Cbl Binding Research Articles

Interaction of Glutathione with MMACHC Arginine-Rich Pocket Variants Associated with Cobalamin C Disease: Insights from Molecular Modeling.

Methylmalonic aciduria and homocystinuria type C protein (MMACHC) is required by the body to metabolize cobalamin (Cbl). Due to its complex structure and cofactor forms, Cbl passes through an extensive series of absorptive and processing steps before being delivered to mitochondrial methyl malonyl-CoA mutase and cytosolic methionine synthase. Depending on the cofactor attached, MMACHC performs either flavin-dependent reductive decyanation or glutathione (GSH)-dependent dealkylation. The alkyl groups of Cbl have to be removed in the presence of GSH to produce intermediates that can later be converted into active cofactor forms. Pathogenic mutations in the GSH binding site, such as R161Q, R161G, R206P, R206W, and R206Q, have been reported to cause Cbl diseases. The impact of these variations on MMACHC's structure and how it affects GSH and Cbl binding at the molecular level is poorly understood. To better understand the molecular basis of this interaction, mutant structures involving the MMACHC-MeCbl-GSH complex were generated using in silico site-directed point mutations and explored using molecular dynamics (MD) simulations. The results revealed that mutations in the key arginine residues disrupt GSH binding by breaking the interactions and reducing the free energy of binding of GSH. Specifically, variations at position 206 appeared to produce weaker GSH binding. The lowered binding affinity for GSH in the variant structures could impact metabolic pathways involving Cbl and its trafficking.

Transforming properties of MET receptor exon 14 skipping can be recapitulated by loss of the CBL ubiquitin ligase binding site.

MET is a receptor tyrosine kinase that is activated in many cancers through various mechanisms. MET exon 14 (Ex14) skipping occurs in 3% of nonsmall cell lung tumors. However, the contribution of the regulatory sites lost upon this skipping, which include a phosphorylated serine (S985) and a binding site for the E3 ubiquitin ligase CBL (Y1003), remains elusive. Sequencing of 2808 lung tumors revealed 71 mutations leading to MET exon 14 skipping and three mutations affecting Y1003 or S985. In addition, MET exon 14 skipping and MET Y1003F induced similar transcriptional programs, increased the activation of downstream signaling pathways, and increased cell mobility. Therefore, the MET Y1003F mutation is able to fully recapitulate responses induced by MET exon 14 skipping, suggesting that loss of the CBL binding site is the main contributor of cell transformation induced by MET Ex14 mutations.

Investigation on a MMACHC mutant from cblC disease: The c.394C>T variant

The cblC disease is an inborn disorder of the vitamin B12 (cobalamin, Cbl) metabolism characterized by methylmalonic aciduria and homocystinuria. The clinical consequences of this disease are devastating and, even when early treated with current therapies, the affected children manifest symptoms involving vision, growth, and learning. The illness is caused by mutations in the gene codifying for MMACHC, a 282aa protein that transports and transforms the different Cbl forms. Here we present data on the structural properties of the truncated protein p.R132X resulting from the c.394C > T mutation that, along with c.271dupA and c.331C > T, is among the most common mutations in cblC. Although missing part of the Cbl binding domain, p.R132X is associated to late-onset symptoms and, therefore, it is supposed to retain residual function. However, to our knowledge structural-functional studies on c.394C > T mutant aimed at verifying this hypothesis are still lacking. By using a biophysical approach including Circular Dichroism, fluorescence, Small Angle X-ray Scattering, and Molecular Dynamics, we show that the mutant protein MMACHC-R132X retains secondary structure elements and remains compact in solution, partly preserving its binding affinity for Cbl. Insights on the fragile stability of MMACHC-R132X-Cbl are provided.

