CYLD) Deubiquitinase Is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor

University of Southern Denmark

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

Sanchez-Quiles, Virginia; Akimov, Vyacheslav; Osinalde, Nerea; Francavilla, Chiara; Puglia, Michele; Barrio-Hernandez, Inigo; Kratchmarova, Irina; Olsen, Jesper V; Blagoev, Blagoy

Published in: Molecular and Cellular Proteomics

DOI: 10.1074/mcp.M116.066423

Publication date: 2017

Document version: Final published version

Citation for pulished version (APA): Sanchez-Quiles, V., Akimov, V., Osinalde, N., Francavilla, C., Puglia, M., Barrio-Hernandez, I., Kratchmarova, I., Olsen, J. V., & Blagoev, B. (2017). Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor. Molecular and Cellular Proteomics, 16(8), 1433-1446. https://doi.org/10.1074/mcp.M116.066423

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© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. This paper is available on line at http://www.mcponline.org Cylindromatosis Tumor Suppressor Protein (CYLD) Deubiquitinase is Necessary for Proper Ubiquitination and Degradation of the Epidermal Growth Factor Receptor*□S

Virginia Sanchez-Quiles‡¶, Vyacheslav Akimov‡, Nerea Osinalde‡**, Chiara Francavilla§, Michele Puglia‡, Inigo Barrio-Hernandez‡, Irina Kratchmarova‡, Jesper V. Olsen§, and Blagoy Blagoev‡ ‡‡

Cylindromatosis tumor suppressor protein (CYLD) is a Epidermal Growth Factor Receptor (EGFR)1 belongs to the deubiquitinase, best known as an essential negative reg- family of receptor tyrosine kinases (RTKs) and plays a crucial ulator of the NFkB pathway. Previous studies have sug- role in the maintenance of a correct cellular homeostasis, gested an involvement of CYLD in epidermal growth fac- controlling central processes such as cell proliferation, migra- tor (EGF)-dependent signal transduction as well, as it tion, differentiation or survival (reviewed in (1)). EGFR consists was found enriched within the tyrosine-phosphorylated of an extracellular domain for the recognition of the ligands, a complexes in cells stimulated with the growth factor. EGF single pass transmembrane region and an intracellular tyro- receptor (EGFR) signaling participates in central cellular sine kinase- containing domain. The binding of a ligand, such processes and its tight regulation, partly through ubiquitination cascades, is decisive for a balanced cellular as Epidermal Growth Factor (EGF), promotes the dimerization homeostasis. Here, using a combination of mass spec- of the receptor and the subsequent activation of its kinase trometry-based quantitative proteomic approaches with activity, which further leads to the autophosphorylation of biochemical and immunofluorescence strategies, we tyrosine residues on the intracellular region of the EGFR (1, 2). demonstrate the involvement of CYLD in the regulation These modified residues act as docking sites for recruiting of the ubiquitination events triggered by EGF. Our data SH2 or PTB domain- containing signaling proteins (3, 4), show that CYLD regulates the magnitude of ubiquitina- hence the stimulation triggers the association of large intra- tion of several major effectors of the EGFR pathway by cellular complexes that support rapid spread and amplifica- assisting the recruitment of the ubiquitin ligase Cbl-b to tion of the signal, eventually resulting in a specific cellular the activated EGFR complex. Notably, CYLD facilitates output (5). the interaction of EGFR with Cbl-b through its Tyr15 To facilitate an adequate response in intensity and duration, phosphorylation in response to EGF, which leads to fine-tuning of the receptor’s ubiquitination and subsequent the downstream events following the activation of the recep- degradation. This represents a previously uncharacterized tor necessitate tight negative regulation that counteracts the strategy exerted by this deubiquitinase and tumors sup- positive signals. In this regard, the attachment of ubiquitin pressor for the negative regulation of a tumorigenic signal- moieties to the EGFR plays a key role in directing its internal- ing pathway. Molecular & Cellular Proteomics 16: ization and further endocytic trafficking that eventually leads 10.1074/mcp.M116.066423, 1433–1446, 2017. to its lysosomal degradation or recycling (6, 7). Hence, the regulated turnover of EGFR is pivotal for a correct cellular From the ‡Department of Biochemistry and Molecular Biology, output. E3 ubiquitin ligases of the casitas B-lineage lym- University of Southern Denmark, 5230 Odense M, Denmark; §Pro- phoma (Cbl) family play a crucial part in this chain of events. teomics Program, The Novo Nordisk Foundation Center for Protein Cbl proteins are recruited to activated receptors, either bind- Research, Faculty of Health and Medical Sciences, University of Copenhagen, DK-2200 Copenhagen, Denmark ing directly to their phosphorylated tyrosine residues or Received December 14, 2016, and in revised form, May 8, 2017 through the assistance of other adaptor proteins (8), thereby Published, MCP Papers in Press, June 1, 2017, DOI 10.1074/ mcp.M116.066423 Author contributions: V.S. and B.B. designed the project and wrote 1 The abbreviations used are: EGFR, Epidermal Growth Factor the paper; V.S., V.A., N.O., M.P., I.B., I.K., J.O., and B.B. analyzed Receptor; Cbl, Casitas B-lineage Lymphoma; DUB, deubiquitinase; data; V.S. performed the experiments resulting in figures 1–5; V.S. EGF, Epidermal Growth Factor; GO, Gene ontology; PRM, Parallel and V.A. performed the StUbEx analyses; V.S. and C.F. performed the Reaction Monitoring; pY, pTyr, phosphorylated tyrosine; RTK, Re- experiments resulting in figure 6; V.S., V.A., and M.P. performed ceptor Tyrosine Kinease; shCYLD, CYLD-silenced; StUbEx, Stable the experiments resulting in figure 7; C.F., I.K., and J.O. edited the Tagged Ubiquitin Exchange System; SILAC, Stable isotope labeling manuscript. by amino acids in cell culture; WT, wild type.

