Cbl Activation Research Articles

SYK ubiquitination by CBL E3 ligases restrains cross-presentation of dead cell-associated antigens by type 1 dendritic cells

Cross-presentation of dead cell-associated antigens by conventional dendritic cells type 1 (cDC1s) is critical for CD8+ Tcells response against many tumors and viral infections. It is facilitated by DNGR-1 (CLEC9A), an SYK-coupled cDC1 receptor that detects dead cell debris. Here, we report that DNGR-1 engagement leads to rapid activation of CBL and CBL-B E3 ligases to cause K63-linked ubiquitination of SYK and terminate signaling. Genetic deletion of CBL E3 ligases or charge-conserved mutation of target lysines within SYK abolishes SYK ubiquitination and results in enhanced DNGR-1-dependent antigen cross-presentation. We also find that cDC1 deficient in CBL E3 ligases are more efficient at cross-priming CD8+ Tcells to dead cell-associated antigens and promoting host resistance to tumors. Our findings reveal a role for CBL-dependent ubiquitination in limiting cross-presentation of dead cell-associated antigens and highlight an axis of negative regulation of cDC1 activity that could be exploited to increase anti-tumor immunity.

Abstract 2548: Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity

Abstract Casitas B-lineage lymphoma (CBL) is a family of RING-type E3 ligases that target proteins for degradation via the proteasomal pathway. CBL-b specifically functions as a negative effector of T cell activation by down regulating the T cell receptor, whereas c-CBL interacts with various receptor tyrosine kinases involved in cell signaling and activation, targeting them for degradation. Therefore, the CBL family regulates the biology of immune cells and targeting CBL-b or c-CBL is a potential therapeutic strategy for the treatment of cancer, infection, or autoimmune diseases. In this study, we used TR-FRET (Time-Resolved Fluorescence Resonance Energy Transfer) to uncover new mechanistic aspects of CBL activation. Our assays were designed and optimized using purified components (E1/E2/c-CBL or CBL-b E3 ligase and kinase substrate Tyro3 or Axl) to recreate the polyubiquitination cascade in vitro. The assays use a Europium cryptate-labeled Ubiquitin (donor) and a Cy5-labeled Ubiquitin (acceptor) to complete the TR-FRET pairing. Since both the TR-FRET donor and acceptor are incorporated into poly-ubiquitin chains formed on the E3 ligase, the technique measures only poly-ubiquitination and not mono-ubiquitination. This FRET-based assay is homogeneous, making it especially suitable for screening applications and real-time kinetics. Validation of the assay indicated that the presence of a kinase substrate (Tyro3 or Axl) activates CBL-b and c-CBL, which are known to display a similar mechanism of activation. Importantly, our assay design allowed us to show that known CBL-B inhibitor CBL-B-IN-1 is not strictly selective toward CBL-B but also affects c-CBL E3 ligase activity to similar extend since the IC50 of the compound for c-CBL and CBL-B differ by only 2-fold. Finally, it has been proposed that CBL-b phosphorylation at Y106, Y133, and Y363 by Tyro3 kinase is required for its activation. Indeed, in our assay, we observed that CBL-b mutation at Y363F decreases TYRO3 ubiquitination drastically. Surprisingly, phospho-mimetic mutation Y363E did not restore CBL-b-mediated ubiquitination of Tyro3, indicating that substrate binding close to Y363 is the predominant requirement for CBL activation. The weak E3 ligase activity of CBL-b Y363E mutant was insensitive to CBL-B-IN-1 treatment.In conclusion, our data indicates that no significant difference in the parameters of substrate ubiquitination exists between CBL-B and c-CBL, and that ubiquitination of receptor tyrosine kinase Tyro3 by either c-CBL or CBL-B depends on the kinase binding to CBL but not on the phosphorylation of CBL. Citation Format: Kasia Zientara-Rytter, Veronique Baron, Pavel Shashkin, Henry Zhu. Substrate binding, not phosphorylation, activates CBL poly-ubiquitination activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2548.