P85.05 MET Exon 14 Skipping Mutation Positive Non-Small Cell Lung Cancer: A Population-Based Cohort

MET exon 14 skipping is a potentially targetable molecular alteration. The goals of this study were to identify patients with MET exon 14 skipping to understand prevalence, biology and response to treatment, and to identify molecular signatures that may predict for response or resistance to targeted MET therapy. British Columbia Cancer provides care for a population of 5.1 million. A laboratory-developed focused DNA-based next generation sequencing (NGS) panel was performed on advanced non-squamous non-small cell lung cancer (NSCLC) and selected never-smoking squamous patients. Patients with advanced NSCLC found to have MET exon 14 skipping alterations between January 2016-September 2019 were identified. Retrospective chart review was completed to collect baseline characteristics, response to systemic treatments, and outcomes. Median overall survival (OS) was calculated using the Kaplan Meier method. Forty-three patients out of 1,934 tested were identified with MET exon 14 skipping (2.2%). Forty-one patients had metastatic disease: median age 77 years, female 46%, non-squamous/squamous/mixed adenosquamous 85/12/2%, current or former/never smoking status 61/39%, median number of pack years 30. Thirty-three patients received systemic therapy; 24 patients treated with crizotinib in the first/second line setting 87/13%. Response rate was PR/SD/PD/NE 21/33/25/21% for a disease control rate (DCR) of 54.2% in the evaluable patients. Median time to treatment discontinuation (TTD) for crizotinib was 2.2 months with 5 patients still on therapy and 3 stopping due to toxicity. Eleven patients received platinum-based chemotherapy with PR/SD/PD/NE 9/64/18/9% and DCR of 73% in the evaluable patients. Median time to platinum discontinuation of 2.8 months. Fourteen patients received immunotherapy (IO) with PR/SD/PD/NE 7/43/21/29% and DCR of 50% in the evaluable patients. Median time to IO treatment discontinuation 2.4 months. Median OS for metastatic patients treated with any systemic therapy was 14.6 months. Molecular details of 41 patients: PDL1<1%/PDL1 1-49%/PDL1≥50%/unknown status was 7/17/63/12%. MET variant frequency <50/≥50% 76/24%. MET alteration type: 2% CBL binding domain mutation, 34% poly- pyrimidine tract deletion, 63% splice donor mutation or deletion. Co-mutations: 20% TP53 alterations, 22% other, 59% none. In this small cohort there were no clear correlations between molecular aberrations and response, TTD or OS. The prevalence of MET exon 14 skipping in a North American population was 2.2%. Unlike other targeted mutations, patients were older and more commonly current or former smokers. Patients with MET exon 14 skipping alteration demonstrate disease control with crizotinib, platinum-based chemotherapy and immunotherapy. Co-mutations with p53 were commonly noted. Correlation between co-mutations and efficacy of therapy were not identified in this cohort.

Abstract 3683: MET exon 14 skipping mutations in lung cancer: Screening, functional and clinical impact

Abstract Mutations affecting exon 14 splice sites of the gene encoding the MET receptor have been recently revealed in non-small cell lung cancer (NSCLC). These mutations induce MET exon 14 skipping (METex14), leading to receptor activation through deletion of a regulatory domain. Importantly, these mutations represent a promising therapeutic opportunity since MET tyrosine kinase inhibitors (TKI) are available. Nevertheless, these mutations raise several scientific and clinical questions. (i) Their functional consequences are still poorly understood,(ii) these mutations are highly heterogeneous which makes them difficult to detect, and (iii) efficacy of MET-TKI seems limited by largely unknown resistances. To address these issues, we first created by genome editing a panel of pulmonary cells expressing either METex14 or MET receptor mutated in each known active site of the regulatory domain. Comparison of signalling pathways, transcriptional landscapes and cellular responses revealed that METex14 activation is recapitulated by mutation of the CBL binding site involved in MET internalization, but provide also an unexpected resistance to apoptosis through abrogating its caspase cleavage. Second, in order to detect METex14 mutations in clinical routine practice, we developed an optimized targeted next generation sequencing panel covering the METex14 in addition to the usual targets. This panel revealed METex14 alterations in 2.2% NSCLC patients and presence of various concurrent alterations. Third, by further characterization of the concurrent alterations, we found high rate of PI3K pathway alterations in METex14 patients. In addition, MET-TKI treatment in 3 patients harboring these alterations had shown progressive disease, suggesting their involvement in resistance. Using a patient-derived cell line with primary resistance and cell lines in which MET or PI3K alterations were inserted, we confirmed involvement of PI3K activation in the resistance process, which was overcome with PI3K inhibitor. Overall, our integrated study reveals that METex14 mutations induce an original activation involving cooperation between regulatory mechanisms, but offering sensitivity to MET-TKI. Therefore, these mutations, now detectable in routine practice, are druggable by MET-TKI providing a novel therapeutic line for NSCLC, but have to face to innate resistance including PI3K alterations. Citation Format: Marie Fernandes, Philippe Jamme, Sonia Paget, Angela Morabito, Frédéric Leprêtre, Martin Figeac, Clotilde Descarpentries, Fabienne Escande, Simon Baldacci, Anne Chotteau-Lelièvre, Luca Grumolato, Marie-Christine Copin, Zoulika Kherrouche, Alexis B. Cortot, David Tulasne. MET exon 14 skipping mutations in lung cancer: Screening, functional and clinical impact [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3683.

