Cbl family proteins are required to maintain the proteome homeostasis in mammary epithelial cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE67301 | GEO | 2016/03/25
SECONDARY ACCESSION(S): PRJNA279459
REPOSITORIES: GEO
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Project description:EGFR degradation is delayed in Cbl, Cbl-b double-deficient MCF10A but EGF stimulation does not enhance their growth. We performed a transcriptome analysis to gain insights into biological consequences of Cbl, Cbl-b loss in mammary epithelial cells. We compared the transcriptome of MCF10A cells expressing shRNA against Cbl and Cbl-b with control shRNA-expressing cells using Affymetrix U133 Plus 2.0. Each sample was prepared in duplicates. Data were analyzed by GSEA.
2016-03-25 | E-GEOD-67301 | biostudies-arrayexpress
Project description:Epidermal growth factor receptor (EGFR) signaling is frequently dysregulated in a variety of cancers. The ubiquitin ligase Cbl regulates degradation of activated EGFR through ubiquitination and acts as an adaptor to recruit proteins required for trafficking. We used Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) mass spectrometry (MS) to compare Cbl complexes in the absence and presence of EGF stimulation. We identified over a hundred novel Cbl interactors, and a secondary siRNA screen found that knockdown of Flotillin-2 (FLOT2) led to increased phosphorylation and degradation of EGFR upon EGF stimulation in HeLa cells. In H441 cells, FLOT2 knockdown increased EGF-stimulated EGFR phosphorylation, ubiquitination, and downstream signaling, reversible by the EGFR inhibitor erlotinib. CRISPR knockout of FLOT2 in HeLa cells confirmed EGFR downregulation, increased signaling, and increased dimerization and trafficking to the early endosome. FLOT2 interacts with both Cbl and EGFR. Downregulation of EGFR upon FLOT2 loss is Cbl-dependent, as co-knockdown of Cbl and Cbl-b restored EGFR levels. Stable overexpression of FLOT2 in HeLa cells decreased EGF-stimulated EGFR phosphorylation and ubiquitination. Overexpression of wild type (WT) FLOT2, but not the soluble G2A FLOT2 mutant, inhibited EGFR phosphorylation upon EGF stimulation in HEK293T cells. FLOT2 loss induces EGFR-dependent proliferation and anchorage-independent cell growth. Lastly, FLOT2 knockout increases tumor formation and tumor volume in nude mice and NSG mice, respectively. These data demonstrate that FLOT2 negatively regulates EGFR activation and dimerization, as well as its subsequent ubiquitination, endosomal trafficking, and degradation. FLOT2 negatively regulates proliferation in vitro and in vivo.
2023-03-11 | PXD031988 | Pride
Project description:MCF10A cells derived from spontaneously immortalized normal human mammary epithelia were subjected to EGF/SERUM stimulation for 0,20,40,60,120,240 and 480 minutes. We used microarrays to understand the temporal regulation of the cellular EGFR cascade. Experiment Overall Design: MCF10A cells were grown in DME:F12 medium supplemented with antibiotics, [10 mg/ml insulin, 0.1 mg/ml cholera toxin, 0.5 mg/ml hydrocortisone, heat-inactivated horse serum (5%); defined as 'serum' in the text] and 10ng/ml EGF. Cells were serum deprived for 24 hours. Following stimulation with EGF/SERUM for 0,20,40,60,120,240 and 480 minutes.
2007-11-01 | E-GEOD-6784 | biostudies-arrayexpress
Project description:Previously, analysis of EGF-induced EGFR ubiquitination by mass spectrometry revealed that ubiquitinated lysine residues were located in the TKD of EGFR. However, the specific lysine residues for the CBL-mediated polyubiquitination have not been identified. To investigate the differences in EGFR ubiquitination by ZNRF1 and CBL, we sought to identify the acceptor residues for polyubiquitin chains mediated by these two E3 ubiquitin ligases. We co-transfected HEK293T cells with EGFR and ZNRF1 or CBL, and immunoprecipitated EGFR for liquid chromatography-tandem mass spectrometry analysis.
