Cbl Family Of Ubiquitin Ligases Research Articles

The Cbl family of ubiquitin ligases regulates FcεRI expression and mast cell activation

Antigen interaction with specific IgE bound to the high-affinity Fc receptor for IgE, constitutively expressed on the cell-surface of mast cells, generates signals that cause a shift in the resting state equilibrium of phosphorylation and dephosphorylation events that serves to maintain homeostasis. The outcome of this activated state is the release of a wide array of preformed and newly synthesized pro-inflammatory mediators. During the past few years, the existence of a negative feedback loop initiated upon FcεRI engagement has also been envisaged. This negative signal involves the coordinated action of adaptors, phosphatases and ubiquitin ligases that limits the intensity and duration of positive signals, thus modulating mast cell functions. Relevant to this, others and we have demonstrated that Cbl family proteins control the amplitude of FcεRI-generated signals by specific ubiquitin modification of activated receptor subunits and associated protein tyrosine kinases. In this article, we review advances in our understanding of the molecular mechanisms through which Cbl proteins regulate FcεRI expression and signaling.

Ligand-induced downregulation of TrkA is partly regulated through ubiquitination by Cbl

Nerve growth factor (NGF) binding to its receptor TrkA, which belongs to the family of receptor tyrosine kinases (RTKs), is known to induce its internalization, endosomal trafficking and subsequent lysosomal degradation. The Cbl family of ubiquitin ligases plays a major role in mediating ubiquitination and degradation of RTKs. However, it is not known whether Cbl participates in mediating ubiquitination of TrkA. Here we report that c-Cbl mediates ligand-induced ubiquitination and degradation of TrkA. TrkA ubiquitination and degradation required direct interactions between c-Cbl and phosphorylated TrkA. c-Cbl and ubiquitinated TrkA are found in a complex after NGF stimulation and are degraded in lysosomes. Taken together, our data demonstrate that c-Cbl can induce downregulation of NGF-TrkA complexes through ubiquitination and degradation of TrkA. Structured summary of protein interactionsTrkAphysically interacts with c-Cbl by anti bait coimmunoprecipitation(View interaction)c-Cblphysically interacts with TrkA by anti bait coimmunoprecipitation(View interaction)

Differential Role of Class 1A PI 3-Kinase Regulatory Subunits in Mast Cell Growth, Maturation and Survival.