Termination of TCR-mediated activation signals is regulated by CrkII-dependent Cbl-mediated ubiquitination and degradation of C3G

Crk adaptor proteins are key players in signal transduction from multiple cell surface receptors, including the T cell antigen receptor (TCR). The involvement of CrkII in the early stages of T cell activation is well documented, but little is known about its role during the termination of the activation response. We substantiated findings showing that CrkII utilizes its SH3N and SH2 domains to constitutively associate with C3G and transiently with Cbl in resting and TCR/CD3-stimulated T cells, respectively. Association of CrkII with Cbl peaks within 1 min post-TCR/CD3 stimulation, and involves the formation of multiple CrkII-containing complexes of different molecular mass. Ubiquitination of C3G commences at ∼5 min post TCR/CD3 stimulation concomitantly with its degradation. This entire process conversely correlates with the levels of expression of CrkII and is dependent on the presence of the CrkII-bound Cbl protein. The data suggest that CrkII functions as a scaffold that brings Cbl into close proximity with C3G in TCR/CD3-stimulated T cells and that tyrosine phosphorylation and activation of Cbl promotes C3G ubiquitination and degradation. We suggest that this mechanism contributes to the termination of the TCR/CD3-induced activation signal and helps tune the length and intensity of T cell-mediated immune responses.

SLAP2 Adaptor Binding Disrupts c-CBL Autoinhibition to Activate Ubiquitin Ligase Function

CBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

Activation of the E3 ligase Cbl by Neutrophil Cathepsin G Impairs CXC chemokine receptor 4 Signaling in Cardiomyocyte Degeneration.

ObjectiveInflammatory cells and their proteases contribute to tissue repair at the site of inflammation. Although beneficial in the early stage, excessive inflammation leads to cell death and tissue damage. Increasing evidence demonstrates the role of inflammatory cells and their proteases in cardiac remodeling during the progression to heart failure; however the mechanisms by which inflammation contribute to myocyte death and subsequent alteration in the geometry and mechanical properties of the heart are still largely unknown. Therefore, we hypothesized that neutrophil‐derived protease Cat.G regulates CXC chemokine receptor (CXCR) 4 protein stability and turnover in myocytes through activation of Casitas b‐lineage lymphoma (c‐Cbl), an adaptor protein with an intrinsic E3 ubiquitin ligase activity.Methods and ResultsWe have previously shown that neutrophil serine protease Cat.G promotes cardiomyocyte death through c‐Cbl activation and interaction with tyrosine receptors that result in their ubiquitination and degradation. We next assessed whether Cbl activation was involved in CXCR4 signaling downregulation and myocyte apoptosis secondary to exposure of Cat.G. Acute stimulation with Cat.G (5 min to 30 min) induced CXCR4 phosphorylation along with its interaction with Cbl. However, chronic stimulation with Cat.G (1–4hrs) increased both CXCR4 ubiquitination and degradation. Inhibition of the ubiquitin proteasome system, with MG132 or lactacystin, or adenoviral expression of dominant negative mutant Cbl significantly reduced CXCR4 degradation induced by Cat.G. In contrast, adenoviral expression of wild type Cbl enhanced CXCR4 ubiquitination and degradation in response to Cat.G. Restoration of CXCR4 signaling, by adenoviral expression of CXCR4, attenuated CXCR4 downstream signaling downregulation, myofibril degeneration and myocyte apoptosis induced by Cat.G.ConclusionThese data support a model in which neutrophil derived serine proteases promote c‐Cbl interaction with CXCR4 protein, resulting in enhanced c‐Cbl‐mediated CXCR4 protein degradation, myofibril degradation and subsequent down‐regulation of myocyte survival signaling.

Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation.

Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved, depicting the protein at different stages of its activation cycle and thus providing mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins-may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than random noncancer mutations. We further tested the ability of these computational models, assessing the changes in CBL stability and its binding to ubiquitin-conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL.

Hydroxytyrosol, a product from olive oil, reduces colon cancer growth by enhancing epidermal growth factor receptor degradation.

