3BP2 positively regulates the high affinity IgE receptor (FcepsilonRI)-mediated activation of degranulation in mast cells. Genetic study identified the point mutations of 3BP2 gene in human-inherited disease cherubism. The multiple cysts in cherubism lesion of jaw bones are filled with the activated osteoclasts and stromal cells, including mast cells. By over-expression study using rat basophilic leukaemia RBL-2H3 mast cells, we have analysed the effect of the point mutations on the function of 3BP2 protein, which plays a positive regulatory role on FcepsilonRI-mediated mast cell activation. Over-expression of 3BP2 mutants suppressed the antigen-induced degranulation and cytokine gene transcription. Antigen-induced phosphorylation of Vav1, activation of Rac1, extracellular signal regulated kinase (ERK), c-Jun N-terminal kinase (JNK), p38 mitogen activated protein kinase (MAPK), inhibitor of nuclear factor kappaB kinase (IKK) and nuclear factor of activated T cells (NFAT) were all impaired in the cells over-expressing the cherubism mutants of 3BP2. Furthermore, cherubism mutations of 3BP2 may abrogate the binding ability to interact with chaperone protein 14-3-3. These results demonstrate that over-expression of the mutant form of 3BP2 inhibits the antigen-induced mast cell activation. It suggests that point mutations of 3BP2 gene cause the dysfunction of 3BP2 in vivo.

Regulation of FcepsilonRI-mediated degranulation by an adaptor protein 3BP2 in rat basophilic leukemia RBL-2H3 cells.

Aggregation of high-affinity IgE receptor FcepsilonRI induces sequential activation of nonreceptor-type protein-tyrosine kinases and subsequent tyrosine phosphorylation of cellular proteins, leading to degranulation in mast cells. A hematopoietic cell-specific adaptor protein, 3BP2, that was originally identified as an Abl SH3-binding protein was rapidly tyrosine phosphorylated by the aggregation of FcepsilonRI on rat basophilic leukemia RBL-2H3 cells. Tyrosine phosphorylation of 3BP2 did not depend on calcium influx from external sources. To examine the role of 3BP2 in mast cells, we overexpressed the SH2 domain of 3BP2 in the RBL-2H3 cells. Overexpression of 3BP2-SH2 domain resulted in a suppression of antigen-induced degranulation as assessed by beta-hexosaminidase release. Even though overall tyrosine phosphorylation of cellular protein was not altered, antigen-mediated tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) and calcium mobilization were significantly suppressed in the cells overexpressing the 3BP2-SH2 domain. Furthermore, antigen stimulation induced the association of 3BP2-SH2 domain with LAT and other signaling molecule complexes in the RBL-2H3 cells. FcepsilonRI-mediated phosphorylation of JNK and ERK was not affected by the overexpression of 3BP2-SH2 domain. These data indicate that 3BP2 functions to positively regulate the FcepsilonRI-mediated tyrosine phosphorylation of PLC-gamma and thereby the signals leading to degranulation.

Negative regulation of FcepsilonRI-mediated mast cell activation by a ubiquitin-protein ligase Cbl-b.

Aggregation of the high-affinity immunoglobulin E (IgE) receptor (FcepsilonRI) on mast cells induces a number of biochemical events, including protein-tyrosine phosphorylation leading to degranulation and multiple cytokine gene transcription. Here, we have demonstrated that a second member of the Cbl family of ubiquitin-protein ligase Cbl-b translocates into the lipid raft after FcepsilonRI engagement. Overexpression of Cbl-b in the lipid raft inhibits FcepsilonRI-mediated degranulation and cytokine gene transcription through the distinct mechanism. A point mutation of Cys373 in the RING finger domain of Cbl-b abrogates the suppression of FcepsilonRI-mediated degranulation but not cytokine gene transcription. The antigen-induced tyrosine phosphorylation of FcepsilonRI, Syk, phospholipase C-gamma (PLC-gamma), activation of c-Jun N-terminal kinase (JNK), extracellular signal regulated kinase (ERK), inhibitor of nuclear factor kappaB kinase (IKK), and Ca++ influx were all suppressed in the cells overexpressing Cbl-b in the lipid raft. In particular, the expression amount of Gab2 protein and thereby its FcepsilonRI-mediated tyrosine phosphorylation were dramatically down-regulated by ubiquitin-protein ligase activity of Cbl-b. These results suggest that Cbl-b is a negative regulator of both Lyn-Syk-LAT and Gab2mediated complementary signaling pathways in FcepsilonRI-mediated mast cell activation.

Negative regulation of Lyn protein-tyrosine kinase by c-Cbl ubiquitin-protein ligase in Fc epsilon RI-mediated mast cell activation.

BACKGROUND: Recent studies have demonstrated that c-Cbl functions as a ubiquitin-protein ligase toward immune receptors and non-receptor protein-tyrosine kinase Syk by facilitating their ubiquitination and subsequent targeting to proteasomes. However, it was not clear whether Src family kinase Lyn is regulated by the Cbl family of ubiquitin-protein ligases. RESULTS: Aggregation of the high affinity IgE receptor (Fc epsilon RI) induces the rapid ubiquitination of Lyn in rat basophilic leukaemia RBL-2H3 cells. Treatment of cells with a proteasome inhibitor enhances the ubiquitination of Lyn. Stimulation of Fc epsilon RI results in the association of Lyn with c-Cbl and Cbl-b, both of which then become tyrosine phosphorylated. Co-transfection study shows that both c-Cbl and Cbl-b could induce the ubiquitination of activated Lyn in COS cells. Furthermore, over-expression of membrane-anchored form of c-Cbl inhibits the Fc epsilon RI-mediated degranulation and cytokine gene production in RBL-2H3 cells by the down-regulation of the kinase activity of Lyn through the enhanced ubiquitination. CONCLUSIONS: These results demonstrate that Lyn is down-regulated by c-Cbl-mediated ubiquitination and subsequent degradation in proteasome after Fc epsilon RI stimulation in mast cells. Targeting of c-Cbl in the lipid raft results in the inhibition of Fc epsilon RI-mediated mast cell activation.