E3 Ubiquitin Ligase c-Cbl Research Articles

PSMD9 promotes the malignant progression of hepatocellular carcinoma by interacting with c-Cbl to activate EGFR signaling and recycling

BackgroundMounting evidences shows that the ubiquitin‒proteasome pathway plays a pivotal role in tumor progression. The expression of 26S proteasome non-ATPase regulatory subunit 9 (PSMD9) is correlated with recurrence and radiotherapy resistance in several tumor types. However, the role and mechanism of PSMD9 in hepatocellular carcinoma (HCC) progression remain largely unclear.MethodsPSMD9 was identified as a prognosis-related biomarker for HCC based on analysis of clinical characteristics and RNA-seq data from The Cancer Genome Atlas (TCGA), Gene Expression Omnibus (GEO) and the JP Project of the International Cancer Genome Consortium (ICGC-LIRI-JP). PSMD9 expression was analyzed in cancer tissues and adjacent noncancerous tissues via immunohistochemistry and Western blotting. Multiple in vivo and in vitro experimental techniques (such as CCK-8, colony formation, EdU, and Transwell assays; flow cytometry; Western blotting; quantitative RT-PCR; Coimmunoprecipitation assay and immunofluorescence confocal imaging) were used to assess the functions of PSMD9 in the pathogenesis of HCC.ResultsWe found that the expression of PSMD9 was upregulated and associated with a poor prognosis in HCC patients. PSMD9 promoted HCC cell proliferation, migration, invasion and metastasis. Knockdown of PSMD9 significantly inhibited HCC cell proliferation by inducing G1/S cell cycle arrest and apoptosis. Mechanistically, we demonstrated that PSMD9 promoted HCC cell proliferation and metastasis via direct interaction with the E3 ubiquitin ligase c-Cbl, suppresses EGFR ubiquitination, influenced EGFR endosomal trafficking and degradation and subsequently activated ERK1/2 and Akt signaling. In addition, we showed that PSMD9 knockdown sensitized HCC cells to the tyrosine kinase inhibitor erlotinib in vitro and in vivo.ConclusionsCollectively, our results indicate that PSMD9 drives HCC progression and erlotinib resistance by suppressing c-Cbl mediated EGFR ubiquitination and therefore can be a potential therapeutic target for HCC.

Abstract 4042: Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade

Abstract INTRODUCTION: Herein, we describe a series of experiments to define the unique biology of dual Cbl-b/c-Cbl (Cbl) inhibition in amplifying T cell activation and anti-tumor responses. Through their E3 ubiquitin ligase activity, Cbl-b and c-Cbl have been shown to negatively regulate multiple signaling proteins downstream of the T cell receptor (TCR), suggesting that inhibition may yield enhanced anti-tumor T cell activity. Indeed, using a dual Cbl-b/c-Cbl small molecule inhibitor, we demonstrate that Cbl blockade enhances T cell activity under standard and sub-optimal stimulation conditions. Moreover, Cbl inhibition increases mouse syngeneic tumor control alone or in combination with α-PD-1, as described below. METHODS: Human CD8+ T cells were isolated from healthy human blood and activated using α-CD3 or α-CD3/CD28 stimulation. Mouse OT-I splenocytes were stimulated with SIINFEKL or lower affinity ovalbumin peptides and cytokine production was assessed. For tumor studies, mice were inoculated with syngeneic cancer cells and were treated with Cbl inhibitor (30 mg/kg, QD) +/- α-PD-1 (10 mg/kg, Q5D) starting at a tumor volume of 75 mm3. RESULTS: Human CD8+ T cells, stimulated in the presence of a Cbl inhibitor, demonstrated a concentration-dependent increase in activation hallmarks including cytokine secretion (IL-2, IFN-γ and granzyme B), proliferation, and the presence of highly polyfunctional cells. Furthermore, using serially stimulated CD8+ T cells with reduced overall activity, Cbl inhibition showed a marked enhancement of IFN-γ production and cytotoxicity compared to controls upon re-stimulation. We then used an antigen-specific T cell assay where OT-I splenocytes were stimulated by peptides with optimal or reduced TCR affinities, Cbl inhibition produced significantly higher IFN-γ levels with all peptides relative to controls demonstrating that Cbl inhibition can increase the response of T cells to sub-optimal stimulation. Using mouse CT26 and MC38 tumor models, Cbl inhibition was able to significantly enhance tumor control in combination with α-PD-1 and as a monotherapy in CT26 tumors. Notably, a greater number of tumor antigen-specific cytotoxic T cells were found in Cbl inhibitor-treated tumors. Higher expression levels of the co-stimulatory molecule CD226 and the TCR in these anti-tumor cells were also observed, demonstrating the unique biology of Cbl inhibition in enhancing anti-tumor responses. Finally, Cbl inhibition had no effect on the growth or survival of cancer cells in vitro consistent with the notion that enhanced tumor control was driven by immune activity. CONCLUSIONS: Altogether, these data demonstrate that pharmacologic Cbl inhibition robustly enhances T cell activity in vitro and in vivo and provide a mechanistic rationale for targeting Cbl-b/c-Cbl to amplify anti-tumor immune responses. Citation Format: Livia Yamashiro, Sachie Marubayashi, Rameshwari Rayaji, Anudari Battsooj, Karim Mrouj, Hema Singh, Yue Tong Lee, Ada Chen, Lunda Shen, Artur Mailyan, Kai Yu, Masha Podunavac, Julie Yu, Dongdong Liu, Clayton Hardman, Johnny Yin, Srikanth Gangam, Ken Lawson, Manmohan Leleti, Matthew Walters, Daniel DiRenzo. Cbl inhibition increases the response of cytotoxic T cells to sub-optimal stimulation and drives enhanced anti-tumor responses alone or in combination with PD-1 blockade [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4042.