Regulation of EGFR Endocytosis by CBL During Mitosis.

The overactivation of epidermal growth factor (EGF) receptor (EGFR) is implicated in various cancers. Endocytosis plays an important role in EGFR-mediated cell signaling. We previously found that EGFR endocytosis during mitosis is mediated differently from interphase. While the regulation of EGFR endocytosis in interphase is well understood, little is known regarding the regulation of EGFR endocytosis during mitosis. Here, we found that contrary to interphase cells, mitotic EGFR endocytosis is more reliant on the activation of the E3 ligase CBL. By transfecting HeLa, MCF-7, and 293T cells with CBL siRNA or dominant-negative 70z-CBL, we found that at high EGF doses, CBL is required for EGFR endocytosis in mitotic cells, but not in interphase cells. In addition, the endocytosis of mutant EGFR Y1045F-YFP (mutation at the direct CBL binding site) is strongly delayed. The endocytosis of truncated EGFR Δ1044-YFP that does not bind to CBL is completely inhibited in mitosis. Moreover, EGF induces stronger ubiquitination of mitotic EGFR than interphase EGFR, and mitotic EGFR is trafficked to lysosomes for degradation. Furthermore, we showed that, different from interphase, low doses of EGF still stimulate EGFR endocytosis by non-clathrin mediated endocytosis (NCE) in mitosis. Contrary to interphase, CBL and the CBL-binding regions of EGFR are required for mitotic EGFR endocytosis at low doses. This is due to the mitotic ubiquitination of the EGFR even at low EGF doses. We conclude that mitotic EGFR endocytosis exclusively proceeds through CBL-mediated NCE.

Loss of PRMT5 Promotes PDGFRα Degradation during Oligodendrocyte Differentiation and Myelination

The oligodendrocyte lineage is responsible for myelination of the central nervous system. Post-translational modifications are known to regulate oligodendrocyte precursor cell (OPC) differentiation into mature myelinating oligodendrocytes. The role of arginine methylation during oligodendrocyte differentiation and myelination is still poorly understood. We generated mice depleted of PRMT5 in OPCs using Olig2-Cre, and these mice developed severe hypomyelination and died at the third post-natal week. PRMT5-deficient cells have lower levels of PDGFRα at the plasma membrane due to increased degradation by the Cbl E3 ligase. Mechanistically, the loss of arginine methylation at R554 of the PDGFRα intracellular domain unmasks a Cbl binding site at Y555. We observed the progressive decrease in PRMT5 during oligodendrocyte differentiation, and we show that one role of this decrease is to downregulate growth signals provided by PDGFRα to initiate oligodendrocyte differentiation and myelination. More broadly, the inhibition of PRMT5 may be used therapeutically to manipulate PDGFRα bioavailability.

Organometallic DNA-B12 Conjugates as Potential Oligonucleotide Vectors: Synthesis and Structural and Binding Studies with Human Cobalamin-Transport Proteins.