Molecular & Cellular Proteomics 16.8 1433 CYLD Contributes to Ubiquitination and Degradation of EGFR ubiquitinating the EGFR, which can in turn be recognized and hence reinforcing and expanding the mechanisms underlying sorted by the endocytic machinery (9, 10). In this context, its role as a tumor suppressor. further level of signaling modulation can be reached by ubiquitin hydrolases (deubiquitinases, DUBs), which may oppose EXPERIMENTAL PROCEDURES the activity of Cbl ligases by removing ubiquitin moieties from Cell Culture—Human cervix epithelial adenocarcinoma HeLa cells the receptor (11, 12). These latter enzymes can thereby have were grown in Dulbecco’s modified Eagle’s medium (DMEM, Lonza, Basel, Switzerland) supplemented with 10% Fetal Bovine Serum, 1% a decisive role for the final cellular response. Penicillin-Streptomycin, and 1% L-Glutamine. Ovarian and laryngeal Cylindromatosis protein (CYLD) is a tumor suppressor that carcinoma cells (Skov3 and Hep2, respectively) were cultured using displays a specific ubiquitin hydrolase activity for K63-linked the same medium. For labeling experiments, cells were grown in light, polyubiquitin chains (13). Negative regulatory actions of CYLD medium or heavy DMEM media, containing either L-arginine (Arg0) 13 14 2 and L-lysine (Lys0), L-arginine- C6 N4 (Arg6) and L-lysine- H4 (Lys4) have been reported for several signaling paradigms, the most 13 15 13 15 or L-arginine- C6 N4 (Arg10) and L-lysine- C6 N2 (Lys8), respec- explored to date being its role in the NF-kB pathway (14–16). tively, as previously described (27). For EGF stimulation, cells were It has also been shown an involvement of CYLD in the control grown to a confluency of 70%, starved without serum for 16 h and of essential cellular processes as apoptosis (14–16), inflam- stimulated with 150 ng/ml of EGF for the indicated times. When using mation (17), proliferation (18) and tumorigenesis (19). The inhibitors, those were added to the starving media 30 min prior the stimulation and present while treating with the growth factor. EGFR downregulation of this DUB has been linked with oncogenesis inhibitors (Iressa and CI1033) and Akt inhibitor MK2206 were pur- in different cellular contexts including melanoma (20), my- chased from Selleckchem (Houston, TX) whereas MEK inhibitor eloma (21), uterine cervix carcinoma (22), hepatocellular and U0126 was from Promega (Madison, WI). Where indicated, 10 ␮g/ml colon carcinoma (23). cycloheximide (Sigma-Aldrich, St. Louis, MO) was used to block the de novo protein synthesis. To our knowledge, the investigation of CYLD and its spec- RNAi-based Silencing and Rescue Expression of Wild Type and trum of actions have mostly been carried out through targeted Y15F Mutant of CYLD—DNA constructs for RNAi silencing of CYLD approaches that explored, one at a time, the different regula- and generation of stable cell lines were performed using a lentiviral tory strategies of this DUB on specific, discrete targets. How- system as described before (33). We used the following targeting Ј Ј ever, advances in quantitative mass spectrometry (MS)-based sequences for RNAi: 5 -GCAATATGACGAGTTAGTA-3 for shControl and 5Ј-GGGTAGAACCTTTGCTAAA-3Ј for shCYLD. Depletion of proteomics allow studies of signaling cascades in a system- CYLD was confirmed by real-time PCR and immunoblotting (see wide, unbiased manner for uncovering novel molecular char- Supplemental Data for details). A sequence-verified cDNA clone of acters with a key part in the transduction mechanisms. Cur- CYLD was acquired from