Abstract 77Stem cell factor (SCF) mediated c-Kit receptor activation plays a pivotal role in mast cell growth, maturation and survival. However, the signaling events downstream from c-Kit are poorly understood. Mast cells express multiple regulatory subunits of class 1A PI 3-kinase including p85α, p85β, p50α, and p55α. While it is known that PI 3-kinase plays an essential role in mast cells; the precise mechanism by which these regulatory subunits impact specific mast cell functions including maturation, growth, and survival are not known. Using mice deficient in the expression of p85α or p85β or combination of both p85α/p55α/p50α as well as all four subunits we have examined the role of these subunits in mast cell functions. We show that loss of p85α subunit alone results in impaired bone marrow derived mast cell (BMMC) maturation, growth, and survival compared to wild-type (WT) controls, in spite of the continuous expression of p85β, p55α, and p50α subunits in these cells. Restoring the expression of p85α in p85α deficient mast cells restores the maturation and growth defects. To assess the contribution of p50α and p55α subunits, we generated mice using the Cre lox system that were deficient in the expression of all three subunits (i.e. p85α/p55α/p50α). Deficiency of p85α/p55α/p50α subunits in bone marrow cells completely blocked mast cell maturation and growth, suggesting an essential role for the smaller subunits p50 and p55 in addition to the full length form of p85. Curiously, over-expression of p50α in p85α deficient BMMCs only marginally rescued mast cell maturation and growth, suggesting that the full length form of p85α functions with specificity in regulating mast cell functions. Since the major difference between the shorter isoforms and the full length form of p85α is the absence of the amino terminal SH3 and BH domains, we generated two mutants of p85α lacking either the SH3 or the BH domain and expressed them in p85α−/− BMMCs. While both these mutants completely restored the maturation defect associated with p85α deficiency and showed normal binding to the c-Kit receptor upon SCF stimulation as well as to the p110 catalytic subunits; none of these mutants completely rescued SCF induced proliferation (50% and 70% respectively, n=3, p<0.004). Biochemically, lack of SCF induced growth rescue in p85α−/− BMMCs expressing p85αΔSH3 and p85αΔBH mutants was associated with a lack of rescue in the activation of Akt and Erk, but complete rescue in the activation of JNK (n=3). Consistently, while transplantation of p85α deficient bone marrow cells transduced with p85α into mast cell deficient Wsh mice resulted in complete restoration of gastrointestinal mast cells as well as mast cells in the stomach and spleen, p85αΔSH3 and p85αΔBH mutants restored mast cells only partially. These results indicate that other domains (SH3 and BH) of p85α are required for mast cell growth. In contrast to p85α, deficiency of p85β alone resulted in increased BMMC maturation, growth and survival compared to controls (1.2 fold, n=3, p<0.003). Consistently, over-expression of p85β in WT bone marrow cells resulted in a profound reduction in the maturation of mast cells as well as proliferation. We studied whether reduced maturation and proliferation due to the loss or over-expression of p85β was a result of altered c-Kit receptor internalization and degradation. Our results revealed significantly more c-Kit receptor internalization and degradation in p85β expressing cells compared to p85α expressing cells (2 fold, n=5, p<0.001). Since Cbl family of ubiquitin ligases are involved in the down-regulation of tyrosine kinase receptors, we analyzed whether c-Cbl is involved in p85β mediated c-Kit receptor internalization and degradation. Phosphorylation of c-Cbl and ubiquitination of c-Kit receptor was more in p85β expressing cells compared to p85 expressing cells (n=3). In conclusion, while the current dogma in the field of PI3Kinase signaling suggests that all regulatory subunits of PI3Kinase function in a similar manner; we provide genetic and biochemical evidence to suggest that p85 regulatory subunits differentially regulate growth and maturation of mast cells. Disclosures:Munugalavadla:Genentech: Employment, Patents & Royalties.

The role of cbl family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells

Background. Exosomes are nanometer-sized vesicles with immunomodulatory functions, which are released by a diverse range of living cells. Although recent studies have shown that tumor-derived exosomes can suppress the function of T cells, the molecular mechanisms are not well understood. In the present study, we investigated the role of the Casitas B lineage lymphoma (cbl) family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells. Materials and methods. By serial centrifugation and sucrose gradient ultracentrifugation, we isolated and purified the exosomes from gastric cancer SGC7901 cells, and identified them by electron microscopy and Western blotting. Cell apoptosis was detected using propidium iodide staining. Western blotting and RT-PCR was exploited to evaluate the expression of proteins and mRNA, respectively. Results. Gastric cancer exosomes induced Jurkat T cell apoptosis in a time- and dose-dependent manner and activated caspases 3, 8 and 9. The expression of Cbl-b and c-Cbl was up-regulated during exosome-induced apoptosis of cells. Meanwhile, exosomes induced ubiquitination of the p85 subunit of phosphoinositide 3-kinase (PI3K) and reduced downstream Akt activity. Inhibition of proteasome led to partial restoration of Akt activity and cell apoptosis. Discussion and Conclusions. The Cbl family of ubiquitin ligases might be involved in regulation of exosome-induced apoptosis of Jurkat T cells by increasing PI3K proteasome degradation, inactivation of PI3K/Akt signaling, thus mediating some effects of caspase activation.

The role of cbl family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells

The role of cbl family of ubiquitin ligases in gastric cancer exosome-induced apoptosis of Jurkat T cells

Negative regulation of lymphocyte development and function by the Cbl family of proteins

Negative regulation of intracellular signaling delivered by the antigen receptors and coreceptors plays an important role in lymphocyte development and activation. Recent data from our laboratory and others have identified the Cbl family of ubiquitin ligases as important negative regulators in both T-cell and B-cell antigen receptor and coreceptor signaling. We show that c-Cbl and Cbl-b, two members of the Cbl family of proteins, play a redundant role in establishing the major histocompatibility complex-dependent development of thymocytes and in thymic selection. They also control the activation threshold and CD28 costimulatory signaling in peripheral T cells. In B cells, c-Cbl and Cbl-b set the B-cell receptor signaling threshold critical for proper B-cell maturation and anergy induction. Biochemical studies indicate that the immune regulation by Cbl proteins correlate with their ubiquitin ligase function. Inactivation of Cbl-b also renders mice resistant to both transplanted and spontaneous tumors due to an enhanced anti-tumor immunity of CD8(+) T cells. These findings thus place Cbl proteins at the center of a complex immune network regulation and suggest that modulation of this signaling pathway may be beneficiary to the treatment of autoimmunity and cancer.