Knockout of c-Cbl/Cbl-b slows c-Met trafficking resulting in enhanced signaling in corneal epithelial cells

In many cell types, the E3 ubiquitin ligases c-Cbl and Cbl-b induce ligand-dependent ubiquitylation of the HGF-stimulated c-Met receptor and target it for lysosomal degradation. This study determines whether c-Cbl/Cbl-b are negative regulators of c-Met in the corneal epithelium (CE), and if their inhibition can augment c-Met-mediated CE homeostasis. Immortalized human corneal epithelial cells were transfected with Cas9 only (Cas9, control cells) or with Cas9 and c-Cbl/Cbl-b guide RNAs to knockout each gene singularly (-c-Cbl or -Cbl-b cells) or both genes (DKO cells) and monitored for their responses to HGF. Cells were assessed for ligand-dependent c-Met ubiquitylation via immunoprecipitation, magnitude and duration of c-Met receptor signaling via immunoblot, and receptor trafficking by immunofluorescence. Single knockout cells displayed a modest decrease in receptor ubiquitylation and a slight increase in phosphorylation as compared to control. DKO cells did not have detectable ubiquitylation, had delayed receptor trafficking, and a 2.3-fold increase in c-Met phosphorylation. Based on the observed changes in receptor trafficking and signaling, we examined HGF-dependent in vitro wound healing via live cell timelapse microscopy in control and DKO cells. HGF-treated DKO cells healed at approximately twice the rate of untreated cells. From these data, we have generated a model in which c-Cbl/Cbl-b mediate the ubiquitylation of c-Met, which targets the receptor through the endocytic pathway toward lysosomal degradation. In the absence of ubiquitylation, the stimulated receptor stays phosphorylated longer and enhances in vitro wound healing. We propose that c-Cbl and Cbl-b are two promising pharmacologic targets for enhancing c-Met-mediated CE re-epithelialization.

