An affinity prediction approach for the ligand of E3 ligase Cbl-b and an insight into substrate binding pattern.

Protein-protein interactions (PPIs) are essentially fundamental to all cellular processes, so that developing small molecule inhibitors of PPIs have great significance despite representing a huge challenge. Studying PPIs with the help of peptide motifs could obtain the structural information and reference significance to reduce the difficulty in the development of small molecules. Computational methods are powerful tools to characterize peptide-protein interactions, especially molecular dynamics simulation and binding free energy calculation. Here, we established an affinity prediction model suitable for Casitas B lymphoma-b (Cbl-b) and phosphorylated motif system. According to the affinity data set of multiple truncated peptides, the force field, solvent model, and internal dielectric constant of molecular mechanics/generalized Born surface area (MM/GBSA) method were optimized. Further, we predicted the affinity of the rationally designed new sequences through this model and obtained a new 6-mer motif with a 7-fold increase in affinity and the comprehensive structure-activity relationship. Moreover, we proposed an insight of unexpected activity of the truncated 5-mer peptide and revealed the possible binding mode of the new highly active 6-mer motif by extended simulation. Our results showed that the activity enhancement of the truncated peptide was caused by the acetyl-mediated conformation change. The side chain of Arg and pTyr in the 6-mer motif co-occupied the site p1 to form numerous hydrogen bond interactions and increased hydrophobic interaction formed with Tyr266, leading to the higher affinity. The present work provided a reference to investigate the PPI of Cbl-b and phosphorylated substrates and guided the development of Cbl-b inhibitors.

c-Cbl-mediated neddylation antagonizes ubiquitination and degradation of the TGF-ß type II receptor.

Transforming growth factor ß (TGF-ß) is a potent antiproliferative factor in multiple types of cells. Deregulation of TGF-ß signaling is associated with the development of many cancers, including leukemia, though the molecular mechanisms are largely unclear. Here, we show that Casitas B-lineage lymphoma (c-Cbl), a known proto-oncogene encoding an ubiquitin E3 ligase, promotes TGF-ß signaling by neddylating and stabilizing the type II receptor (TßRII). Knockout of c-Cbl decreases the TßRII protein level and desensitizes hematopoietic stem or progenitor cells to TGF-ß stimulation, while c-Cbl overexpression stabilizes TßRII and sensitizes leukemia cells to TGF-ß. c-Cbl conjugates neural precursor cell-expressed, developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, to TßRII at Lys556 and Lys567. Neddylation of TßRII promotes its endocytosis to EEA1-positive early endosomes while preventing its endocytosis to caveolin-positive compartments, therefore inhibiting TßRII ubiquitination and degradation. We have also identified a neddylation-activity-defective c-Cbl mutation from leukemia patients, implying a link between aberrant TßRII neddylation and leukemia development.

Exploring Molecular Contacts of MUC1 at CIN85 Binding Interface to Address Future Drug Design Efforts.

The modulation of protein-protein interactions (PPIs) by small molecules represents a valuable strategy for pharmacological intervention in several human diseases. In this context, computer-aided drug discovery techniques offer useful resources to predict the network of interactions governing the recognition process between protein partners, thus furnishing relevant information for the design of novel PPI modulators. In this work, we focused our attention on the MUC1-CIN85 complex as a crucial PPI controlling cancer progression and metastasis. MUC1 is a transmembrane glycoprotein whose extracellular domain contains a variable number of tandem repeats (VNTRs) regions that are highly glycosylated in normal cells and under-glycosylated in cancer. The hypo-glycosylation fosters the exposure of the backbone to new interactions with other proteins, such as CIN85, that alter the intracellular signalling in tumour cells. Herein, different computational approaches were combined to investigate the molecular recognition pattern of MUC1-CIN85 PPI thus unveiling new structural information useful for the design of MUC1-CIN85 PPI inhibitors as potential anti-metastatic agents.