Up-regulation of the Cbl family of ubiquitin ligases is involved in ATRA and bufalin-induced cell adhesion but not cell differentiation

The Casitas B-lineage Lymphoma (Cbl) family of ubiquitin ligases is multifunctional proteins that play important roles in different cell signaling pathways. It has been reported that c-Cbl and Cbl-b mRNAs are up-regulated during TPA-induced U937 and HL-60 cell differentiation. But the mechanism of the up-regulation and the roles of the Cbl family of ubiquitin ligases still remain unclear. In the present study, we demonstrated that bufalin enhanced all- trans retinoic acid (ATRA) induced differentiation of HL-60 cells, accompanied by up-regulation of the Cbl family of ubiquitin ligases. CsA, an inhibitor of calcium mobilization, reversed this up-regulation. Pretreatment with CsA and PS-341 did not affect the expression of CD11b, but suppressed the percentage of adherent cells. Lipid raft localization of Cbl-b enhanced cell adhesion, while C-terminal deletion partially suppressed the effect. Moreover, the expression of the adhesion-related kinases Pyk2 and Paxillin was up-regulated in parallel with the increase of Cbl proteins. These results suggested that up-regulation of c-Cbl and Cbl-b was involved in the regulation of ATRA and bufalin-induced HL-60 cell adhesion rather than cell differentiation, which might be mediated by lipid raft localization, ubiquitin ligase activity and C-terminal structure of Cbl proteins. Meanwhile, up-regulation of proline-rich tyrosine kinase (Pyk2) and Paxillin might also be implicated in this regulation.

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

The overexpression or mutation of tyrosine kinases (TKs), such as the epidermal growth factor receptor (EGFR), can lead to the development of cancer. The most common mutation of the EGFR in glioblastomas is the deletion of exons 2-7 known as the EGFRvIII. This mutant receptor cannot bind EGF but, instead, is constitutively active. The Cbl family of ubiquitin ligases (Cbl, Cbl-b, and Cbl-c) targets the activated EGFR for degradation. As the EGFRvIII is transforming, we investigated whether it could be downregulated by the Cbl proteins. The overexpression of all three Cbl proteins resulted in the ubiquitination and degradation of the EGFRvIII. As with the wild-type EGFR, the TK-binding domain and the RING finger of Cbl-b are sufficient for the downregulation of the EGFRvIII. Also, we found that Cbl-b is recruited to the EGFRvIII and inhibits the transformation of NIH 3T3 cells by the EGFRvIII. Mutation of the Cbl-binding site (Y1045F) in the EGFRvIII inhibits its ubiquitination and downregulation by Cbl-b and enhances its ability to transform. Furthermore, the EGFR TK inhibitor, AG 1478, prevents the downregulation of the EGFRvIII by the Cbl proteins and antagonizes the ability of an immunotoxin directed against the EGFRvIII to kill cells expressing this receptor. In conclusion, the EGFRvIII does not transform by escaping regulation by Cbl proteins and this activation-induced downregulation of the EGFRvIII has an important role in mediating the toxicity of anti-EGFRvIII immunotoxins.