Free fatty acids stabilize integrin β1via S-nitrosylation to promote monocyte–endothelial adhesion

Hyperlipidemia characterized by high blood levels of free fatty acids (FFAs) is important for the progression of inflammatory cardiovascular diseases. Integrin β1 is a transmembrane receptor that drives various cellular functions, including differentiation, migration, and phagocytosis. However, the underlying mechanisms modifying integrin β1 protein and activity in mediating monocyte/macrophage adhesion to endothelium remain poorly understood. In this study, we demonstrated that integrin β1 protein underwent S-nitrosylation in response to nitrosative stress in macrophages. To examine the effect of elevated levels of FFA on the modulation of integrin β1 expression, we treated the macrophages with a combination of oleic acid and palmitic acid (2:1) and found that FFA activated inducible nitric oxide synthase/nitric oxide and increased the integrin β1 protein level without altering the mRNA level. FFA promoted integrin β1 S-nitrosylation via inducible nitric oxide synthase/nitric oxide and prevented its degradation by decreasing binding to E3 ubiquitin ligase c-Cbl. Furthermore, we found that increased integrin α4β1 heterodimerization resulted in monocyte/macrophage adhesion to endothelium. In conclusion, these results provided novel evidence that FFA-stimulated N--O stabilizes integrin β1via S-nitrosylation, favoring integrin α4β1 ligation to promote vascular inflammation.

Targeting the PTP1B-Bcr-Abl1 interaction for the degradation of T315I mutant Bcr-Abl1 in chronic myeloid leukemia.

Small-molecule-induced degradation of mutant Bcr-Abl1 provides a potential approach to overcome Bcr-Abl1 tyrosine kinase inhibitor (TKI)-resistant chronic myeloid leukemia (CML). Our previous study reported that a synthetic steroidal glycoside SBF-1 showed remarkable anti-CML activity by inducing the degradation of native Bcr-Abl1 protein. Here, we observed the comparable growth inhibition for SBF-1 in CML cells harboring T315I mutant Bcr-Abl1 in vitro and in vivo. SBF-1 triggered its degradation through disrupting the interaction between protein-tyrosine phosphatase 1B (PTP1B) and Bcr-Abl1. Using SBF-1 as a tool, we found that Tyr46 in the PTP1B catalytic domain and Tyr852 in the Bcr-Abl1 pleckstrin-homology (PH) domain are critical for their interaction. Moreover, the phosphorylation of Tyr1086 within the Bcr-Abl1 SH2 domain recruited the E3 ubiquitin ligase c-Cbl to catalyze K27-linked ubiquitin chains, which serve as a recognition signal for p62-dependent autophagic degradation. PTP1B dephosphorylated Bcr-Abl1 at Tyr1086 and prevented the recruitment of c-Cbl, leading to the stability of Bcr-Abl1. This study unravels the action mechanism of PTP1B in stabilizing Bcr-Abl1 protein and indicates that the PTP1B-Bcr-Abl1 interaction might be one of druggable targets for TKI-resistant CML with point mutations.

Fluctuations in AKT and PTEN Activity Are Linked by the E3 Ubiquitin Ligase cCBL.

3-Poly-phosphoinositides (PIP3) regulate cell survival, division, and migration. Both PI3-kinase (phosphoinositide-3-kinase) and PTEN (phosphatase and tensin-homolog in chromosome 10) control PIP3 levels, but the mechanisms connecting PI3-kinase and PTEN are unknown. Using non-transformed cells, the activation kinetics of PTEN and of the PIP3-effector AKT were examined after the addition of growth factors. Both epidermal growth factor and serum induced the early activation of AKT and the simultaneous inactivation of PTEN (at ~5 min). This PIP3/AKT peak was followed by a general reduction in AKT activity coincident with the recovery of PTEN phosphatase activity (at ~10–15 min). Subsequent AKT peaks and troughs followed. The fluctuation in AKT activity was linked to that of PTEN; PTEN reconstitution in PTEN-null cells restored AKT fluctuations, while PTEN depletion in control cells abrogated them. The analysis of PTEN activity fluctuations after the addition of growth factors showed its inactivation at ~5 min to be simultaneous with its transient ubiquitination, which was regulated by the ubiquitin E3 ligase cCBL (casitas B-lineage lymphoma proto-oncogene). Protein-protein interaction analysis revealed cCBL to be brought into the proximity of PTEN in a PI3-kinase-dependent manner. These results reveal a mechanism for PI3-kinase/PTEN crosstalk and suggest that cCBL could be new target in strategies designed to modulate PTEN activity in cancer.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

USP25 Regulates EGFR Fate by Modulating EGF-Induced Ubiquitylation Dynamics.