The synthesis and structural characterization of Co-(dN)25 -Cbl (Cbl: cobalamin; dN: deoxynucleotide) and Co-(dN)39 -Cbl, which are organometallic DNA-B12 conjugates with single DNA strands consisting of 25 and 39 deoxynucleotides, respectively, and binding studies of these two DNA-Cbl conjugates to three homologous human Cbl transporting proteins, transcobalamin (TC), intrinsic factor (IF), and haptocorrin (HC), are reported. This investigation tests the suitability of such DNA-Cbls for the task of eventual in vivo oligonucleotide delivery. The binding of DNA-Cbl to TC, IF, and HC was investigated in competition with either a fluorescent Cbl derivative and Co-(dN)25 -Cbl, or radiolabeled vitamin B12 (57 Co-CNCbl) and Co-(dN)25 -Cbl or Co-(dN)39 -Cbl. Binding of the new DNA-Cbl conjugates was fast and tight with TC, but poorer with HC and IF, which extends a similar original finding with the simpler DNA-Cbl, Co-(dN)18 -Cbl. The contrasting affinities of TC versus IF and HC for the DNA-Cbl conjugates are rationalized herein by a stepwise mechanism of Cbl binding. Critical contributions to overall affinity result from gradual conformational adaptations of the Cbl-binding proteins to the DNA-Cbl, which is first bound to the respective β domains. This transition is fast with TC, but slow with IF and HC, with which weaker binding results. The invariably tight interaction of the DNA-Cbl conjugates with TC makes the Cbl moiety a potential natural vector for the specific delivery of oligonucleotide loads from the blood into cells.

MTE12.01 Clinically Relevant Signal Transduction Pathways

MTE12.01 Clinically Relevant Signal Transduction Pathways

Cell Type-Specific Modulation of Cobalamin Uptake by Bovine Serum.

Tracking cellular 57Co-labelled cobalamin (57Co-Cbl) uptake is a well-established method for studying Cbl homeostasis. Previous studies established that bovine serum is not generally permissive for cellular Cbl uptake when used as a supplement in cell culture medium, whereas supplementation with human serum promotes cellular Cbl uptake. The underlying reasons for these differences are not fully defined. In the current study we address this question. We extend earlier observations by showing that fetal calf serum inhibits cellular 57Co-Cbl uptake by HT1080 cells (a fibrosarcoma-derived fibroblast cell line). Furthermore, we discovered that a simple heat-treatment protocol (95°C for 10 min) ameliorates this inhibitory activity for HT1080 cell 57Co-Cbl uptake. We provide evidence that the very high level of haptocorrin in bovine serum (as compared to human serum) is responsible for this inhibitory activity. We suggest that bovine haptocorrin competes with cell-derived transcobalamin for Cbl binding, and that cellular Cbl uptake may be minimised in the presence of large amounts of bovine haptocorrin that are present under routine in vitro cell culture conditions. In experiments conducted with AG01518 cells (a neonatal foreskin-derived fibroblast cell line), overall cellular 57Co-Cbl uptake was 86% lower than for HT1080 cells, cellular TC production was below levels detectable by western blotting, and heat treatment of fetal calf serum resulted in only a modest increase in cellular 57Co-Cbl uptake. We recommend a careful assessment of cell culture protocols should be conducted in order to determine the potential benefits that heat-treated bovine serum may provide for in vitro studies of mammalian cell lines.

Abstract 1150: Targeting MET Exon 14 mutations with the selective small molecule inhibitor Savolitinib