We studied the effects and mechanism of 2-(3,4-dihydroxyphenil)ethanol (or hydroxytyrosol, HT), a polyphenol from extra virgin olive oil, investigating the regulation of epidermal growth factor receptor (EGFR) expression in colon tumour cells. We demonstrate that HT significantly downregulates EGFR expression in human colorectal adenocarcinoma cells HT-29, CaCo2, and WiDr, and in HT-29 xenografts. HT accelerates EGFR degradation by reducing its half-life. Specifically, HT induces EGFR ubiquitination that is mediated by phosphorylation at pY1045, the docking site for Cbl, thereby enabling receptor ubiquitination and degradation. Pretreatment with either the lysosomal inhibitor chloroquine, or the proteasomal inhibitor MG132 blocks HT-induced EGFR downregulation. In colon cancer cells, EGFR downregulation by HT is associated with reduced cell proliferation. Tumour growth and EGFR expression levels are also decreased by HT treatment in HT-29 xenograft. We conclude that HT downregulates EGFR expression via lysosomal and proteasomal degradation, activated by HT-induced EGFR phosphorylation at pY1045 and increased Cbl activity. Cbl activation induces, in turn, EGFR ubiquitination. Our results reveal a new mechanism for HT's antitumour effects that may be important for colon tumour prevention and treatment.

Testosterone stimulates glucose uptake and GLUT4 translocation through LKB1/AMPK signaling in 3T3-L1 adipocytes.

Decreases in serum testosterone concentrations in aging men are associated with metabolic disorders. Testosterone has been reported to increase GLUT4-dependent glucose uptake in skeletal muscle cells and cardiomyocytes. However, studies on glucose uptake occurring in response to testosterone stimulation in adipocytes are currently not available. This study was designed to determine the effects of testosterone on glucose uptake in adipocytes. Glucose uptake was assessed with 2-[(3)H] deoxyglucose in 3T3-L1 adipocytes. GLUT4 translocation was evaluated in plasma membrane (PM) sheets and PM fractions by immunofluorescence and immunoblotting, respectively. Activation of GLUT4 translocation-related protein kinases, including Akt, AMPK, LKB1, CaMKI, CaMKII, and Cbl was followed by immunoblotting. Expression levels of androgen receptor (AR) mRNA and AR translocation to the PM were assessed by real-time RT-PCR and immunoblotting, respectively. The results showed that both high-dose (100nM) testosterone and testosterone-BSA increased glucose uptake and GLUT4 translocation to the PM, independently of the intracellular AR. Testosterone and testosterone-BSA stimulated the phosphorylation of AMPK, LKB1, and CaMKII. The knockdown of LKB1 by siRNA attenuated testosterone- and testosterone-BSA-stimulated AMPK phosphorylation and glucose uptake. These results indicate that high-dose testosterone and testosterone-BSA increase GLUT4-dependent glucose uptake in 3T3-L1 adipocytes by inducing the LKB1/AMPK signaling pathway.

F604S exchange in FIP1L1-PDGFRA enhances FIP1L1-PDGFRA protein stability via SHP-2 and SRC: a novel mode of kinase inhibitor resistance.

FIP1L1-PDGFRA is a constitutively activated kinase described in chronic eosinophilic leukemia (CEL) and hypereosinophilic syndrome (HES). Imatinib is clinically active in FIP1L1-PDGFRA-positive diseases. Using in vitro screening to identify imatinib-resistant mutations, we frequently detected a Phe to Ser exchange at position 604 (F604S) of FIP1L1-PDGFRA alone or in combination with other exchanges. Surprisingly, FIP1L1-PDGFRA/F604S did not increase the biochemical or cellular IC50 value of imatinib when compared with unmutated FIP1L1-PDGFRA. However, FIP1L1-PDGFRA/F604S more efficiently induced growth factor independence in cell lines and primary mouse bone marrow cells. Pulse chase analysis revealed that the F604S exchange strongly stabilized FIP1L1-PDGFRA/F604S. The F604S mutation creates a binding site for the phosphatase domain of SHP-2, leading to lower autophosphorylation of FIP1L1-PDGFRA/F604S. This is associated with a reduced activation of SRC and CBL by FIP1L1-PDGFRA/F604S compared with the unmutated oncogene. As SRC inhibition and knockdown resulted in FIP1L1-PDGFRA stabilization, this explains the extended half-life of FIP1L1-PDGFRA/F604S. Interestingly, FIP1L1-PDGFRA/L629P, a recently identified mutation in an imatinib-resistant CEL patient, also showed protein stabilization similar to that observed with FIP1L1-PDGFRA/F604S. Therefore, resistance mutations in FIP1L1-PDGFRA that do not interfere with drug binding but rather increase target protein stability seem to be one of the drug-resistance mechanisms in FIP1L1-PDGFRA-positive disease.

