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Identification ubiquitination of EGFR by LC-MS/MS

ABSTRACT: 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.

INSTRUMENT(S): LTQ FT, Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

SUBMITTER: Pang-Hung Hsu

LAB HEAD: Pang-Hung Hsu

PROVIDER: PXD024279 | Pride | 2021-09-10

REPOSITORIES: Pride

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			Action	DRS
	20160402_egfr-cbl_t.raw	Raw
	20160402_egfr-cbl_tc.raw	Raw
	20160402_egfr-vec_t.raw	Raw
	20160402_egfr-vec_tc.raw	Raw
	20160402_egfr-znrf1_t.raw	Raw

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Publications

ZNRF1 Mediates Epidermal Growth Factor Receptor Ubiquitination to Control Receptor Lysosomal Trafficking and Degradation.

Shen Chia-Hsing CH Chou Chih-Chang CC Lai Ting-Yu TY Hsu Jer-En JE Lin You-Sheng YS Liu Huai-Yu HY Chen Yan-Kai YK Ho I-Lin IL Hsu Pang-Hung PH Chuang Tsung-Hsien TH Lee Chih-Yuan CY Hsu Li-Chung LC

Frontiers in cell and developmental biology 20210429

Activation of the epidermal growth factor receptor (EGFR) is crucial for development, tissue homeostasis, and immunity. Dysregulation of EGFR signaling is associated with numerous diseases. EGFR ubiquitination and endosomal trafficking are key events that regulate the termination of EGFR signaling, but their underlying mechanisms remain obscure. Here, we reveal that ZNRF1, an E3 ubiquitin ligase, controls ligand-induced EGFR signaling via mediating receptor ubiquitination. Deletion of ZNRF1 inhi ...[more]

PMID: 33996800

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Flotillin-2 regulates EGFR activation, degradation, and cancer growth

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

Capuani2015 - Binding of Cbl and Grb2 to EGFR (Early Activation Model - EAM)

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

Capuani2015 - Binding of Cbl and Gbr2 to EGFR (Multisite Phosphorylation Model - MPM)

2024-09-02 | BIOMD0000000594 | BioModels

EGF-dependent ubiquitinome, phosphorylation and interactome analysis in control and CYLD-deficient cells

Project description:Cylindromatosis tumor suppressor protein (CYLD) is deubiquitinase, best known as an essential negative regulator of the NFkB pathway. Previous studies have suggested an involvement of CYLD in epidermal growth factor (EGF)-dependent signal transduction as well, as it was found enriched within the tyrosine-phosphorylated complexes in cells stimulated with the growth factor. EGF receptor (EGFR) signaling participates in central cellular processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular homeostasis. Here, using a combination of mass spectrometry-based quantitative proteomic approaches with biochemical and immunofluorescence strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events triggered by EGF. Our data show that CYLD regulates the magnitude of ubiquitination of several major effectors of the EGFR pathway by assisting the recruitment of the ubiquitin ligase Cbl-b to the activated EGFR complex. Notably, the deficiency of CYLD impairs the interaction of EGFR with Cbl-b, which leads to a reduced ubiquitination of the receptor followed by its decreased degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors pressor for the negative regulation of a tumorigenic signaling pathway.

2017-06-05 | PXD006390 | Pride

Magnitude of ubiquitination determines the fate of activated epidermal growth factor receptor and downstream cellular effects

Project description:Receptor tyrosine kinases (RTK) bind growth factors and are critical for cell proliferation and differentiation. Their dysregulation leads to a loss of growth control, often resulting in cancer. Epidermal growth factor receptor (EGFR) is the prototypic RTK and can bind several ligands exhibiting distinct mitogenic potentials. Whereas the phosphorylation on individual EGFR sites and their roles for downstream signaling have been extensively studied, less is known about ligand-specific ubiquitination events on EGFR, which are crucial for signal attenuation and termination. We used a proteomics-based workflow for absolute quantitation combined with mathematical modelling to unveil potentially decisive ubiquitination events on EGFR from the first 30 seconds to 15 minutes of stimulation. Four ligands were used for stimulation: epidermal growth factor (EGF), heparin-binding-EGF like growth factor, transforming growth factor- and epiregulin. Whereas only little differences in the kinetic profiles of individual ubiquitination sites were observed, the overall amount of modified receptor differed depending on the used ligand, indicating that absolute magnitude of EGFR ubiquitination, and not distinctly regulated ubiquitination sites, is a major determinant for signal attenuation and the subsequent cellular outcomes.

2022-05-31 | PXD019621 | Pride

Quantitative Analysis of Wild Type CBL, CBL 70Z and Y371S 3T3 Stable Cell Transcriptomes

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

The target genes of EGFR activity in glioma cells

Project description:As a first step towards identifying the target genes of EGFR activity in glioma cells, genome-wide expression analyses were performed using the Affymetrix GeneChip Human Genome U133A array. To accomplish this, mRNA expression levels of these genes were measured in the glioblastoma cell lines, U87 and U178, engineered with EGFR by retrovirus transduction (termed U87-EGFR and U178-EGFR respectively), with or without 20 ng/mL EGF treatment for 3 h. U87 and U178 cells engineered to express EGFR were stimulated with or without EGF. The experiment was replicated twice for each U87 and U178 cells.

2011-11-05 | E-GEOD-33442 | biostudies-arrayexpress

Cbl family proteins are required to maintain the proteome homeostasis in mammary epithelial cells

2016-03-25 | GSE67301 | GEO

Cbl family proteins are required to maintain the proteome homeostasis in mammary epithelial cells

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

Expression Data From HCMV-Infected Human Monocytes: Role of EGFR

Project description:Human cytomegalovirus (HCMV) induces pro-inflammatory monocytes following infection and we have evidence that EGFR is a key mediator in this early activation. To begin to address how this signalling pathway is responsible for the rapid activation of infected monocytes, we examined the role this pathway played in the transcriptome of infected monocytes. Global transcriptional profiling using cDNA microarrays revealed a significant number of genes, including inflammatory genes, were regulated in a EGFR-dependent manner, identifying this pathway as a key cellular control point in the conversion of monocytes to an activated pro-inflammatory state following HCMV infection. Keywords: Disease state To begin to globally define how EGFR is involved in the HCMV-induced changes in monocyte function, we performed a transcriptome analysis in the presence of inhibitors to the EGFR signalling pathway. Specifically, a cDNA microarray containing 12,625 unique probe sets was utilized to assess the modulation of the monocyte transcriptome at 24 hours post infection in the presence of blocking anti-EGFR antibody and pharmacological agent AG1478 (AG; an EGFR inhibitor). A total of 4 replicates from mock-infected, HCMV-infected, anti-EGFR antibody-pretreated infected and AG-pretreated infected monocytes were analyzed in this study.

2009-09-03 | E-GEOD-17948 | biostudies-arrayexpress

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