Deregulated epidermal growth factor receptor (EGFR) signaling is a key feature in different stages of oncogenesis. One important mechanism whereby cancer cells achieve increased and uncontrolled EGFR signaling is escaping down-modulation of the receptor. Ubiquitylation of the EGFR plays a decisive role in this process, as it regulates receptor internalization, trafficking and degradation. Deubiquitinating enzymes (DUBs) may oppose the ubiquitylation process, antagonizing or even promoting receptor degradation. Here, we use qualitative and quantitative assays to measure EGFR internalization and degradation after Ubiquitin Specific Peptidase 25 (USP25) depletion. We show that, by acting at the early steps of EGFR internalization, USP25 restrains the degradation of the EGFR by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR, thereby modulating the amplitude of ubiquitylation on the receptor. This study establishes USP25 as a negative regulator of the EGFR down-modulation process and suggests that it is a promising target for pharmacological intervention to hamper oncogenic growth signals in tumors that depend on the EGFR.

Hematopoietic Stem and Progenitor Cells Exhibit Stage-Specific Translational Programs via mTOR- and CDK1-Dependent Mechanisms.

Hematopoietic stem cells (HSCs) require highly regulated rates of protein synthesis, but it is unclear if they or lineage-committed progenitors preferentially recruit transcripts to translating ribosomes. We utilized polysome profiling, RNA sequencing, and whole-proteomic approaches to examine the translatome in LSK (Lin-Sca-1+c-Kit+) and myeloid progenitor (MP; Lin-Sca-1-c-Kit+) cells. Our studies show that LSKs exhibit low global translation but high translational efficiencies (TEs) of mRNAs required for HSC maintenance. In contrast, MPs activate translation in an mTOR-independent manner due, at least in part, to proteasomal degradation of mTOR by the E3 ubiquitin ligase c-Cbl. In the near absence of mTOR, CDK1 activates eIF4E-dependent translation in MPs through phosphorylation of 4E-BP1. Aberrant activation of mTOR expression and signaling in c-Cbl-deficient MPs results in increased mature myeloid lineage output. Overall, our data demonstrate that hematopoietic stem and progenitor cells (HSPCs) undergo translational reprogramming mediated by previously uncharacterized mechanisms of translational regulation.

Local Delivery of Gemcitabine Inhibits Pancreatic and Cholangiocarcinoma Tumor Growth by Promoting Epidermal Growth Factor Receptor Degradation.

Gemcitabine is clinically used to treat certain types of cancers, including pancreatic and biliary cancer. We investigated the signal transduction pathways underlying the local antitumor effects of gemcitabine-eluting membranes (GEMs) implanted in pancreatic/biliary tumor-bearing nude mice. Here, we report that GEMs increased the E3 ubiquitin ligase c-CBL protein level, leading to degradation of epidermal growth factor receptor (EGFR) in SCK and PANC-1 cells. GEMs decreased the RAS and PI3K protein levels, leading to a reduction in the protein levels of active forms of downstream signaling molecules, including PDK, AKT, and GSK3β. GEM reduced proliferation of cancer cells by upregulating cell cycle arrest proteins, particularly p53 and p21, and downregulating cyclin D1 and cyclin B. Moreover, GEMs reduced the levels of proangiogenic factors, including VEGF, VEGFR2, CD31, and HIF-1α, and inhibited tumor cell migration and invasion by inducing the expression of E-cadherin and reducing that of N-cadherin, snail, and vimentin. We demonstrated that local delivery of gemcitabine using GEM implants inhibited tumor cell growth by promoting c-CBL-mediated degradation of EGFR and inhibiting the proliferation, angiogenesis, and epithelial–mesenchymal transition of pancreatic/biliary tumors. Use of gemcitabine-eluting stents can improve stent patency by inhibiting the ingrowth of malignant biliary obstructions.