Abstract Alterations in the MET oncogene occurs across a broad range of tumor indications. Amplification or mutations in MET lead to increased activity of downstream pathways including PI3K and MAPK, eventually resulting in tumor formation. Several small molecule inhibitors are currently in clinical trials, including the selective inhibitor Savolitinib (HMP-504, Volitinib, AZD6094), which shows single digit nanomolar activity in MET-amplified cell lines. Newly emerging data suggest mutations in MET causing complete skipping of Exon 14 occur in approximately 4% of non-small cell lung cancer (NSCLC), and are more rare in other indications [1, 2]. MET exon 14 skipping mutations were shown to be mutually exclusive from EGFRm, ALK and KRAS and can occur in the context of MET gene amplification [3]. Exon 14 harbors the CBL binding site (Y1003), which is critical for receptor degradation after binding of its ligand, HGF, and suppression of downstream signaling events. Clinical trial results with less potent, pan RTK inhibitors Crizotinib (31nM GI50 vs 3nM for Savolitinib) and Cabozantinib show promising early results, but fall short in long term responses. Therefore, better therapies targeting MET are needed. Human cell line models with Exon 14 deletions are rare. Therefore, we used engineered cell lines to test the effect of Savolitinib on these mutations. To do this, we expressed MET-Y1003F mutants in NIH-3T3 and HEK293T cells. We found that Savolitinib potently inhibited phospho-MET in both models expressing this mutant (100% phospho-MET inhibition). In addition, we tested whether or not Savolitinib could inhibit HGF-dependent signaling and growth of a NSCLC cell line, NCI-H596. In the presence of FBS (10%), Savolitinib had no effect on the growth rate of these cells, however was highly efficient at blocking HGF-dependent growth in the absence of FBS. To test the effect of this mutation in the background of amplification, we also tested the gastric cancer cell line Hs746T, which harbors exon 14 skipping in addition to MET amplification. Savolitinib was highly efficacious at blocking the growth of this cell line. Future studies are aimed at looking at the in vivo effect of Savolitinib targeting exon 14 mutants. These data provide a platform of evidence for using Savolitinib to target exon 14 mutant MET in patients. 1. Paik, P.K., et al., Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping. Cancer Discov, 2015. 5(8): p. 842-9. 2. Frampton, G.M., et al., Activation of MET via diverse exon 14 splicing alterations occurs in multiple tumor types and confers clinical sensitivity to MET inhibitors. Cancer Discov, 2015. 5(8): p. 850-9. 3. Cancer Genome Atlas Research, N., Comprehensive molecular profiling of lung adenocarcinoma. Nature, 2014. 511(7511): p. 543-50. Citation Format: Evan Barry, Elizabeth Maloney, Ryan Henry, Alexandra Borodovsky, Edwin Clark, Melanie Frigault, Michael Zinda, Celina D’Cruz. Targeting MET Exon 14 mutations with the selective small molecule inhibitor Savolitinib. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1150.

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.

P130Cas scaffold protein regulates ErbB2 stability by altering breast cancer cell sensitivity to autophagy.

Overexpression of the ErbB2/HER2 receptor tyrosine kinase occurs in up to 20% of human breast cancers and correlates with aggressive disease. Several efficacious targeted therapies, including antibodies and kinase inhibitors, have been developed but the occurring of resistance to these agents is often observed. New therapeutic agents targeting the endocytic recycling and intracellular trafficking of membrane in tumor cells overexpressing ErbB2 are actually in clinical development. Nevertheless the mechanisms underlying ErbB2 downregulation are still obscure. We have previously demonstrated that the overexpression of the p130Cas adaptor protein in ErbB2 positive breast cancer, promotes tumor aggressiveness and progression. Here we demonstrate that lowering p130Cas expression in breast cancer cells is sufficient to induce ErbB2 degradation by autophagy. Conversely, p130Cas overexpression protects ErbB2 from degradation by autophagy. Furthermore, this autophagy-dependent preferential degradation of ErbB2 in absence of p130Cas is due to an increased ErbB2 ubiquitination. Indeed, the overexpression of p130Cas impairs ErbB2 ubiquitination by inhibiting the binding of Cbl and CHIP E3 ligases to ErbB2. Finally, our results indicate that p130Cas-dependent ErbB2 protection from degradation by autophagy may alter the sensitivity to the humanized monoclonal antibody trastuzumab. Consistently, in human ErbB2 positive breast cancers that develop resistance to trastuzumab, p130Cas expression is significantly increased suggesting that elevated levels of p130Cas can be involved in trastuzumab resistance.

Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells.

Ubiquitination of the epidermal growth factor receptor (EGFR) that occurs when Cbl and Grb2 bind to three phosphotyrosine residues (pY1045, pY1068 and pY1086) on the receptor displays a sharp threshold effect as a function of EGF concentration. Here we use a simple modelling approach together with experiments to show that the establishment of the threshold requires both the multiplicity of binding sites and cooperative binding of Cbl and Grb2 to the EGFR. While the threshold is remarkably robust, a more sophisticated model predicted that it could be modulated as a function of EGFR levels on the cell surface. We confirmed experimentally that the system has evolved to perform optimally at physiological levels of EGFR. As a consequence, this system displays an intrinsic weakness that causes—at the supraphysiological levels of receptor and/or ligand associated with cancer—uncoupling of the mechanisms leading to signalling through phosphorylation and attenuation through ubiquitination.

An Intra-Molecular Disulfide Cross-Link Stabilizes an Inward-Oriented Transport Intermediate Conformation of the Tonb-Dependent Transporters

The Gram negative bacteria TonB-dependent transporter (TBDT) BtuB translocates vitamin B12 (cobalamin or Cbl) across the outer membrane. The amino-terminal lumenal domain fits within the 22-stranded s-barrel and contains several determinants for Cbl binding. Current models suggest that the TBDT carries out active transport driven by energy from the proton motive force (pmf), which is transmitted via direct interactions between the inner membrane protein TonB and the BtuB amino-terminal Ton Box motif. The lumenal domain must undergo large conformational changes to accommodate passage of the substrate through the barrel, but the molecular features of the mechanism are unknown. We show that disulfide bonds can be induced to form in whole cells between three pairs of cysteines introduced at barrel-lumenal domain contact points, consistent with the x-ray crystallographic structures. However, we also find spontaneously formed, stoichiometric cross-links between cysteines in the Ton Box motif and in place of Ser120 that indicate a conformation different from the x-ray structures. The TBDT family members FecA and FhuA with cysteines in equivalent positions also form stoichiometric disulfide bonds. Cbl uptake through BtuB is blocked by the Ton Box Cys-S120C cross-link and activity is recovered upon reduction. Significantly, Cbl binds to the cross-linked BtuB in isolated outer membrane fragments but not to whole cells, indicating that the Cbl binding site of the cross-linked form is oriented towards the periplasmic side. In contrast, the transporter is oriented outwards in all other conditions, included in the absence of a pmf. These results suggest an alternating access transport mechanism.

Cbl and Itch binding sites in ERBB4 CYT-1 and CYT-2 mediate K48- and K63-polyubiquitination, respectively

ERBB receptors have an important function in mammalian development and normal physiology, but overexpression and poor downregulation of ERBB receptors have been associated with malignant growth. Ligand-induced ERBB receptor signaling is terminated by clathrin-dependent receptor endocytosis, followed by incorporation of activated receptor complexes into multi-vesicular bodies and subsequent degradation in lysosomes. In the case of ERBB1, also known as the EGF receptor, it has been shown that ubiquitination serves as a signal to facilitate internalization and subsequent endosomal sorting, but little is known about the role of ubiquitination of other ERBB receptors. In the present study we investigated the regulation of ubiquitination and deubiquitination of the ERBB4 CYT-1 and CYT-2 isoforms in the context of chimeric EGFR-ERBB4 receptors. We demonstrate that EGFR-ERBB4 CYT-2 chimera shows decreased ligand-induced downregulation and EGF-degradation, as well as enhanced EGF recycling, when compared to EGFR-ERBB4 CYT-1. Moreover we show that the mutation Y1103F in the binding site for Cbl which is present in both CYT-1 and CYT-2, does not influence ERBB4 endosomal trafficking. We further demonstrate that total ligand-induced ubiquitination of CYT-1 is higher than that of CYT-2, whereby CYT-1 ubiquitination is mainly dependent on the PPXY1056 Itch binding site for the E3-ligase Itch which is only present in CYT-1, while that of CYT-2 is dependent on the Y1103 Cbl binding site. The E3-ligase c-Cbl is more efficiently phosphorylated upon EGF stimulation of the CYT-2 than the CYT-1 isoform. Moreover our data show that the pY1103 Cbl binding site is required for K48-polyubiquitination of both CYT-1 and CYT-2, whereas the PPXY1056 Itch binding site is required for K63-polyubiquitination of CYT-1. We further demonstrate that EGF stimulation of EGFR-ERBB4 CYT-1 and CYT-2 does not result in efficient binding to and tyrosine phosphorylation of the ESCRT-0 subunit Hrs. Finally, even though CYT-1 shows ligand-induced K63-polyubiquitination, it is not subjected to deubiquitination by the K63 polyubiquitin-specific AMSH deubiquitinating enzyme, while CYT-1 is slightly deubiquitinated by USP8. We conclude that Cbl and Itch binding sites in ERBB4 CYT-1 and CYT-2 mediate K48- and K63-polyubiquitination, respectively.