Chlorocob(II)alamin Formation Which Enhances the Thiol Oxidase Activity of the B12-Trafficking Protein CblC.

CblC is a chaperone that catalyzes removal of the ß-axial ligand of cobalamin (or B12), generating cob(II)alamin in an early step in the cofactor trafficking pathway. Cob(II)alamin is subsequently partitioned to support cellular needs for the synthesis of active cobalamin cofactor derivatives. In addition to the ß-ligand transferase activity, the Caenorhabdiitis elegans CblC (ceCblC) and clinical R161G/Q variants of the human protein exhibit robust thiol oxidase activity, converting glutathione to glutathione disulfide while concomitantly reducing O2 to H2O2. The chemical efficiency of the thiol oxidase side reaction during ceCblC-catalyzed dealkylation of alkylcobalamins is noteworthy in that it effectively scrubs ambient oxygen from the reaction mixture, leading to air stabilization of the highly reactive cob(I)alamin product. In this study, we report that the enhanced thiol oxidase activity of ceCblC requires the presence of KCl, which explains how the wasteful thiol oxidase activity is potentially curtailed inside cells where the chloride concentration is low. We have captured an unusual chlorocob(II)alamin intermediate that is formed in the presence of potassium chloride, a common component of the reaction buffer, and have characterized it by electron paramagnetic resonance, magnetic circular dichroism, and computational analyses. The ability to form a chlorocob(II)alamin intermediate could represent an evolutionary vestige in ceCblC, which is structurally related to bacterial B12-dependent reductive dehalogenases that have been proposed to form halogen cob(II)alamin intermediates in their catalytic cycle.

C-Cbl negatively regulates TRAF6-mediated NF-κB activation by promoting K48-linked polyubiquitination of TRAF6.

BACKGROUND: In its RING domain, tumor necrosis factor receptor-associated factor 6 (TRAF6) has ubiquitin E3 ligase activity that facilitates the formation of lysine 63-linked polyubiquitin chains. This activity is required to activate nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) and plays an important role in the IκB kinase (IKK) complex. METHODS: An in vitro ubiquitination assay was used to establish whether c-Cbl could promote TRAF6 ubiquitination. We assessed direct binding and performed fine mapping between c-Cbl and TRAF6 based on the results of an immunoprecipitation assay with cultured 293 T cells. The luciferase reporter assay was applied to establish if c-Cbl-mediated ubiquitination affected NF-κB activation after stimulus from various TRAF-mediated signals: tumor necrosis factor-α (TNF-α), receptor activator of NF-κB ligand (RANKL), and interleukin-1ß (IL-1ß). An in vivo ubiquitination assay was performed using endogenous immunoprecipitation of TRAF6 in bone marrow macrophages (BMMs) and osteoclasts. RESULTS: Here, we report on a form of TRAF6 ubiquitination that is mediated by c-Cbl, leading to the formation of lysine 48-linked polyubiquitin chains. The NF-κB activity induced by RANKL and IL-1ß treatment is inhibited when c-Cbl is overexpressed, while the NF-κB activity induced by TNFα treatment is not. c-Cbl inhibits NF-κB activity mediated by TRAF6, but not by TRAF2. These findings show that c-Cbl ubiquitin ligase activity is essential for TRAF6 ubiquitination and negative regulation of NF-κB activity. Fine mapping revealed that the proline-rich domain of c-Cbl is critical for interaction with TRAF6. Stimulation with RANKL or interferon-γ (IFN-γ) caused c-Cbl to bind to polyubiquitinated TRAF6. CONCLUSIONS: These findings indicate that the interaction of TRAF6 with c-Cbl causes lysine 48-linked polyubiquitination for both negative feedback regulation and signaling cross-talk between RANKL and IFN-γ.

Biochemical and Structural Studies of the Interaction between ARAP1 and CIN85.