Unique Regulatory Pathways Dictate the Translational Program in Hematopoietic Stem and Progenitor Cells

Prior studies in numerous biological systems have shown that alterations in mRNA expression frequently fail to predict changes in protein expression. This may be due to many regulatory mechanisms that occur post-transcriptionally including mRNA recruitment to ribosomes, translational initiation, ribosome processivity, and protein stability, among others. Indeed, several examples of selective translation of mRNAs has been described both in malignant and normal cells. To determine the extent and potential impact of translational reprogramming on early hematopoietic development, we performed an integrated analysis of total RNA, polysome RNA, and whole proteome data generated from HSC-enriched LSK (Lin-Sca-1+c-Kit+) and MP (Lin-Sca-1-c-Kit+) cells from mouse. Our studies revealed that although LSK cells show lower global translation than MPs, they exhibited significantly higher translational efficiency (TE = polysome/total RNA abundance) of mRNAs supporting processes required for HSC maintenance (e.g. glycolysis, fatty acid metabolism, oxidative phosphorylation, mTOR signaling) (Fig 1A). Additionally, integrated analysis of proteomic and RNA expression data showed that, TE changes better predicted protein expression changes for these pathways, than total RNA expression (Fig1B). Biochemical characterization of MP cells revealed markedly decreased mTOR protein expression and signaling in MP cells, especially in GMP and MEP. This is mediated through proteasomal degradation of mTOR protein. An E3 ligase prediction algorithm, identified c-Cbl as a potential candidate, targeting mTOR, which was confirmed by demonstrating the aberrant expression of mTOR in MPs in c-Cbl KO mice. In vitro and in vivo mTOR inhibition studies confirm that the MPN-like phenotype of c-Cbl KO mice, is due to aberrant activation of mTOR signaling in committed myeloid progenitors. Intriguingly, despite decreased expression of mTOR protein in MP cells, 4E-BP1, a known target of mTOR, was still phosphorylated at Ser-65- a critical step for initiating cap-dependent translation. Through a combination of prediction algorithms and candidate gene experimental approaches, we show that the critical phosphorylation event at Ser-65 is mediated by , as immunoprecipitation studies show physical association between CDK1 and 4E-BP, and pharmacological inhibition of CDK1 activity, reduced 4E-BP P-Ser-65 levels. Overall, our data provide the first comprehensive characterization of the translatome in early hematopoiesis and demonstrated that the LSK to MP transition is characterized by significant translational reprogramming. This is, in part, mediated by the activation of a unique, mTOR-independent pathway to activate cap-dependent translation through the concerted action of c-Cbl and CDK1 to induce degradation of mTOR and phosphorylate 4E-BP to activation translation, respectively. Abrogation of the downregulation of mTOR signaling in myeloid progenitors, results in expansions of numerous myeloid lineages including neutrophils, monocytes and platelets (Fig 1C). Thus, our studies demonstrate the importance of proper translational reprogramming in early hematopoiesis. Figure legend. (A) Heatmap showing pathways significantly enriched in LSK and or MP cells based on TE. (B) Comparison of TE to protein expression in LSK cells for genes involved in the indicated biological processes (Blue dots: mRNAs that showed an anticorrelation between total RNA and protein expression; Red dots: mRNAs that showed a positive correlation between total RNA and protein expression). (C) Model for translational reprogramming in early hematopoiesis. Despite lower rates of global translation, LSK cells show preferential translation of mRNAs sensitive to mTOR inhibition and required for HSC maintenance. In contrast, in highly translating MP cells, loss of mTOR expression is mediated by the E3 ubiquitin ligase c-Cbl. When c-Cbl is deleted and mTOR protein is aberrantly expressed, this results in increased mature myeloid output. In the absence of mTOR, eIF4E-cap-dependent translation is maintained through the action of CDK1, which phosphorylates the S65 residue of 4E-BP1 to release eIF4E. Figure Disclosures No relevant conflicts of interest to declare.

Unique-region phosphorylation targets LynA for rapid degradation, tuning its expression and signaling in myeloid cells.