Recycling of EGFR and ErbB2 is associated with impaired Hrs tyrosine phosphorylation and decreased deubiquitination by AMSH

ErbB receptors play an important role in normal cellular growth, differentiation and development, but overexpression or poor downregulation can result in enhanced signaling and cancerous growth. ErbB signaling is terminated by clathrin-dependent receptor-mediated endocytosis, followed by incorporation in multi-vesicular bodies and subsequent degradation in lysosomes. In contrast to EGFR, ErbB2 displays poor ligand-induced downregulation and enhanced recycling, but the molecular mechanisms underlying this difference are poorly understood. Given our previous observation that both EGFR and an EGFR–ErbB2 chimera undergo Cbl-mediated K63-polyubiquitination, we investigated in the present study whether activation of the EGFR and the EGFR–ErbB2 chimera is associated with tyrosine phosphorylation of the ESCRT-0 complex subunit Hrs and AMSH-mediated deubiquitination. EGF stimulation of the EGFR resulted in efficient Hrs tyrosine phosphorylation and deubiquitination by the K63-polyubiquitin chain-specific deubiquitinating enzyme AMSH. In contrast, EGF activation of EGFR–ErbB2 showed significantly decreased Hrs tyrosine phosphorylation and deubiquitination by AMSH. To test whether this phenotype is the result of endosomal recycling, we induced recycling of the EGFR by stimulation with TGFα. Indeed, even though TGFα-stimulation of EGFR is associated with efficient ligand-stimulated K63-polyubiquitination, we observed that Hrs tyrosine phosphorylation as well as AMSH-mediated deubiquitination is significantly reduced under these conditions. Using various EGFR–ErbB2 chimeras, we demonstrate that enhanced recycling, decreased Hrs tyrosine phosphorylation and decreased AMSH mediated deubiquitination of EGFR–ErbB2 chimeras is primarily due to the presence of ErbB2 sequences or the absence of EGFR sequences C-terminal to the Cbl binding site. We conclude that endosomal recycling of the EGFR and ErbB2 receptors is associated with significantly impaired tyrosine phosphorylation of the ESCRT-0 subunit Hrs as well as decreased deubiquitination by AMSH, which is consistent with the finding that recycling receptors are not efficiently incorporated in the MVB pathway.

Structure of MMACHC Reveals an Arginine-Rich Pocket and a Domain-Swapped Dimer for Its B12 Processing Function