Arf-GAP with Rho-GAP domain, ANK repeat and PH domain-containing protein 1 (ARAP1), Cbl-interacting protein of 85 kDa (CIN85), and casitas B-lineage lymphoma (Cbl) play important roles in epidermal growth factor receptor (EGFR) internalization and recycling. In previous studies, ARAP1 was found to interact with CIN85, and their interaction attenuated the ubiquitination of EGFR. However, the molecular mechanism was still unclear. In this study, we first biochemically and structurally characterized the interaction between ARAP1 and CIN85, and found that the CIN85 SH3B domain bound to the ARAP1 PXPXXRX (except P) XXR/H/K motif with high affinity and specificity. Based on this binding model, we further predicted other potential CIN85 binding partners and tested their interactions biochemically. Moreover, our swapping data and structure alignment analysis suggested that the ß2-ß3 loops of the CIN85 SH3 domains and the H87ARAP1/E132CIN85 interaction were critical for ARAP1 binding specificity. Finally, our competitive analytical gel-filtration chromatography and isothermal titration calorimetry (ITC) results showed that ARAP1 could compete with Cbl for CIN85 binding, which provides a biochemical basis for the regulatory roles of ARAP1 in the CIN85-mediated EGFR internalizing process.

Structural Determinants of the Gain-of-Function Phenotype of Human Leukemia-associated Mutant CBL Oncogene.

Mutations of the tyrosine kinase-directed ubiquitin ligase CBL cause myeloid leukemias, but the molecular determinants of the dominant leukemogenic activity of mutant CBL oncogenes are unclear. Here, we first define a gain-of-function attribute of the most common leukemia-associated CBL mutant, Y371H, by demonstrating its ability to increase proliferation of hematopoietic stem/progenitor cells (HSPCs) derived from CBL-null and CBL/CBL-B-null mice. Next, we express second-site point/deletion mutants of CBL-Y371H in CBL/CBL-B-null HSPCs or the cytokine-dependent human leukemic cell line TF-1 to show that individual or combined Tyr → Phe mutations of established phosphotyrosine residues (Tyr-700, Tyr-731, and Tyr-774) had little impact on the activity of the CBL-Y371H mutant in HSPCs, and the triple Tyr → Phe mutant was only modestly impaired in TF-1 cells. In contrast, intact tyrosine kinase-binding (TKB) domain and proline-rich region (PRR) were critical in both cell models. PRR deletion reduced the stem cell factor (SCF)-induced hyper-phosphorylation of the CBL-Y371H mutant and the c-KIT receptor and eliminated the sustained p-ERK1/2 and p-AKT induction by SCF. GST fusion protein pulldowns followed by phospho-specific antibody array analysis identified distinct CBL TKB domains or PRR-binding proteins that are phosphorylated in CBL-Y371H-expressing TF-1 cells. Our results support a model of mutant CBL gain-of-function in which mutant CBL proteins effectively compete with the remaining wild type CBL-B and juxtapose TKB domain-associated PTKs with PRR-associated signaling proteins to hyper-activate signaling downstream of hematopoietic growth factor receptors. Elucidation of mutant CBL domains required for leukemogenesis should facilitate targeted therapy approaches for patients with mutant CBL-driven leukemias.

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 ß 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.

Pathogenic mutations differentially affect the catalytic activities of the human B12-processing chaperone CblC and increase futile redox cycling.

Human CblC catalyzes the elimination of the upper axial ligand in cobalamin or B12 derivatives entering the cell from circulation. This processing step is critical for assimilation of dietary cobalamin into the active cofactor forms that support the B12-dependent enzymes, methionine synthase and methylmalonyl-CoA mutase. Using a modified nitroreductase scaffold tailored to bind cobalamin and glutathione, CblC exhibits versatility in the mechanism by which it removes cyano versus alkyl ligands in cobalamin. In this study, we have characterized the effects of two pathogenic missense mutations at the same residue, R161G and R161Q, which are associated with early and late onset of the CblC disorder, respectively. We find that the R161Q and R161G CblC mutants display lower protein stability and decreased dealkylation but not decyanation activity, suggesting that cyanocobalamin might be therapeutically useful for patients carrying mutations at Arg-161. The mutant proteins also exhibit impaired glutathione binding. In the presence of physiologically relevant glutathione concentrations, stabilization of the cob(II)alamin derivative is observed, which occurs at the expense of increased oxidation of glutathione. Futile redox cycling, which is suppressed in wild-type human CblC, explains the reported increase in oxidative stress levels associated with the CblC disorder.