PTEN Is Essential for Normal Cytokine Signaling and Oncogenic Transformation of Pre-B Cells

Background: PTEN is a negative regulator of PI3K-AKT signaling and a potent tumor suppressor throughout all main types of human cancer. Recent work demonstrated that loss of PTEN in T cell acute lymphoblastic leukemia (ALL) confers glucocorticoid resistance. To develop a model for glucocorticoid resistance in pre-B ALL, we studied Cre-mediated deletion of Pten.Results: Surprisingly, loss of one or both alleles of Pten caused rapid cell death in pre-B ALL cells and was sufficient to cure transplant recipient mice from leukemia. Furthermore, deletion of Pten in pre-B cells disabled oncogenic transformation by BCR-ABL1 and NRASG12D oncogenes. A reanalysis of genetic lesions of PTEN in human cancer revealed a high frequency across all main subtypes of mature B cell lymphoma, myeloid leukemias and T cell lineage leukemia/lymphoma (11.3% in 1,697 samples). Strikingly, however, lesions of PTEN were not detected in any of 670 pre-B ALL patient samples. Whereas high expression levels of PTEN predict favorable outcomes for patients with T-ALL, pre-B ALL patients with high PTEN expression levels at the time of diagnosis have particularly poor clinical outcomes (P=0.01). Studying the unexpected role of PTEN in B cell lineage ALL, we developed a genetic model for Cre-mediated deletion of Pten in mouse pre-B ALL cells carrying BCR-ABL1 or NRASG12D oncogenes. Deletion of one or both alleles of Pten reduced the colony forming ability (p < 10E-06) and proliferation rate (p < 10E-04) of pre-B ALL cells while strongly increasing cellular senescence (P < 10E-03). Since immediate effects of Pten deletion include hyperactivation of PI3K-AKT signaling, we hypothesized that small molecule inhibitors that limit PI3K-AKT pathway activity may rescue the pre-B ALL cells from cell death when Pten is inducibly deleted. Therefore, we tested the effect of AKT and PI3K inhibitors AZD5363 and BKM120 on Pten deleted cells and confirmed that chemical inhibition of PI3K-AKT pathway indeed rescued cell death of pre-B ALL cells upon Pten deletion. Also, global tyrosine phosphoprofiling by quantitative mass spectrometry revealed that Cre-mediated deletion of Pten strongly induced activation of CBL, a ubiquitin ligase that induces degradation of the IL7 receptor in pre-B cells and oncogenic tyrosine kinases in leukemia. Genetic experiments confirmed that deletion of Pten and hyperactivation of AKT engaged a novel, CBL-dependent pathway of negative feedback inhibition of STAT5 and tyrosine kinase signaling. Consistent with these finding, Reverse Phase Protein Array (RPPA) measurements for 155 newly diagnosed cases of pre-B ALL revealed a strong negative correlation between STAT5 protein levels and phospho-ATKT308 (P=0.0021) and phospho-AKTS473 (P=0.0005).Unlike pre-B ALL, deletion of Pten in CML like cells showed no toxicity. To further study the lineage specifity of Pten dependency we used a doxycycline-inducible CEBPa construct to differentiate pre-B cell ALL into myeloid lineage leukemia cells. Similar to our finding in CML like cells, unlike the pre-B ALL, myeloid lineage Leukemia showed no dependency on Pten and the deletion of Ptencaused no significant change in the cells viability. Studying the basal level of PTEN, pre-B cells showed ~40-fold higher expression levels than myeloid lineage cells. Consistently, genetic knockdown of PTEN using two different shRNA was only toxic in patient-derived pre-B but not myeloid leukemia cells.Clinical relevance: To find out the clinical potential of our finding, we tested the effects of SF1670 a small molecule inhibitor of PTEN on a panel of pre-B ALL and CML. Interestingly, the pre-B ALL showed more sensitivity to PTEN inhibition compared to CML cells.Conclusion: Our analysis uncovered an unexpected vulnerability of human Pre-B ALL cells toward PTEN inhibition and hyperactivation of the PI3K-AKT pathway, which provides an explanation why activating mutations of this pathway are negatively selected in human pre-B ALL. Since short (few hours) and profound inhibition of Pten is sufficient to commit human pre-B ALL cells to cell death, transient inhibition of this pathway or transient hyperactivation of PI3K-AKT may be a promising strategy to target multi-drug resistant human ALL. DisclosuresNo relevant conflicts of interest to declare.