I.M.A.G.E. consortium (IRAUp969D0565D). rently, MS-based quantitative proteomics represents a powerful RNAi-resistant cDNA of CYLD was created by mutating 8 nucleotides (indicated in capitals below) in the sequence targeted by the shRNA, tool for the comprehensive characterization of signaling net- but maintaining the amino acid code. The following primer was used works (24, 25), including their dynamics changes (26–28), for this purpose: 5Ј-ttgaatattctgtttcatcatattttaagAgtTgaGccCCtCTtG- protein interactions (29, 30) and numerous post-translational aaaataagatcagcaggtcaaaagg-3Ј. Using methods of conventional modifications (31). Indeed, the first evidences pointing to an cloning, the RNAi-resistant cDNA was then sub-cloned into the shCYLD vector with the Flag-epitope sequence at the N terminus and involvement of CYLD in the molecular events downstream under control of EF1a promoter (construct named shCYLD-Flag-WT- the EGFR were obtained by MS-based proteomics (32). CYLD). A mutant expressing CYLD with tyrosine exchanged with Following addition of EGF, the presence of CYLD among the phenylalanine at position 15 was created by conventional methods of tyrosine-phosphorylated complexes showed a very rapid site-directed DNA mutagenesis (construct named shCYLD-Flag- and strong increase, which could indicate a role for this Y15F-CYLD). HeLa cells with CYLD KD (shCYLD) were used to create clones expressing shCYLD-Flag-WT-CYLD and shCYLD-Flag-Y15F- deubiquitinase in the RTK signaling and make CYLD-related CYLD constructs with the lentiviral delivery method as described ubiquitination events in EGFR pathway worth investigating. above. Here, we conducted a global analysis of the cellular ubiq- Immunoblotting, Immunoprecipitation, and Immunofluorescence— uitinome by employing a recently described approach termed Antibodies used for immunoblotting were from Santa Cruz Biotech- nology (Dallas, TX) (CYLD, Cbl-b), BD Transduction Laboratories (San StUbEx (Stable Tagged Ubiquitin Exchange) followed by MS- Jose, CA) (c-Cbl), Millipore (Billerica, MA) (EGFR), Enzo Life Sciences based quantitative proteomics (33). This, in combination with (Farmingdale, NY) (Ubiquitin), Sigma (␣-Tubulin), or Cell Signaling protein and peptide pull-down assays, uncovered an unex- Technology (Danvers, MA) (phospho-Akt Ser473, Akt, phosphor- pected role for CYLD as a key factor for proper ubiquitination Erk1/2 Thr202/Tyr204, Erk). of the EGFR and downstream signaling components. Namely, For immunoprecipitation experiments, proteins were extracted in ice-cold modified RIPA buffer (50 mM Tris/HCl pH 8.0, 150 mM NaCl, ubiquitination state of the activated receptor is compromised 1% (v/v) Nonidet P-40, 0.25% Sodium Deoxycholate) supplemented in the absence of CYLD, which results in a decreased receptor with proteases and phosphatases inhibitors (both from Roche). The degradation. We demonstrate herein that CYLD interacts with antibodies used for immunoprecipitation of phosphorylated-tyrosine the ubiquitin ligase Cbl-b, contributing to the recruitment of containing complexes were from Millipore (clone 4G10) and from Cell Signaling Technology (P-Tyr-100). Antibodies sc-120 and 06-847, the later to the EGFR in response to EGF stimulation. Our from Santa Cruz Biotechnology and Millipore respectively, were used findings provide a yet uncharacterized and intriguing strategy for the immunoprecipitation of the EGFR. CYLD and Cbl-b proteins of CYLD for the negative modulation of signaling pathways, were immunoprecipitated using the antibodies sc-137139, sc-1704