Structural analysis of the TKB domain of ubiquitin ligase Cbl-b complexed with its small inhibitory peptide, Cblin.

Cbl-b is a RING-type ubiquitin ligase. Previously, we showed that Cbl-b-mediated ubiquitination and proteosomal degradation of IRS-1 contribute to muscle atrophy caused by unloading stress. The phospho-pentapeptide DGpYMP (Cblin) mimics Tyr612-phosphorylated IRS-1 and inhibits the Cbl-b-mediated ubiquitination and degradation of IRS-1 in vitro and in vivo. In this study, we confirmed the direct interaction between Cblin and the TKB domain of Cbl-b using NMR. Moreover, we showed that the shortened tripeptide GpYM also binds to the TKB domain. To elucidate the inhibitory mechanism of Cblin, we solved the crystal structure of the TKB-Cblin complex at a resolution of 2.5 Å. The pY in Cblin inserts into a positively charged pocket in the TKB domain via hydrogen-bond networks and hydrophobic interactions. Within this complex, the Cblin structure closely resembles the TKB-bound form of another substrate-derived phosphopeptide, Zap-70-derived phosphopeptide. These peptides lack the conserved intrapeptidyl hydrogen bond between pY and a conserved residue involved in TKB-domain binding. Instead of the conserved interaction, these peptides specifically interact with the TKB domain. Based on this binding mode of Cblin to the TKB domain, we can design drugs against unloading-mediated muscle atrophy.

Cell surface epidermal growth factor receptors increase Src and c-Cbl activity and receptor ubiquitylation.

There is an established role for the endocytic pathway in regulation of epidermal growth factor receptor (EGFR) signaling to downstream effectors. However, because ligand-mediated EGFR endocytosis utilizes multiple "moving parts," dissecting the spatial versus temporal contributions has been challenging. Blocking all endocytic trafficking can have unintended effects on other receptors as well as give rise to compensatory mechanisms, both of which impact interpretation of EGFR signaling. To overcome these limitations, we used epidermal growth factor (EGF) conjugated to polystyrene beads (EGF beads). EGF beads simultaneously activate the EGFR while blocking its endocytosis and allow analysis of EGFR signaling from the plasma membrane. Human telomerase immortalized corneal epithelial (hTCEpi) cells were used to model normal epithelial cell biology. In hTCEpi cells, both cell surface and intracellular EGFRs exhibited dose-dependent increases in effector activity after 15 min of ligand stimulation, but only the serine phosphorylation of signal transducer and activator of transcription 3 (STAT3) was statistically significant when accounting for receptor phosphorylation. However, over time with physiological levels of receptor phosphorylation, cell surface receptors produced either enhanced or sustained mitogen-activated protein kinase kinase (MEK), Casitas B-lineage lymphoma (c-Cbl), and the pro-oncogene Src activity. These increases in effector communication by cell surface receptors resulted in an increase in EGFR ubiquitylation with sustained ligand incubation. Together, these data indicate that spatial regulation of EGFR signaling may be an important regulatory mechanism in receptor down-regulation.

Juvenile myelomonocytic leukemia due to a germline CBL Y371C mutation: 35-year follow-up of a large family.