Berberine Inhibits Proliferation and Down-Regulates Epidermal Growth Factor Receptor through Activation of Cbl in Colon Tumor Cells

Berberine, an isoquinoline alkaloid, is an active component of Ranunculaceae and Papaveraceae plant families. Berberine has been found to suppress growth of several tumor cell lines in vitro through the cell-type-dependent mechanism. Expression and activation of epidermal growth factor receptor (EGFR) is increased in colonic precancerous lesions and tumours, thus EGFR is considered a tumour promoter. The aim of this study was to investigate the effects and mechanisms of berberine on regulation of EGFR activity and proliferation in colonic tumor cell lines and in vivo. We reported that berberine significantly inhibited basal level and EGF-stimulated EGFR activation and proliferation in the immorto Min mouse colonic epithelial (IMCE) cells carrying the APC min mutation and human colonic carcinoma cell line, HT-29 cells. Berberine acted to inhibit proliferation through inducing G1/S and G2/M cell cycle arrest, which correlated with regulation of the checkpoint protein expression. In this study, we also showed that berberine stimulated ubiquitin ligase Cbl activation and Cbl's interaction with EGFR, and EGFR ubiquitinylation and down-regulation in these two cell lines in the presence or absence of EGF treatment. Knock-down Cbl expression blocked the effects of berberine on down-regulation of EGFR and inhibition of proliferation. Furthermore, berberine suppressed tumor growth in the HT-29 cell xenograft model. Cell proliferation and EGFR expression level was decreased by berberine treatment in this xenograft model and in colon epithelial cells of APC min/+ mice. Taken together, these data indicate that berberine enhances Cbl activity, resulting in down-regulation of EGFR expression and inhibition of proliferation in colon tumor cells.

Abstract 214: Cbl Inhibition Improves Cardiac Function and Survival in Response to Myocardial Ischemia

Introduction and Hypothesis: Proteasome degradation of ubiquitin-targeted proteins plays an important role in the development of cardiomyopathy and heart failure. The mechanisms that control proteasome function and protein ubiquitination and turnover remain poorly defined. The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets tyrosine kinases, resulting in their ubiquitination and down-regulation. Although Cbl is a crucial signaling molecule in many cells, the role of Cbl in the heart is unknown. Methods and Results: We show increased Cbl expression in human failing hearts and in response to pathological stress stimuli in mice. Cbl deficient mice demonstrated a more robust functional recovery after ischemia reperfusion injury, as well as significantly reduced myocyte apoptosis and epidermal growth factor receptor and focal adhesion (FA) protein ubiquitination and downregulation. Cbl Knockdown in-vitro protected cardiomyocytes against H2O2-induced apoptosis. Cbl deficient mice also show greater functional recovery after myocardial ischemia. This cardioprotective effect of Cbl deletion can be attributed, at least in part, to enhanced neoangiogenesis in the infarcted region via upregulation of vascular endothelial growth factor-a. Conclusions: Cbl activation regulates myocyte apoptosis and angiogenesis and contributes to adverse cardiac remodeling post-MI. These findings point to Cbl as a potential therapeutic target for the maintenance of cardiac function and remodeling post-MI.