The activity of Src-family kinases (SFKs), which phosphorylate immunoreceptor tyrosine-based activation motifs (ITAMs), is a critical factor regulating myeloid-cell activation. We reported previously that the SFK LynA is uniquely susceptible to rapid ubiquitin-mediated degradation in macrophages, functioning as a rheostat regulating signaling (Freedman et al., 2015). We now report the mechanism by which LynA is preferentially targeted for degradation and how cell specificity is built into the LynA rheostat. Using genetic, biochemical, and quantitative phosphopeptide analyses, we found that the E3 ubiquitin ligase c-Cbl preferentially targets LynA via a phosphorylated tyrosine (Y32) in its unique region. This distinct mode of c-Cbl recognition depresses steady-state expression of LynA in macrophages derived from mice. Mast cells, however, express little c-Cbl and have correspondingly high LynA. Upon activation, mast-cell LynA is not rapidly degraded, and SFK-mediated signaling is amplified relative to macrophages. Cell-specific c-Cbl expression thus builds cell specificity into the LynA checkpoint.

Abstract 4293: Analyzing the redox/fyn/c-Cbl pathway in glioblastoma multiforme: A mechanistic approach

Abstract Glioblastoma multiforme (GBM) is the most common aggressive tumor of the central nervous system (CNS) with 10,000 newly diagnosed cases annually, and an incidence rate of 3 in 100,000 individuals in the United States. Unfortunately, the current standard of care of radiotherapy with concomitant Temozolomide (TMZ) has not been effective in consistently controlling tumor growth, as the median age of survival for patients is 14.6 months after treatment, with inevitable recurrence beyond this time. One of the difficulties that emerge when treating GBM is tumor heterogeneity. For example, GBM cells upregulate different receptor tyrosine kinases (RTKs). Signaling through RTKs enables these cells to maintain their proliferative status to ensure their survival. The E3 ubiquitin ligase c-Cbl is a negative regulator of RTKs, and our lab has found it to be physically sequestered by ARGHEF7 (Cool-1) in GBM. We identified a combination of drugs, a c-Cbl restorative agent (CRA01) and an oxidizing agent (Tamoxifen [TMX]), that decreased c-Cbl/Cool-1 complex formation and activated c-Cbl through the Redox/Fyn/c-Cbl pathway (RFC) in GBM cells. Treatment with CRA01+TMX led to degradation of RTKs. Little is known about the RFC pathway in cancer regulation or its ability to prevent cell growth in TMZ resistant GBM cells. Here, we examine ways to regulate c-Cbl in GBM cells and seek to identify the mechanism(s) resulting in c-Cbl/Cool-1 complex formation. Furthermore, we aim to use a mouse model of TMZ resistance to determine the effects of CRA01+TMX in vivo. These results will provide important knowledge on the RFC pathway that can help properly identify patients with GBM and different cancers with c-Cbl dysregulation for whom this treatment could prove beneficial. Citation Format: Neal Shah, Jennifer Stripay. Analyzing the redox/fyn/c-Cbl pathway in glioblastoma multiforme: A mechanistic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4293.

Ascorbic acid improves thrombotic function of platelets during living donor liver transplantation by modulating the function of the E3 ubiquitin ligases c-Cbl and Cbl-b.

ObjectiveTo investigate the effect of ascorbic acid (AA) on hemostatic function during living donor liver transplantation (LDLT).MethodsBlood samples from 21 LDLT recipients were taken within 30 minutes after induction and at 120 minutes after reperfusion. Rotational thromboelastography (TEG) and western blot analysis were used to analyze for fibrinolysis and functional changes in c-Cbl and Cbl-b, respectively. TEG test samples were prepared as one of three groups: C group (0.36 mL of blood), N group (0.324 mL of blood + 0.036 mL of 0.9% normal saline), and A group (0.324 mL of blood + 0.036 mL of 200 µmol/L-AA dissolved in 0.9% normal saline).ResultsAA decreased fibrinolysis and increased clot rigidity at baseline and 120 minutes after reperfusion. Cbl-b expression was significantly increased at baseline and 120 minutes after reperfusion in the A group compared with the C and N groups. However, c-Cbl phosphorylation was most significantly decreased in the A group at baseline and 120 minutes after reperfusion.ConclusionAA can significantly decrease fibrinolysis and improve clot rigidity in LT recipients during LDLT, and functional changes in Cbl-b and c-Cbl might represent the underlying mechanism. AA may be considered for use during LDLT to decrease hyperfibrinolysis.