1434 Molecular & Cellular Proteomics 16.8 CYLD Contributes to Ubiquitination and Degradation of EGFR

and sc-8006, from Santa Cruz Biotechnology. For control immuno- as variable modification for the searches from the peptide-pull-downs precipitations, an antibody against the GFP protein (sc-9996, Santa and CYLD-IP experiments, whereas di-glycyl variable modification on Cruz Biotechnology) was used. For immunoprecipitation of the Flag- lysine was set in the StUbEx searches to account for protein ubiq- tagged WT and Y15F CYLD, lysates were prepared in RIPA buffer, uitination. Carbamidomethylation of cysteine was set as fixed modi- further incubated for 30 min with 1% SDS to disrupt protein-protein fication for all experiments. The maximum number of modifications interactions and then diluted to 0.05% SDS with RIPA buffer to accepted per peptide was five and the minimum peptide length was perform the immunoprecipitation using Anti-Flag M2 Affinity Agarose set to seven amino acids. Trypsin was used as protease with a Gel (Sigma-Aldrich). maximum of two missed cleavages. Peptides were identified with For immunofluorescence, HeLa cells were transfected with GFP- mass tolerance of 7 ppm for precursors and 20 ppm for fragment Rab7 plasmid and lipofectamine as described previously (34). Anti- ions. Both peptide and protein maximum false discovery rates (FDR) body sc-120 from Santa Cruz was used for detection of EGFR (see were set to 0.01 based on the target-decoy approach. Known com- supplemental Data for details). mon contaminants, as specified in MaxQuant, we excluded from the StUbEx and Peptide Pull-down Analyses—For the enrichment of identification. ubiquitinated proteins, shControl and shCYLD HeLa cells containing Parallel Reaction Monitoring (PRM) Analysis—All PRM analyses the StUbEx construct (33) were grown in the presence of doxycycline were performed on a Q Exactive HF (Thermo Scientific) using the 60 h prior the experiment. Cells were subjected to serum starvation same LC-MS/MS system previously described with the following and stimulated with 150 ng/ml of EGF as indicated. To study the four method modifications. Precursor m/z of proteotypic target peptides conditions of interest, two paralleled triple SILAC experiments were for EGFR, Cbl-b, CYLD, and FLNA (supplemental Table S8) have been performed per replica (we carried out two biological replicas, thus selected from previous top 12 data-dependent runs and added to the corresponding to a total of four triple SILAC experiments; the exper- instrument inclusion list. MS/MS spectra of target peptides were imental design of one replica is exemplified in Fig. 2A). Cells were acquired in unscheduled PRM mode using a resolution of 60,000 (at lysed in a 50 mM phosphate buffer pH 8.0 containing 6 M Guanidine- m/z 200) and an AGC target of 5e5 with a maximum injection time of HCl and 500 mM NaCl. The subsequent enrichment of ubiquitinated 150–200ms (37, 38). Fragmentation was performed in HCD mode proteins was performed as described previously (33). with normalized collision energy of 28. PRM data analysis was carried For immunoprecipitation and peptide pull-down of CYLD, cells out on Skyline™ 3.6.0 software (39). Spectral libraries were built using MaxQuant ms/ms search files. Resolving power and mass analyzer were lysed in ice-cold modified RIPA buffer (50 mM Tris/HCl pH 8.0, were set respectively to 60.000 and Orbitrap. Area under the curve 150 mM NaCl, 1% (v/v) Nonidet P-40, 0.25% Sodium Deoxycholate) containing proteases and phosphatases inhibitors (both from Roche). (AUC) values relative to the 4–6 most intense product ions for each Lysates were cleared and protein extracts from the differently labeled peptide were exported and plotted in a matrix which was used for conditions were quantified. CYLD antibody (Santa Cruz Biotechnol- further statistical analysis (supplemental Table S8). Total MS2 frag- ogy) coupled to protein A Sepharose beads was used for the immu- ments AUC values for EGFR recycling (3 biological replicas) and