Juvenile myelomonocytic leukemia (JMML) is a pediatric myeloproliferative neoplasm that arises from malignant transformation of the stem cell compartment and results in increased production of myeloid cells. Somatic and germline variants in CBL (Casitas B-lineage lymphoma proto-oncogene) have been associated with JMML. We report an incompletely penetrant CBL Y371C mutation discovered by whole-exome sequencing in three individuals with JMML in a large pedigree with 35 years of follow-up. The Y371 residue is highly evolutionarily conserved among CBL orthologs and paralogs. In silico bioinformatics prediction programs suggested that the Y371C mutation is highly deleterious. Protein structural modeling revealed that the Y371C mutation abrogated the ability of the CBL protein to adopt a conformation that is required for ubiquitination. Clinically, the three mutation-positive JMML individuals exhibited variable clinical courses; in two out of three, primary hematologic abnormalities persisted into adulthood with minimal clinical symptoms. The penetrance of the CBL Y371C mutation was 30% for JMML and 40% for all leukemia. Of the 8 mutation carriers in the family with available photographs, only one had significant dysmorphic features; we found no evidence of a clinical phenotype consistent with a "CBL syndrome". Although CBL Y371C has been previously reported in familial JMML, we are the first group to follow a complete pedigree harboring this mutation for an extended period, revealing additional information about this variant's penetrance, function and natural history.

Gain-of-function of mutated C-CBL tumour suppressor in myeloid neoplasms.

Acquired uniparental disomy (aUPD) is a common feature of cancer genomes, leading to loss of heterozygosity. aUPD is associated not only with loss-of-function mutations of tumour suppressor genes, but also with gain-of-function mutations of proto-oncogenes. Here we show unique gain-of-function mutations of the C-CBL (also known as CBL) tumour suppressor that are tightly associated with aUPD of the 11q arm in myeloid neoplasms showing myeloproliferative features. The C-CBL proto-oncogene, a cellular homologue of v-Cbl, encodes an E3 ubiquitin ligase and negatively regulates signal transduction of tyrosine kinases. Homozygous C-CBL mutations were found in most 11q-aUPD-positive myeloid malignancies. Although the C-CBL mutations were oncogenic in NIH3T3 cells, c-Cbl was shown to functionally and genetically act as a tumour suppressor. C-CBL mutants did not have E3 ubiquitin ligase activity, but inhibited that of wild-type C-CBL and CBL-B (also known as CBLB), leading to prolonged activation of tyrosine kinases after cytokine stimulation. c-Cbl(-/-) haematopoietic stem/progenitor cells (HSPCs) showed enhanced sensitivity to a variety of cytokines compared to c-Cbl(+/+) HSPCs, and transduction of C-CBL mutants into c-Cbl(-/-) HSPCs further augmented their sensitivities to a broader spectrum of cytokines, including stem-cell factor (SCF, also known as KITLG), thrombopoietin (TPO, also known as THPO), IL3 and FLT3 ligand (FLT3LG), indicating the presence of a gain-of-function that could not be attributed to a simple loss-of-function. The gain-of-function effects of C-CBL mutants on cytokine sensitivity of HSPCs largely disappeared in a c-Cbl(+/+) background or by co-transduction of wild-type C-CBL, which suggests the pathogenic importance of loss of wild-type C-CBL alleles found in most cases of C-CBL-mutated myeloid neoplasms. Our findings provide a new insight into a role of gain-of-function mutations of a tumour suppressor associated with aUPD in the pathogenesis of some myeloid cancer subsets.

Autoinhibition and phosphorylation-induced activation mechanisms of human cancer and autoimmune disease-related E3 protein Cbl-b.

Cbl-b is a RING-type E3 ubiquitin ligase that functions as a negative regulator of T-cell activation and growth factor receptor and nonreceptor-type tyrosine kinase signaling. Cbl-b dysfunction is related to autoimmune diseases and cancers in humans. However, the molecular mechanism regulating its E3 activity is largely unknown. NMR and small-angle X-ray scattering analyses revealed that the unphosphorylated N-terminal region of Cbl-b forms a compact structure by an intramolecular interaction, which masks the interaction surface of the RING domain with an E2 ubiquitin-conjugating enzyme. Phosphorylation of Y363, located in the helix-linker region between the tyrosine kinase binding and the RING domains, disrupts the interdomain interaction to expose the E2 binding surface of the RING domain. Structural analysis revealed that the phosphorylated helix-RING region forms a compact structure in solution. Moreover, the phosphate group of pY363 is located in the vicinity of the interaction surface with UbcH5B to increase affinity by reducing their electrostatic repulsion. Thus, the phosphorylation of Y363 regulates the E3 activity of Cbl-b by two mechanisms: one is to remove the masking of the RING domain from the tyrosine kinase binding domain and the other is to form a surface to enhance binding affinity to E2.