I787 provides signals for c-Kit receptor internalization and functionality that control mast cell survival and development

AbstractMast cell (MC) differentiation, survival, and activation are controlled by the membrane tyrosine kinase c-Kit upon interaction with stem cell factor (SCF). Here we describe a single point mutation induced by N-ethyl-N-nitrosurea (ENU) mutagenesis in C57BL/6J mice—an A to T transversion at position 2388 (exon 17) of the c-Kit gene, resulting in the isoleucine 787 substitution by phenylalanine (787F), and analyze the consequences of this mutation for ligand binding, signaling, and MC development. The Kit787F/787F mice carrying the single amino acid exchange of c-Kit lacks both mucosal and connective tissue-type MCs. In bone marrow-derived mast cells (BMMCs), the 787F mutation does not affect SCF binding and c-Kit receptor shedding, but strongly impairs SCF-induced cytokine production, degranulation enhancement, and apoptosis rescue. Interestingly, c-Kit downstream signaling in 787F BMMCs is normally initiated (Erk1/2 and p38 activation as well as c-Kit autophosphorylation) but fails to be sustained thereafter. In addition, 787F c-Kit does not efficiently mediate Cbl activation, leading to the absence of subsequent receptor ubiquitination and impaired c-Kit internalization. Thus, I787 provides nonredundant signals for c-Kit internalization and functionality.

Insulin-mimetic action of conglutin-γ, a lupin seed protein, in mouse myoblasts

Background and aims Lupin seed is referred to as an antidiabetic product in traditional medicine. Conglutin-γ, a lupin seed glycoprotein, was found to cause a significant plasma glucose reduction when orally administered to rats in glucose overload trials. Conglutin-γ was identified as being responsible for the claimed biological activity, and the aim of this work was to envisage its hypothetical insulin-mimetic cellular mechanism of action. Insulin is responsible for proteosynthesis control through IRS/AKT/P70S6k/PHAS1 pathways modulation, glucose homeostasis through PKC/Flotillin-2/caveolin-3/Cbl activation and muscle differentiation/hypertrophy via muscle-specific MHC gene transcription control. Methods and results To assess whether conglutin-γ modulates the same insulin-activated kinases, myoblastic C2C12 cells were incubated after 72 h of differentiation with 100 nM insulin or 0.5 mg/mL (∼10 μM) conglutin-γ. Metformin-stimulated cells were used as a positive control. The effect on the above mentioned pathways was evaluated after 5, 10, 20 and 30 min. In the control cells medium insulin, conglutin-γ and metformin were not added. We demonstrated that insulin or conglutin-γ cell stimulation resulted in the persistent activation of protein synthetic pathway kinases and increased glucose transport, glut4 translocation and muscle-specific gene transcription regulation. Conclusions Our results indicate that conglutin-γ may regulate muscle energy metabolism, protein synthesis and MHC gene transcription through the modulation of the same insulin signalling pathway, suggesting the potential therapeutic use of this natural legume protein in the treatment of diabetes and other insulin-resistant conditions, as well as the potential conglutin-γ influence on muscle cells differentiation and regulation of muscle growth.

Abstract 1457: Cbl, E3 Ubiquitin Ligase, is a Regulator of Focal Adhesion Protein Proteasomal Degradation during Cardiomyocyte Anoikis