noprecipitation. The peptide pull-down experiments were performed Cbl-b Co-IP (2 biological replicas) analysis were normalized using the housekeeping protein Filamin-A or the Co-IP antigen Cbl-b, respec- as described (35), using the following sequences: SGSQEKVTSP-pY- tively. All the Skyline data, MaxQuant searches, and .raw files have WEERIF and the unmodified counterpart SGSQEKVTSPYWEERIF. An been deposited to the ProteomeXchange Consortium via the PRIDE empty-beads pull-down was carried out in the light condition to partner repository. discriminate unspecific background proteins (see supplemental Data Experimental Design and Statistical Rationale—Four triple SILAC for details). experiments corresponding to two full biological replicas were per- NanoLC Tandem Mass Spectrometry (LC-MS/MS) and Data Anal- formed for the described StUbEx experiments. Two biological repli- ysis—Peptides were separated by reversed-phase in an EASY-nLC cates were performed for peptide pull-down experiments per exper- 1000 (Thermo Scientific) coupled to a mass spectrometer equipped iment, swapping medium and heavy SILAC conditions to avoid any with a nanoelectrospray ion source. The samples were analyzed in technical bias. A control condition, in which the light SILAC protein either an LTQ Orbitrap Velos or a Q Exactive (Thermo Scientific). For extract was incubated with empty beads, was included in both pull- the chromatographic separation, solvent A was 0.5% acetic acid and down experiments, helping to discriminate background and contam- solvent B was 80% acetonitrile in 0.5% acetic acid. The peptides inant signals. In the case of CYLD immunoprecipitation, two biological were eluted from an analytical in-house packed column of ReproSil replicates we performed as well. Pur C -AQ, 3 ␮m resin (Dr Maisch GmbH), at a flow rate of 250 18 The visualization of overlapping protein identifications was per- nL/min. The mass spectrometers were operated in positive ionization formed using the BioVenn web application (http://www.cmbi.ru.nl/ mode, in a top 12 data-dependent manner, at a resolution of 70,000 cdd/biovenn/), whereas the significance of the changes in SILAC (at m/z 400) and AGC target of 1e6 for the MS survey, scanning from protein ratios were based on significance B calculations inbuilt in the 300 to 1750 m/z. For the MS/MS analysis, resolution was set to MaxQuant/Perseus software and visualized with GProX (40). The 35,000 (at m/z 400), AGC target to 1e5, minimum intensity to 4e4 and analysis of functional groups and GO term categories were carried out isolation window to 2.0 m/z. To minimize the repeated fragmentation in MetaCore (Thomson Reuters), which relies on Fisher statistical test of ions, an exclusion time of 45 s was programmed. The maximum with an FDR adjusted p values. injection time values for survey and MS/MS scans were 120ms and 124ms, respectively. RESULTS Raw data files were analyzed with MaxQuant (36) version 1.3.0.5, which includes the Andromeda search engine. Peak lists were CYLD is Enriched in Early Tyr-phosphorylated Complexes searched against human UniProt database version 2014.01 (88479 Upon EGF Stimulation—EGFR pathway drives central cellular sequence entries). Three SILAC labeling channels were set, corre- processes and, hence, its fine-tuned regulation is crucial for sponding to Lys0/Arg0, Lys4/Arg6 and Lys8/Arg10 for the light, me- the maintenance of a correct cellular homeostasis. Therefore, dium and heavy conditions, respectively. Variable modifications for the searches included N-terminal protein acetylation, methionine ox- the characterization of the molecular events downstream this idation, deamidation of asparagine and glutamine. In addition, phos- RTK constitutes a powerful strategy for understanding both phorylation on serine, threonine and tyrosine residues was included healthy and pathological mechanisms of the cell. In previous

Molecular & Cellular Proteomics 16.8 1435 CYLD Contributes to Ubiquitination and Degradation of EGFR

A B Input (4%) Input (4%)