As4S4 targets RING-type E3 ligase c-CBL to induce degradation of BCR-ABL in chronic myelogenous leukemia.

Arsenic, a curative agent for acute promyelocytic leukemia, induces cell apoptosis and degradation of BCR-ABL in chronic myelogenous leukemia (CML). We demonstrated that ubiquitination and degradation of BCR-ABL was mediated by c-CBL, a RING-type E3 ligase that was also shown to be involved in ubiquitination for many other receptor/protein tyrosine kinases. Our data showed that c-CBL protein was considerably up-regulated by arsenic sulfide (As(4)S(4)). Interestingly, arsenic directly bound the RING finger domain of c-CBL to inhibit its self-ubiquitination/degradation without interfering with the enhancement of ubiquitination and subsequent proteolysis of its substrate BCR-ABL. Degradation of BCR-ABL due to c-CBL induction as a result of arsenic treatment was also observed in vivo in CML mice. These findings provide insight into the molecular mechanisms of arsenic and further support its therapeutic applications in CML in combination with tyrosine kinase inhibitors and potentially also in other malignancies involving aberrant receptor/protein tyrosine kinase signaling.

Structural basis for a novel intrapeptidyl H-bond and reverse binding of c-Cbl-TKB domain substrates.

The c-Cbl tyrosine kinase binding domain (Cbl-TKB), essentially an 'embedded' SH2 domain, has a critical role in targeting proteins for ubiquitination. To address how this domain can bind to disparate recognition mofits and to determine whether this results in variations in substrate-binding affinity, we compared crystal structures of the Cbl-TKB domain complexed with phosphorylated peptides of Sprouty2, Sprouty4, epidermal growth factor receptor, Syk, and c-Met receptors and validated the binding with point-mutational analyses using full-length proteins. An obligatory, intrapeptidyl H-bond between the phosphotyrosine and the conserved asparagine or adjacent arginine is essential for binding and orients the peptide into a positively charged pocket on c-Cbl. Surprisingly, c-Met bound to Cbl in the reverse direction, which is unprecedented for SH2 domain binding. The necessity of this intrapeptidyl H-bond was confirmed with isothermal titration calorimetry experiments that also showed Sprouty2 to have the highest binding affinity to c-Cbl; this may enable the selective sequestration of c-Cbl from other target proteins.

Activating CBL mutations are associated with a distinct MDS/MPN phenotype.

Activating point mutations in CBL have recently been identified in diverse subtypes of myeloid neoplasms. Because detailed clinical and hematological characteristics of CBL-mutated cases is lacking, we screened 156 BCR-ABL and JAK2 V617F negative patients with myeloproliferative neoplasms (MPN) and overlap syndromes between myelodysplastic syndrome (MDS) and MPN (MPS/MPN) for mutations in exons 8 and 9 of CBL by denaturing high-performance liquid chromatography and direct sequencing. CBL mutations were identified in 16/156 patients (10%), of which five also carried mutations in EZH2 (n = 3) and TET2 (n = 2). Comprehensive clinical and hematological characteristics were available from 13/16 patients (81%). In addition to splenomegaly (77%), striking common hematological features were CML-like left-shifted leukocytosis (85%) with monocytosis (85%), anemia (100%), and thrombocytopenia (62%). Thrombocytosis was not observed in any patient. Relevant bone marrow features (n = 12) included hypercellularity (92%) with marked granulopoiesis (92%), nonclustered microlobulated megakaryocytes (83%), and marrow fibrosis (83%). Nine deaths (progression to secondary acute myeloid leukemia/blast phase, n = 7; cytopenia complications, n = 2) were recorded. Three-year survival rate was 27%, possibly indicating poor prognosis of CBL mutated MDS/MPN patients.