Proteasome degradation of ubiquitin-targeted proteins is an important mechanism that negatively controls activated signaling pathways. The proto-oncogene Casitas b-lineage lymphoma (Cbl) is an adaptor protein with an intrinsic E3 ubiquitin ligase activity that targets receptor tyrosine kinase signaling, resulting in their ubiquitination and down-regulation. We have shown previously that neutrophil derived protease cathepsin G (Cat.G) induced focal adhesion (FA) protein degradation and myocyte apoptosis by anoikis. We hypothesized that Cbl is involved in myocyte apoptosis in response to Cat.G through ubiquitination and downregulation of FA proteins. Results : Immunoprecitpitation and western blot studies showed increased Cbl tyrosine phosphorylation in response to Cat.G that was associated with its interaction with FA proteins, FAK and paxillin. This led to an increase in FAK and Paxillin ubiquitination and degradation at 2 hrs after Cat.G addition. Inhibition of the ubiquitin proteasome system, with MG132 or lactacystin, or adenoviral expression of dominant negative mutant Cbl significantly reduced FAK and paxillin degradation. In contrast, adenoviral expression of wild type Cbl enhanced FAK and paxillin ubiquitination and degradation in response to Cat.G. Interestingly, Cbl activation was involved in Cat.G-induced myocyte apoptosis as overexpression of dominant negative Cbl significantly reduced caspase-3 activation and DNA fragmentation induced by Cat.G. Concomitant with these changes in vitro, heart tissue samples from patients with ischemic or dilated cardiomyopathy showed a marked increase in Cbl accumulation and FAK and paxillin degradation compared to heart controls. Conclusion : These studies show that Cbl abundance is increased in human ischemic and dilated cardiomyopathy and that activation of Cbl increases FA protein degradation and myocyte apoptosis. These results indicate that Cbl is a positive regulator of FA protein degradation and myocyte apoptosis and may play an important role in mediating the effect of neutrophil derived proteases during the progression of congestive heart failure.

KIT oncoprotein interactions in gastrointestinal stromal tumors: therapeutic relevance

Most gastrointestinal stromal tumors (GISTs) express oncogenic and constitutively active forms of the KIT or platelet-derived growth factor receptor alpha (PDGFRA) receptor tyrosine kinase proteins, and these kinase oncoproteins serve as targets for effective therapies. Given that mutant KIT oncoproteins serve crucial transforming roles in GISTs, we evaluated interactions with the KIT oncoproteins and determined signaling pathways that are dependent on KIT oncogenic activation in GISTs. Tyrosine-phosphorylated KIT oncoproteins interacted with PDGFRA, PDGFRB, phosphatidylinositol 3-kinase (PI3-K) and PKCtheta in GIST cells, and these interactions were abolished by KIT inhibition with imatinib or PKC412 or KIT RNAi. Notably, tyrosine-phosphorylated PDGFRA was prominent in frozen GIST tumors expressing KIT oncoproteins, suggesting that KIT-mediated PDGFRA phosphorylation is an efficient and biologically consequential mechanism in GISTs. Activated signaling intermediates were identified by immunoaffinity purification of tyrosine-phosphorylated proteins in GIST cells before and after treatment with KIT inhibitors, and these analyses show that GRB2, SHC, CBL and MAPK activation are largely KIT dependent in GISTs, whereas PI3-K, STAT1 and STAT3 activation are partially KIT dependent. In addition, we found that phosphorylation of several tyrosine kinase proteins - including JAK1 and EPHA4 - did not depend on KIT activation. Likewise, paxillin activation was independent of the KIT oncogenic signal. These studies identify signaling pathways that can provide both KIT-dependent and KIT-independent therapeutic synergies in GIST, and thereby highlight clinical strategies that might consolidate GIST therapeutic response to KIT/PDGFRA inhibition.

P38 kinase regulates epidermal growth factor receptor downregulation and cellular migration.

Internalization and proteolytic degradation of epidermal growth factor (EGF) receptor (R) following ligand binding is an important mechanism for regulating EGF-stimulated signals. Using pharmacological and RNA interference inhibition of p38 mitogen-activated protein kinase, we show that p38 is required for efficient EGF-induced EGFR destruction but not internalization. In the absence of p38 activity, EGF fails to stimulate the ubiquitin ligase Cbl or ubiquitinylation of EGFR, and internalized EGFR accumulates in intracellular vesicles containing caveolin-1. These effects are accompanied by loss of EGFR phosphorylation on Y1045, a phosphorylation site required for Cbl activation. Furthermore, similar to cells treated with p38 inhibitors, intestinal epithelial cells expressing Y1045F EGFR mutants show increased proliferation but not migration in response to EGF, thus uncoupling these biological responses. Together these data position p38 as a modulator of ligand-stimulated EGFR processing and demonstrate that this processing has a profound impact on the cellular outcome of EGFR signaling.