Defects in the MMACHC gene represent the most common disorder of cobalamin (Cbl) metabolism, affecting synthesis of the enzyme cofactors adenosyl-Cbl and methyl-Cbl. The encoded MMACHC protein binds intracellular Cbl derivatives with different upper axial ligands and exhibits flavin mononucleotide (FMN)-dependent decyanase activity toward cyano-Cbl as well as glutathione (GSH)-dependent dealkylase activity toward alkyl-Cbls. We determined the structure of human MMACHC·adenosyl-Cbl complex, revealing a tailor-made nitroreductase scaffold which binds adenosyl-Cbl in a "base-off, five-coordinate" configuration for catalysis. We further identified an arginine-rich pocket close to the Cbl binding site responsible for GSH binding and dealkylation activity. Mutation of these highly conserved arginines, including a replication of the prevalent MMACHC missense mutation, Arg161Gln, disrupts GSH binding and dealkylation. We further showed that two Cbl-binding monomers dimerize to mediate the reciprocal exchange of a conserved "PNRRP" loop from both subunits, serving as a protein cap for the upper axial ligand in trans and required for proper dealkylation activity. Our dimeric structure is supported by solution studies, where dimerization is triggered upon binding its substrate adenosyl-Cbl or cofactor FMN. Together our data provide a structural framework to understanding catalytic function and disease mechanism for this multifunctional enzyme.

The Loss of Cbl-Phosphatidylinositol 3-Kinase Interaction Perturbs RANKL-mediated Signaling, Inhibiting Bone Resorption and Promoting Osteoclast Survival

Cbl is an adaptor protein and an E3 ligase that plays both positive and negative roles in several signaling pathways that affect various cellular functions. Tyrosine 737 is unique to Cbl and is phosphorylated by Syk and Src family kinases. Phosphorylated Cbl Tyr(737) creates a binding site for the p85 regulatory subunit of PI3K, which also plays an important role in the regulation of bone resorption by osteoclasts. To investigate the role of Cbl-PI3K interaction in bone homeostasis, we examined the knock-in mice (Cbl(YF/YF)) in which the PI3K binding site in Cbl is ablated due to the mutation in the regulatory tyrosine. We report that in Cbl(YF/YF) mice, despite increased numbers of osteoclasts, bone volume is increased due to defective osteoclast function. Additionally, in ex vivo cultures, mature Cbl(YF/YF) osteoclasts showed an increased ability to survive in the presence of RANKL due to delayed onset of apoptosis. RANKL-mediated signaling is perturbed in Cbl(YF/YF) osteoclasts, and most interestingly, AKT phosphorylation is up-regulated, suggesting that the lack of PI3K sequestration by Cbl results in increased survival and decreased bone resorption. Cumulatively, these in vivo and in vitro results show that, on one hand, binding of Cbl to PI3K negatively regulates osteoclast differentiation, survival, and signaling events (e.g. AKT phosphorylation), whereas on the other hand it positively influences osteoclast function.

ERBB2 is a target for USP8-mediated deubiquitination

Overexpression and poor downregulation of ErbB receptor tyrosine kinases are associated with enhanced signaling and tumorigenesis. Attenuation of EGF-receptor (EGFR) signaling is mediated by endocytosis and ubiquitination by the E3-ligase Cbl. En route to lysosomes, but before incorporation of the EGFR into internal vesicles of MVBs, the EGFR undergoes Usp8-mediated deubiquitination. ErbB2 displays enhanced recycling back to the cell surface, and therefore we hypothesized that Usp8 is not part of the ErbB2 trafficking pathway. Here, we demonstrate, in the context of a chimeric EGFR-ErbB2 receptor, that (i) EGF induces pY1091 Cbl binding site-dependent K63-polyubiquitination of EGFR-ErbB2, (ii) Cbl is tyrosine phosphorylated upon stimulation of EGFR-ErbB2 wt and Y1091F mutant receptor, (iii) EGF-induced activation of EGFR-ErbB2 induces Usp8 tyrosine phosphorylation, and (iv) ubiquitination of the EGFR-ErbB2 wt and Y1091F mutant is enhanced upon coexpression of catalytically inactive Usp8-C748A in the presence and absence of EGF. We further show that Usp8 tyrosine phosphorylation upon stimulation of EGFR-ErbB2 is (a) independent of Y1091, (b) dependent on Src- and EGFR-ErbB2-kinase activity, (c) enhanced upon coexpression of Usp8-C748A, and (d) partly dependent on the Microtubule Interacting and Transport (MIT) domain of Usp8. Our findings demonstrate that Usp8 is part of the ErbB2 endosomal trafficking pathway.