The N terminus of Cbl-c regulates ubiquitin ligase activity by modulating affinity for the ubiquitin-conjugating enzyme.

Cbl proteins are ubiquitin ligases (E3s) that play a significant role in regulating tyrosine kinase signaling. There are three mammalian family members: Cbl, Cbl-b, and Cbl-c. All have a highly conserved N-terminal tyrosine kinase binding domain, a catalytic RING finger domain, and a C-terminal proline-rich domain that mediates interactions with Src homology 3 (SH3) containing proteins. Although both Cbl and Cbl-b have been studied widely, little is known about Cbl-c. Published reports have demonstrated that the N terminus of Cbl and Cbl-b have an inhibitory effect on their respective E3 activity. However, the mechanism for this inhibition is still unknown. In this study we demonstrate that the N terminus of Cbl-c, like that of Cbl and Cbl-b, inhibits the E3 activity of Cbl-c. Furthermore, we map the region responsible for the inhibition to the EF-hand and SH2 domains. Phosphorylation of a critical tyrosine (Tyr-341) in the linker region of Cbl-c by Src or a phosphomimetic mutation of this tyrosine (Y341E) is sufficient to increase the E3 activity of Cbl-c. We also demonstrate for the first time that phosphorylation of Tyr-341 or the Y341E mutation leads to a decrease in affinity for the ubiquitin-conjugating enzyme (E2), UbcH5b. The decreased affinity of the Y341E mutant Cbl-c for UbcH5b results in a more rapid turnover of bound UbcH5b coincident with the increased E3 activity. These data suggest that the N terminus of Cbl-c contributes to the binding to the E2 and that phosphorylation of Tyr-341 leads to a decrease in affinity and an increase in the E3 activity of Cbl-c.

Sleuthing biochemical evidence to elucidate unassigned electron density in a CBL-SLAP2 crystal complex.

The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL E3 ubiquitin ligase to downregulate antigen, cytokine and tyrosine kinase receptor signalling. In contrast to the phosphotyrosine-dependent binding of CBL substrates through its tyrosine kinase-binding domain (TKBD), CBL TKBD associates with the C-terminal tail of SLAP2 in a phospho-independent manner. To understand the distinct nature of this interaction, a purification protocol for SLAP2 in complex with CBL TKBD was established and the complex was crystallized. However, determination of the complex crystal structure was hindered by the apparent degradation of SLAP2 during the crystallization process, such that only the CBL TKBD residues could initially be modelled. Close examination of the CBL TKBD structure revealed a unique dimer interface that included two short segments of electron density of unknown origin. To elucidate which residues of SLAP2 to model into this unassigned density, a co-expression system was generated to test SLAP2 deletion mutants and define the minimal SLAP2 binding region. SLAP2 degradation products were also analysed by mass spectrometry. The model-building and map-generation features of the Phenix software package were employed, leading to successful modelling of the C-terminal tail of SLAP2 into the unassigned electron-density segments.

A novel, germline, deactivating CBL variant p.L493F alters domain orientation and is associated with multiple childhood cancers.

CBL is a mammalian gene encoding the protein CBL, which is an E3 ubiquitin-protein ligase involved in cell signaling and protein ubiquitination. Pathogenic variants in this gene have been implicated in a number of human cancers, particularly acute myeloid leukemia (AML). Here, we present a 5-year-old male patient with a history of AML, diffuse midline glioma, and left brain lesion with histiocytic features. A variant of uncertain significance (VUS): p.L493F was detected in his CBL gene via clinical evaluation. Protein modeling predicts this variant to be pathogenic. Details of the clinical evaluation and modeling assay are discussed.