Biochemical Basis for the Requirement of Kinase Activity for Cbl-dependent Ubiquitinylation and Degradation of a Target Tyrosine Kinase

Members of the Cbl family of ubiquitin ligases have emerged as crucial negative regulators of tyrosine kinase signaling. These proteins preferentially interact with and target activated tyrosine kinases for ubiquitinylation, thereby facilitating the lysosomal sorting of receptor tyrosine kinases or proteasomal degradation of nonreceptor tyrosine kinases. Recent work has indicated a crucial role of the target kinase activity in Cbl-dependent ubiquitinylation and degradation, but the biochemical basis for this requirement is not understood. Here, we have used the Src-family kinase Fyn, a well characterized Cbl target, to address this issue. Using defined Fyn mutants, we demonstrate that the kinase activity of Fyn is crucial for its Cbl-dependent ubiquitinylation and degradation, but a low level of ubiquitinylation and degradation of kinase-inactive Fyn mutants was consistently observed. Mutational induction of an open conformation enhanced the susceptibility of kinase-active Fyn to Cbl but was insufficient to promote the ubiquitinylation and degradation of kinase-inactive Fyn. Notably, the Cbl-dependent degradation of Fyn did not require the Fyn-mediated phosphorylation of Cbl. Finally, we show that the major determinant of the susceptibility of Fyn protein to Cbl-dependent ubiquitinylation and degradation is the extent to which it physically associates with Cbl; kinase activity of Fyn serves as a critical determinant to promote its association with Cbl, which we demonstrate is mediated by multiple protein-protein interactions. Our results strongly suggest that promotion of association with Cbl is the primary mechanism by which the kinase activity of the targets of Cbl contributes to their susceptibility to Cbl.

Scaffolding of antigen receptors for immunogenic versus tolerogenic signaling.

Lymphocyte antigen receptors are responsible for inducing the opposite responses of immunity or tolerance. How the correct polarity of antigen receptor signaling is encoded has been an enduring enigma. Here we summarize recent advances defining key scaffolding molecules, CARMA1 (also known as CARD11) and the Cbl family of ubiquitin ligases, required for either immunogenic or tolerogenic signaling by antigen receptors. These scaffolding proteins may determine the polarity of response to antigen by promoting assembly around antigen receptors of competing multiprotein signal complexes: immunosomes versus tolerosomes. Each of the factors that influence immunogenicity or tolerogenicity--stage of lymphocyte differentiation, concurrent engagement of inhibitory or costimulatory receptors, extent of receptor crosslinking, and prior antigen experience--may be integrated in lymphocytes through their capacity to influence the probability of assembling immunosomes versus tolerosomes.

ErbB2 Degradation Mediated by the Co-chaperone Protein CHIP

ErbB2 overexpression contributes to the evolution of a substantial group of human cancers and signifies a poor clinical prognosis. Thus, down-regulation of ErbB2 signaling has emerged as a new anti-cancer strategy. Ubiquitinylation, mediated by the Cbl family of ubiquitin ligases, has emerged as a physiological mechanism of ErbB receptor down-regulation, and this mechanism appears to contribute to ErbB2 down-regulation induced by therapeutic anti-ErbB2 antibodies. Hsp90 inhibitory ansamycin antibiotics such as geldanamycin (GA) induce rapid ubiquitinylation and down-regulation of ErbB2. However, the ubiquitin ligase(s) involved has not been identified. Here, we show that ErbB2 serves as an in vitro substrate for the Hsp70/Hsp90-associated U-box ubiquitin ligase CHIP. Overexpression of wild type CHIP, but not its U-box mutant H260Q, induced ubiquitinylation and reduction in both cell surface and total levels of ectopically expressed or endogenous ErbB2 in vivo, and this effect was additive with that of 17-allylamino-geldanamycin (17-AAG). The CHIP U-box mutant H260Q reduced 17-AAG-induced ErbB2 ubiquitinylation. Wild type ErbB2 and a mutant incapable of association with Cbl (ErbB2 Y1112F) were equally sensitive to CHIP and 17-AAG, implying that Cbl does not play a major role in geldanamycin-induced ErbB2 down-regulation. Both endogenous and ectopically expressed CHIP and ErbB2 coimmunoprecipitated with each other, and this association was enhanced by 17-AAG. Notably, CHIP H260Q induced a dramatic elevation of ErbB2 association with Hsp70 and prevented the 17-AAG-induced dissociation of Hsp90. Our results demonstrate that ErbB2 is a target of CHIP ubiquitin ligase activity and suggest a role for CHIP E3 activity in controlling both the association of Hsp70/Hsp90 chaperones with ErbB2 and the down-regulation of ErbB2 induced by inhibitors of Hsp90.

