Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor

Abstract

Cylindromatosis tumor suppressor protein (CYLD) is a 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, CYLD facilitates the interaction of EGFR with Cbl-b through its Tyr15 phosphorylation in response to EGF, which leads to fine-tuning of the receptor's ubiquitination and subsequent degradation. This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the negative regulation of a tumorigenic signaling pathway.

Fig. 1.

CYLD engagement in EGFR signaling. A, CYLD enrichment in the tyrosine-phosphorylated complexes upon EGF stimulation in HeLa cells observed by immunoprecipitation (IP) with antiphosphotyrosine (pTyr) antibodies followed by immunoblotting with indicated antibodies. WCL-whole cell lysate. B, Same as in A, in the presence of the EGFR kinase inhibitors Iressa and CI1033. C, Efficiency of CYLD silencing in HeLa cells. The mRNA (left) and protein (right) levels of CYLD in cells expressing scrambled shRNA (shControl) or shRNA specific to CYLD (shCYLD) as estimated by qPCR and Western blotting, respectively. D, Effect of CYLD silencing on the overall ubiquitination profile of the EGF-activated proteins. Lysates from wild type (WT), shControl and shCYLD HeLa cells were subjected to IP with anti-pTyr antibodies, followed by immunoblotting (IB) with indicated antibodies. See also supplemental Fig. S1.

Fig. 2.

SILAC-based quantitative proteomics analysis of ubiquitinated proteins in control and CYLD-silenced cells using the StUbEx system. A, Experimental workflow. SILAC-labeled shControl and shCYLD HeLa cells expressing 6xHis-Flag-tagged ubiquitin instead of endogenous ubiquitin were stimulated with EGF for 6 min, where indicated. Ubiquitinated proteins were enriched by sequential purification using nickel-affinity chromatography and anti-Flag antibodies, digested in-solution and analyzed by LC-MS/MS. Two biological replicas were performed for each of the indicated SILAC experiments (each biological replica corresponding to two paralleled triple SILAC experiments). B, Overlap of the identified and quantified proteins between the two biological replicas (left) and correlation of SILAC values between the channels common to all experiments (medium over light). See also supplemental Fig. S2. C, The group of 11 proteins with most significant ubiquitination changes in response to growth factor stimulation and the comparison of their corresponding EGF/Unstimulated ratios in control and CYLD-silenced cells. D, Ubiquitination status of the 11 proteins from panel C in the shControl and shCYLD cells, relative to their basal levels in the unstimulated shControl cells. See also supplemental Fig. S3.

Fig. 3.

CYLD is phosphorylated on Tyr15 in response to EGF. A, Experimental design for the SILAC-based mass spectrometric approach. Wild Type and CYLD-silenced cells were differentially SILAC encoded as depicted and stimulated with EGF for 6 min where indicated. Immunoprecipitated CYLD from the corresponding cellular lysates was subjected to trypsin digestion and subsequent mass spectrometric analysis. B, SILAC ratios of the immunoprecipitated CYLD protein from the corresponding cellular conditions. C, Annotated MS/MS spectrum for the pTyr15-containing peptide derived from CYLD. D, Signal intensities of the pTyr15-containing peptide from the corresponding cellular conditions. E, Estimation of CYLD Tyr15 phosphorylation stoichiometry.

Fig. 4.

Cbl-b is recruited to CYLD phosphorylated Tyr15- containing peptide. A, Workflow for the SILAC-based peptide pull-down assay using empty beads (light channel; negative control), unmodified (medium) or Tyr15 phosphorylated (heavy) CYLD peptides as bait. Two replicas were performed, swapping the medium and heavy conditions. Overlap (B), SILAC ratios correlation (C) and Ratio versus Intensity plot (D) of the proteins quantified in the two replicate pull-down experiments. E, p-Values, SILAC ratios and MS intensities of the most enriched interactors of the pTyr15-containing CYLD peptide.

Fig. 5.

CYLD facilitates ligand-dependent recruitment of Cbl-b to EGFR and subsequent receptor ubiquitination. A, Immunoprecipitation (IP) of EGFR from shControl and shCYLD HeLa cells unstimulated or stimulated with EGF for 6 min, followed by Western blotting with indicated antibodies. WCL-whole cell lysates B, Anti-phosphotyrosine (pTyr) IP from shControl and shCYLD HeLa cells, followed by Western blotting with antibodies against Cbl-b and c-Cbl. C, IP of Cbl-b from shControl and shCYLD HeLa cells, followed by Western blotting with indicated antibodies. Equivalent experiment using anti-GFP antibodies was carried out in parallel as control. D, IP of EGFR from shControl and shCYLD HeLa cells, followed by Western blotting with anti-CYLD antibodies. E, IP of EGFR and Cbl-b from shControl and shCYLD SKOV3 cells (left) or shControl and shCYLD Hep2 cells (right) followed by Western blotting with indicated antibodies.

Fig. 6.

Impaired EGFR trafficking and degradation in CYLD-silenced cells. A and B, Immunofluorescence images of EGFR (red) in shControl and shCYLD HeLa cells expressing GFP-tagged Rab7 (green) and stimulated with EGF for indicated times. DAPI staining was used to visualize cellular nuclei (blue). Three independent images from the 2 h time point are shown in panel B, where the GFP-Rab7 (green) channel is not shown for better comparison of the EGFR (red) signals. C, Immunoblotting of EGFR on total lysates from shControl and shCYLD cells pre-treated with cycloheximide and stimulated with EGF for indicated times. Three different exposures of the image are shown. D, The mRNA levels of EGFR in shControl and shCYLD cells assessed by qPCR.

Fig. 7.

CYLD phosphoTyr15-deficient mutant mimics the effects of CYLD silencing. A, Immunoblotting of whole cell lysates from shControl, shCYLD and shCYLD cells transfected with either Flag-tagged wild type CYLD (CYLD WT) or Flag-tagged CYLD with tyrosine 15-mutated to phenylalanine (CYLD Y15F). B, Immunoprecipitation of Flag-CYLD WT and Flag-CYLD Y15F mutant using anti-Flag antibodies under stringent conditions, followed by immunoblotting with anti-pTyr antibodies. C, Lysates from cells with reconstituted expression of CYLD WT or CYLD Y15F were subjected to immunoprecipitation of EGFR. Precipitated complexes as well as whole cell lysates (WCL) were subjected to immunoblotting with the indicated antibodies. D, Quantitative PRM analysis of Cbl-b immunoprecipitated complexes from CYLD-silenced cells (shCYLD) and cells reconstituted with either CYLD WT or CYLD Y15F mutant. E, Whole cell lysates from shCYLD cells and cells reconstituted with either CYLD WT or CYLD Y15F mutant were subjected to immunoblotting to investigate the degradation of EGFR upon EGF stimulation for the indicated times. F, Quantitative PRM measurements of EGFR levels from the same cells and conditions as in panel E.