IP EGFR IP EGFR pTyr CYLD pTyr CYLD

EGFR EGFR CYLD WCL CYLD WCL αTubulin αTubulin 6’ 6’ 6’ 0’ 1’ 6’ 0’ 1’ 1’ 1’ 15’ 15’ 15’ 15’ 30’ 60’ EGF 150 ng/ml 120’ EGF 150 ng/ml

DMSO Iressa CI1033

C D

1 CYLD EGF 0’ 6’ 0’ 6’ 0’ 6’ 150 ng/ml αTubulin

0.5 UbiquitinIB shControl +- shCYLD -+ Relative Expression Relative 0 shControl shCYLD IB phospho-Tyr IP pTyr

CYLD WCL αTubulin

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. proteomics studies aiming at gaining insights into the molec- To assess whether the enrichment of CYLD is dependent ular players involved in the EGF-induced response, we re- on EGFR activation, we performed the previous experiments vealed a very strong increment of the deubiquitinase CYLD in the presence of Canertinib (CI1033) or Gefinitib (Iressa), within the tyrosine-phosphorylated complexes formed upon inhibitors of the EGFR kinase activity. CYLD enrichment was EGF stimulation (32). To corroborate this finding, we per- abrogated when the activity of the receptor was abolished formed a protein immunoprecipitation using antibodies (Fig. 1B) but not when inhibiting the two main signaling against phosphorylated tyrosine residues followed by immu- branches downstream of the EGFR: the MAPK and the PI3K- noblotting, thus confirming the involvement of CYLD within Akt pathways (supplemental Fig. S1A and S1B, respectively). the complexes engaged in response to EGF. The presence of These results, together with the very rapid enrichment of this DUB was very rapidly increased, being evident just 1 min CYLD after the stimulation, indicated that the deubiquitinase after the addition of the ligand and declining at 15 min post- could play a role in the very early signaling processes after stimulation (Fig. 1A). Interestingly, this dynamic enrichment EGFR activation, independently of the subsequent feedback profile parallels that of the EGFR itself, which is nevertheless events. stronger. Furthermore, the signal corresponding to the en- CYLD Deficiency Impairs the Ubiquitination of Early Protein riched CYLD after the stimulation appeared to represent only Effectors of the EGF Pathway—Taking into account the ubiq- a small proportion of the total amount of this protein ex- uitin hydrolase activity of CYLD and that many of the proteins pressed in the cell (input lysate, Fig. 1A, first lane on the right), in the EGFR signaling network undergo ubiquitination, includ- indicating that merely a slight fraction of this deubiquitinase is ing K63 polyubiquitination (41–43), we reasoned that CYLD engaged in the process. could be responsible for deubiquitinating EGFR signaling pro-