B7-H3 promotes multiple myeloma cell survival and proliferation by ROS-dependent activation of Src/STAT3 and c-Cbl-mediated degradation of SOCS3.

B7-H3 (CD276) is broadly overexpressed by multiple human cancers. It plays a vital role in tumor progression and has been accepted as one of the inhibitory B7 family checkpoint molecules. To identify the functions and underlying mechanisms of B7-H3 in multiple myeloma, we analyzed B7-H3 expression in myeloma patients and used siRNAs and overexpression plasmid of B7-H3 to investigate its roles and downstream signaling molecules in myeloma cell lines. The results showed that surface expression of B7-H3 was upregulated in myeloma samples and cell lines. Lower expression of B7-H3 in myeloma cells was associated with better progression-free survival. Myeloma cell survival, drug resistance, and tumor growth could be promoted by B7-H3. The molecular basis for these functional roles of B7-H3 involved the activation of JAK2/STAT3 via redox-mediated oxidation and activation of Src. We further identified a STAT3-promoting signaling pathway by which oxidant-mediated Src phosphorylation led to secondary activation of the E3 ubiquitin ligase c-Cbl. Activated c-Cbl subsequently caused specific proteasomal degradation of SOCS3, a negative regulator of JAK2/STAT3. These data indicate B7-H3's important role in the activation of ROS/Src/c-Cbl pathway in multiple myeloma which integrates redox regulation and sustained STAT3 activation at the level of degradation of STAT3 suppressor.

Crk adaptor proteins mediate actin-dependent T cell migration and mechanosensing induced by the integrin LFA-1.

T cell entry into inflamed tissue involves firm adhesion, spreading, and migration of the T cells across endothelial barriers. These events depend on "outside-in" signals through which engaged integrins direct cytoskeletal reorganization. We investigated the molecular events that mediate this process and found that T cells from mice lacking expression of the adaptor protein Crk exhibited defects in phenotypes induced by the integrin lymphocyte function-associated antigen 1 (LFA-1), namely, actin polymerization, leading edge formation, and two-dimensional cell migration. Crk protein was an essential mediator of LFA-1 signaling-induced phosphorylation of the E3 ubiquitin ligase c-Cbl and its subsequent interaction with the phosphatidylinositol 3-kinase (PI3K) subunit p85, thus promoting PI3K activity and cytoskeletal remodeling. In addition, we found that Crk proteins were required for T cells to respond to changes in substrate stiffness, as measured by alterations in cell spreading and differential phosphorylation of the force-sensitive protein CasL. These findings identify Crk proteins as key intermediates coupling LFA-1 signals to actin remodeling and provide mechanistic insights into how T cells sense and respond to substrate stiffness.

Abnormal regulation of BCR signalling by c-Cbl in chronic lymphocytic leukaemia.

Abnormalities of molecules involved in signal transduction pathways are connected to Chronic Lymphocytic Leukemia (CLL) pathogenesis and a critical role has been already ascribed to B-Cell Receptor (BCR)-Lyn axis. E3 ubiquitin ligase c-Cbl, working together with adapter protein CIN85, controls the degradation of protein kinases involved in BCR signaling. To investigate cell homeostasis in CLL, we studied c-Cbl since in normal B cells it is involved in the ubiquitin-dependent Lyn degradation and in the down-regulation of BCR signaling. We found that c-Cbl is overexpressed and not ubiquitinated after BCR engagement. We observed that c-Cbl did not associate to CIN85 in CLL with respect to normal B cells at steady state, nor following BCR engagement. c-Cbl association to Lyn was not detectable in CLL after BCR stimulation, as it happens in normal B cells. In some CLL patients, c-Cbl is constitutively phosphorylated at Y731 and in the same subjects, it associated to regulatory subunit p85 of PI3K. Moreover, c-Cbl is constitutive associated to Cortactin in those CLL patients presenting Cortactin overexpression and bad prognosis. These results support the hypothesis that c-Cbl, rather than E3 ligase activity, could have an adaptor function in turn influencing cell homeostasis in CLL.