2021-09-10 | PXD024279 | Pride
Project description:Cbl (Casitas B-lineage lymphoma) is a prominent post-translational regulator of protein tyrosine kinases (PTKs) that targets activated receptors for ubiquitination-mediated degradation. However, mutations within its E3 ubiquitin ligase activity rendering RING domain are often not sufficient to induce transformation. Rather, a conserved residue (Y371) within the linker helix region (LHR) of Cbl has been a mutational hotspot in myeloproliferative diseases. Interestingly, phosphorylation of Y371 is a prerequisite for Cbl’s ability to ubiquitinate PTKs. We have previously shown that Y371 mutations affect the conformation of Cbl that can be related to its transformation potential. We wanted to investigate for any additional advantage that the Cbl-Y371 mutants could gain over their wild-type counterpart so as to explain their oncogenicity. In our current study, we wanted to investigate the transcriptome profiles of cells overexpressing WT CBL or CBL-70Z, a well-characterized oncogenic CBL mutant lacking residues 366-382 and Y371S a prevalent MDS/MPN CBL mutant. We found an interesting inverse correlationship between the transcriptome of cells expressing WT CBL and the mutant CBLs. The results from this study can be linked to the oncogenic abilities of the mutant CBLs.
2019-10-20 | GSE121176 | GEO
Project description:Fabrizio Capuani, Alexia Conte, Elisabetta Argenzio, Luca Marchetti, Corrado Priami, Simona Polo, Pier Paolo Di Fiore, Sara Sigismund & Andrea Ciliberto. Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells. Nature Communications 6 (2015).
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.
2024-09-02 | BIOMD0000000595 | BioModels
Project description:Fabrizio Capuani, Alexia Conte, Elisabetta Argenzio, Luca Marchetti, Corrado Priami, Simona Polo, Pier Paolo Di Fiore, Sara Sigismund & Andrea Ciliberto. Quantitative analysis reveals how EGFR activation and downregulation are coupled in normal but not in cancer cells. Nature Communications 6 (2015).
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.
2024-09-02 | BIOMD0000000594 | BioModels
Project description:The goal of the transcriptomic profiling is to understand what genes or pathway Cbl and Cbl-b regulate for CSF-1R dependent growth signaling
2019-05-01 | GSE130527 | GEO
Project description:Cbl (Casitas B-lineage lymphoma) is a prominent post-translational regulator of protein tyrosine kinases (PTKs) that targets activated receptors for ubiquitination-mediated degradation. However, mutations within its E3 ubiquitin ligase activity rendering RING domain are often not sufficient to induce transformation. Rather, a conserved residue (Y371) within the linker helix region (LHR) of Cbl has been a mutational hotspot in myeloproliferative diseases. Interestingly, phosphorylation of Y371 is a prerequisite for Cbl’s ability to ubiquitinate PTKs. We have previously shown that Y371 mutations affect the conformation of Cbl that can be related to its transformation potential. We wanted to investigate for any additional advantage that the Cbl-Y371 mutants could gain over their wild-type counterpart so as to explain their oncogenicity. In our current study, we utilized CBL-70Z, a well-characterized oncogenic CBL mutant lacking residues 366-382 that behave similar to the prevalent MDS/MPN CBL mutants, as an example to explore differences in interactomes between Wild type CBl and CBL 70Z. Any divergent interacting abilities between the two can be used to decipher the mechanism by which the mutants elicit oncogenic transformation.
2021-02-26 | PXD011369 | Pride
Project description:Expression of genes that are differentially expressed between c-Cbl control and c-Cbl mutant hematopoietic stem and progenitor cells were identified through this approach. Interestingly, several leukemia associated genes, particularly transcription factors, were differentially expressed between c-Cbl WT and c-Cbl mutant hematopoietic stem and progenitor cells. Hematopoietic stem and progenitor cells of c-Cbl control and c-Cbl mutant mice were sorted based on their defined immunophenotype. Gene expression studies were performed using Illumina microarray chips.
2010-08-12 | E-GEOD-23564 | biostudies-arrayexpress