Direct binding of Cbl to Tyr568 and Tyr936 of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation

The ubiquitin E3 ligase Cbl has been shown to negatively regulate tyrosine kinase receptors, including the stem cell factor receptor/c-Kit. Impaired recruitment of Cbl to c-Kit results in a deregulated positive signalling that eventually can contribute to carcinogenesis. Here, we present results showing that Cbl is activated by the SFKs (Src family kinases) and recruited to c-Kit in order to trigger receptor ubiquitination. We demonstrate that phosphorylated Tyr568 and Tyr936 in c-Kit are involved in direct binding and activation of Cbl and that binding of the TKB domain (tyrosine kinase binding domain) of Cbl to c-Kit is specified by the presence of an isoleucine or leucine residue in position +3 to the phosphorylated tyrosine residue on c-Kit. Apart from the direct association between Cbl and c-Kit, we show that phosphorylation of Cbl by SFK members is required for activation of Cbl to occur. Moreover, we demonstrate that Cbl mediates monoubiquitination of c-Kit and that the receptor is subsequently targeted for lysosomal degradation. Taken together, our findings reveal novel insights into the mechanisms by which Cbl negatively regulates c-Kit-mediated signalling.

Insulin Stimulates the Activation of the Casitas b‐lymphoma (Cbl) Protein in Striated Skeletal Muscle

Insulin activates glucose transport in adipose and striated muscle cells by stimulating the recruitment of intracellular vesicles containing the glucose transporter GLUT4 to the plasma membrane. In adipocytes two insulin signaling cascades have been implicated in this process: the insulin receptor substrate/phosphatidyl inositol 3 kinase-dependent pathway and the proto-oncogene Casitas b- lymphoma (c-Cbl) dependent pathway. The objective of our study was to examine whether the Cbl-dependent pathway is activated by insulin in striated muscle cells. The components of the Cbl pathway were expressed in both cultured L6 myotubes and in muscle tissue in vivo. Insulin induced the phosphosphorylation of Cbl in the tyrosine residues and the recruitment of the protein to the lipid raft microdomains of the plasma membrane in L6 cells. Palmitate-induced insulin resistance inhibited the phosphorylation of Cbl but not Cbl expression in L6 cells. In vivo, insulin administration induced the phosphorylation of Cbl in adipose, heart, and soleus muscle, but no phosphorylation was observed in hindlimb muscle. In agreement with this, activation of the downstream GTP binding protein TC10 was observed in heart and adipose tissue, following 5 minutes of insulin treatment. TC10 was not activated in hindlimb muscle. Streptozotocin-induced insulin deficiency severely downregulated Cbl and CAP expression in heart and moderate reductions in Cbl protein levels were also observed in adipose tissue and soleus muscle. Overall our findings suggest that insulin induces the activation of Cbl in muscle cells and that this signaling pathway is inhibited in insulin-resistant and insulin-deficient states. The regulation of this pathway in cardiac cells may be important for the regulation of glucose uptake in this tissue during normal and pathological conditions. Funding: DAREF and ADA.

Activation of the Cbl insulin signaling pathway in cardiac muscle; Dysregulation in obesity and diabetes

In adipocytes, the Cbl/CAP dependent signaling pathway has been involved in regulating insulin-stimulated glucose uptake. We investigated activation of Cbl and its downstream effector TC10 in cardiac and skeletal muscle of Balb/C mice. Insulin administration resulted in Cbl phosphorylation in cardiac, skeletal muscle, and adipose tissue. Subsequent TC10 activation was detected only in heart and adipose tissue. c-Cbl and CAP gene expression was significantly reduced in the heart tissue of streptozotocin-induced diabetic animals, whereas no change was observed for other components of the pathway. No changes in Cbl expression were detected in hindlimb muscle. In leptin −/− obese mice Cbl expression in heart and adipose tissue was maintained, although insulin-mediated Cbl phosphorylation and subsequent TC10 activation were significantly reduced. In conclusion, our data demonstrate that Cbl/CAP/TC10 insulin signaling pathway is active in cardiac muscle and impaired during obesity and insulin deficiency.