Increased expression of the Cbl family of E3 ubiquitin ligases decreases Interleukin-2 production in a rat model of peripheral neuropathy

BackgroundInterleukin 2 (IL-2) influences the development and severity of pain due to its antinociceptive and immunomodulatory effects. Its production is influenced by the increased expression of c-Cbl (Casitas B-lineage lymphoma proto-oncogene) and Cbl-b E3 ubiquitin ligases. We evaluated the effects on IL-2-mediated changes in c-Cbl and Cbl-b expression in a rat model of chronic neuropathic pain.MethodsPeripheral neuropathy was induced in adult male Sprague-Dawley rats weighing 250–300 g by chronic spinal nerve ligation. Half of the spinal cord ipsilateral to the nerve injury was harvested at 1, 3, and 6 weeks, and the expression levels of IL-2, c-Cbl, Cbl-b, phospholipase C-γ1 (PLC-γ1), ZAP70, and protein kinase Cθ (PKCθ), as well as ubiquitin conjugation, were evaluated.ResultsTotal IL-2 mRNA levels were significantly decreased at 3 and 6 weeks after nerve injury compared to those in sham-operated rats. The mRNA levels of c-Cbl and Cbl-b, as well as the level of ubiquitin conjugation, were significantly increased at 3 and 6 weeks. In contrast, the levels of phosphorylated ZAP70 and PLC-γ1 were decreased at 3 and 6 weeks after spinal nerve ligation. Ubiquitination of PLC-γ1 and PKCθ was increased at 3 and 6 weeks.ConclusionsOur results suggest that ubiquitin and the E3 ubiquitin ligases c-Cbl and Cbl-b function as neuroimmune modulators in the subacute phase of neuropathic pain after nerve injury.

Refinement of the endogenous epitope tagging technology allows the identification of a novel NRAS binding partner in melanoma.

The NRAS oncoprotein is highly mutated in melanoma. However, to date, no comprehensive proteomic study has been reported for NRAS. Here, we utilized the endogenous epitope tagging (EET) approach for the identification of novel NRAS binding partners. Using EET, an epitope tag is added to the endogenously expressed protein, via modification of its genomic coding sequence. Existing EET systems are not robust, suffer from high background, and are labor-intensive. To this end, we present a polyadenylation signal-trap construct for N'-tagging that generates a polycistronic mRNA with the gene of interest. This system requires the integration of the tagging cassette in frame with the target gene to be expressed. Using this design, we demonstrate, for the first time, endogenous tagging of NRAS in melanoma cells allowing the identification of the E3 ubiquitin ligase c-CBL as a novel NRAS binding partner. Thus, our developed EET technology allows the characterization of new RAS effectors, which could be beneficial for the design of future drugs that inhibit constitutive signaling of RAS oncogenic mutants.

IFT20 modulates ciliary PDGFRα signaling by regulating the stability of Cbl E3 ubiquitin ligases.

Primary cilia have pivotal roles as organizers of many different signaling pathways, including platelet-derived growth factor receptor α (PDGFRα) signaling, which, when aberrantly regulated, is associated with developmental disorders, tumorigenesis, and cancer. PDGFRα is up-regulated during ciliogenesis, and ciliary localization of the receptor is required for its appropriate ligand-mediated activation by PDGF-AA. However, the mechanisms regulating sorting of PDGFRα and feedback inhibition of PDGFRα signaling at the cilium are unknown. Here, we provide evidence that intraflagellar transport protein 20 (IFT20) interacts with E3 ubiquitin ligases c-Cbl and Cbl-b and is required for Cbl-mediated ubiquitination and internalization of PDGFRα for feedback inhibition of receptor signaling. In wild-type cells treated with PDGF-AA, c-Cbl becomes enriched in the cilium, and the receptor is subsequently ubiquitinated and internalized. In contrast, in IFT20-depleted cells, PDGFRα localizes aberrantly to the plasma membrane and is overactivated after ligand stimulation because of destabilization and degradation of c-Cbl and Cbl-b.