CIN85 Participates in Cbl-b-mediated Down-regulation of Receptor Tyrosine Kinases

The Cbl family of ubiquitin ligases in mammals contains three members, Cbl, Cbl-b, and Cbl-3, that are involved in down-regulation of receptor tyrosine kinases (RTKs) by mediating receptor ubiquitination and degradation. More recently, a novel pathway has been identified whereby Cbl promotes internalization of EGF receptor via a CIN85/endophilin pathway that is functionally separable from the ubiquitin ligase activity of Cbl (1). Here we show that Cbl-b, but not Cbl-3, utilize the same mechanism to down-regulate multiple RTKs. CIN85 was shown to bind to the minimal binding domain identified in the carboxyl terminus of Cbl-b. Ligand-induced phosphorylation of Cbl-b further increased their interactions and led to a rapid and sustained recruitment of CIN85 in the complex with EGF or PDGF receptors. Inhibition of binding between CIN85 and Cbl-b was sufficient to impair Cbl-b-mediated internalization of EGF receptors, while being dispensable for Cbl-b-directed polyubiquitination of EGF receptors. Moreover, CIN85 and Cbl/Cbl-b were constitutively associated with activated PDGF, EGF, or c-Kit receptors in several tumor cell lines. Our data reveal a common pathway utilized by Cbl and Cbl-b that may have an important and redundant function in negative regulation of ligand-activated as well as oncogenically activated RTKs in vivo.

An essential role of ubiquitination in Cbl-mediated negative regulation of the Src-family kinase Fyn

The Cbl family of ubiquitin ligases function as negative regulators of activated receptor tyrosine kinases by facilitating their ubiquitination and subsequent lysosomal targeting. Here, we have investigated the role of Cbl ubiquitin ligase activity in the negative regulation of a non-receptor tyrosine kinase, the Src-family kinase Fyn. Using primary embryonic fibroblasts from Cbl(+/+) and Cbl(-/-) mice, we demonstrate that endogenous Cbl mediates the ubiquitination of Fyn and dictates the rate of Fyn turnover. By analyzing CHO-TS20 cells with a temperature-sensitive ubiquitin activating enzyme, we demonstrate that intact cellular ubiquitin machinery is required for Cbl-induced degradation of Fyn. Analyses of Cbl mutants, with mutations in or near the RING finger domain, in 293T cells revealed that the ubiquitin ligase activity of Cbl is essential for Cbl-induced degradation of Fyn by the proteasome pathway. Finally, use of a SRE-luciferase reporter demonstrated that Cbl-dependent negative regulation of Fyn function requires the region of Cbl that mediates the ubiquitin ligase activity. Given the conservation of structure between various Src-family kinases and the ability of Cbl to interact with multiple members of this family, Cbl-dependent ubiquitination could serve a general role to negatively regulate activated Src-family kinases.

The Cbl family of ubiquitin ligases: critical negative regulators of tyrosine kinase signaling in the immune system

The Cbl family of proteins are evolutionarily conserved negative regulators of activated tyrosine kinase-coupled receptors. Antigen receptors are prominent targets of negative regulation by the Cbl family members, Cbl and Cbl-b, which proteins function as ubiquitin ligases. Cbl and Cbl-b contain substrate recognition domains that interact specifically with activated protein tyrosine kinases of the Src and Syk/ZAP-70 families. Cbl-mediated ubiquitination of these kinases leads to their degradation, resulting in attenuation of receptor signals. Cbl may also control activation-induced monoubiquitination of antigen receptors, thus facilitating their delivery to lysosomes for subsequent degradation. Finally, the interactions of Cbl proteins with downstream targets of tyrosine kinases, such as PI-3-kinase and Vav, could provide an additional mechanism to attenuate receptor signaling. By targeting multiple components of antigen receptor signaling for degradation, the Cbl protein family provides a critical mechanism to ensure an appropriate immune response. The hyperresponsiveness of Cbl(-/-) and Cbl-b(-/-) lymphocytes and the autoimmune phenotype of Cbl-b(-/-) mice lend strong support for this proposal. The ability to control early receptor signals through regulated protein degradation provides a novel paradigm of immunoregulation.