1436 Molecular & Cellular Proteomics 16.8 CYLD Contributes to Ubiquitination and Degradation of EGFR teins following ligand stimulation. To examine that, we si- and compared their corresponding enrichment to the CYLD- lenced the expression of CYLD, achieving at least 80% de- silenced condition. Applying very stringent criteria (signifi- crease at the mRNA and protein levels (Fig. 1C) and efficiently cance B, p Ͻ 0.001 for each replica), we detected a set of 11 depleted CYLD from the EGF-dependent tyrosine-phosphor- protein groups with consistently increased EGF-dependent ylated complexes (as shown in supplemental Fig. S1C). We ubiquitination in control cells (Fig. 2C, supplemental Fig. S3A, next explored the impact of the silencing on the ubiquitination S3B and supplemental Table S3), in agreement with previous status of the proteins involved in the EGF pathway. Surpris- studies on the EGF-dependent ubiquitinome (33, 43). Accord- ingly, we found that the downregulation of the deubiquitinase ingly, the GO-term annotations for these 11 proteins in re- did not lead to an increase, but rather diminished the overall spect to function and localization were related to the EGFR/ ubiquitination signal of the engaged proteins after EGF addi- ErbB signaling pathways and the corresponding membrane tion (Fig. 1D). To gain deeper insight into this intriguing ob- structures and complexes (supplemental Fig. S3C,S3D and servation, we resolved to explore the EGF-induced ubiquiti- supplemental Table S4). Interestingly, all 11 proteins dis- nation events upon CYLD downregulation in a system-wide, played much lower levels of enrichment in response to EGF unbiased manner. from the CYLD-silenced cells (shCYLD) compared with the We used quantitative MS-based proteomics coupled to cells with normal CYLD expression (shControl) (Fig. 2C). To StUbEx, a strategy that has proven successful for large-scale visualize more clearly the effect of CYLD downregulation on investigation of ubiquitination events (33). Briefly, in the the ubiquitination status of these molecular effectors, we nor- StUbEx system the endogenous ubiquitin is replaced by a dual malized the SILAC ratios from the different conditions to the tagged version containing a tandem 6xHis sequence and a Flag shControl-unstimulated cells (Fig. 2D and supplemental Fig. tag, whereas the total pool of ubiquitin in the cell remains at S3E). The impact of CYLD deficiency on the EGF-induced physiological levels. Ubiquitinated proteins can then be en- ubiquitination changes appeared to be the most significant riched through a two steps procedure, making use of the 6xHis (significance B, p Ͻ 0.001 in each replica) for EGFR, Cbl-b, and Flag tags contained within their ubiquitin moieties (33). UBASH3B and SHC1 (supplemental Table S5). We combined StUbEx with shRNA silencing of CYLD and In summary, the unbiased investigation of the ubiquitination stable isotope labeling by amino acids in cell culture (SILAC) signaling through StUbEx and quantitative proteomics al- (44) to provide a quantitative proteomic view of the changes in lowed us to decipher the effect of CYLD silencing in the protein ubiquitination upon the downregulation of the DUB ubiquitination events following EGF stimulation. Notably, the (Fig. 2A). We performed 4 triple SILAC experiments corre- results suggested an indirect role of CYLD in the RTK path- sponding to two biological replicas (each replica consisted of way, as the downregulation of this DUB triggered an unex- two triple SILAC experiments; Fig. 2A) and consistently quan- pected decrease in the EGF-dependent ubiquitination state of tified 1812 protein groups across them (FDRϽ0.01; Fig. 2B several major molecular characters of the pathway including and supplemental Table S1). Only these proteins quantified in the EGFR, Cbl-b, UBASH3B, and SHC1. all four SILAC experiments were used for the subsequent CYLD is Phosphorylated on Tyr15 upon EGF Stimulation—A analysis. Considering that the ubiquitinated proteome is of great part of the signaling cascades subsequent to the RTKs relatively low abundance in the cell (45), the obtained extent of activation rely on protein-protein interactions mediated through quantified proteins was rather satisfactory. In addition, the the phosphorylation of tyrosine residues and proteins contain- quantitation reproducibility of the measurements was very ing PTB or SH2 domains (46, 47). Our previous results dem- high, with Pearson correlation values between the conditions onstrated the involvement of CYLD within the phospho-tyro- common to all 4 SILAC experiments (medium over light) rang- sine protein complexes following EGF stimulation (Fig. 1A). ing between 0.92 and 0.96 (supplemental Fig. S2A and sup- However, this DUB does not contain any PTB or SH2 domains plemental Table S1). Gene ontology (GO) analysis of the iden- and, hence, we hypothesized that its EGF-dependent enrich- tified proteins demonstrates that there is no bias of the ment was caused by its own tyrosine phosphorylation. To technique for any molecular network or pathway (supplemen- examine that, we used a SILAC-based approach coupled to tal Fig. S2B and supplemental Table S2), as their associated immunoprecipitation of CYLD from wild type (WT) HeLa cells biological processes were mostly metabolic and organizational, that were either left unstimulated or treated with EGF for 6 representing the general functions of the cell. Moreover, the min (Fig. 3A). To discriminate potential background signals, enriched proteins originated from different sub-cellular loca- shCYLD cells were utilized for the light (Lys0/Arg0) condition tions, covering from cytosol to nuclear and membrane organ- in our experimental design. The SILAC ratios corresponding elles (supplemental Fig. S2C and supplemental Table S2), to the CYLD protein were more than 20-fold higher in the WT hence displaying no bias for a particular subcellular fraction. over CYLD-silenced cells, correlating well with our earlier To identify potential regulatory effects of CYLD in the mo- estimation of ϳ80% silencing efficiency, whereas the enrich- lecular characters of the EGF pathway, we focused our anal- ment of CYLD in the WT cells 6ЈEGF/unstimulated remained ysis on proteins whose ubiquitination status significantly unchanged as expected (Fig. 3B and supplemental Table S6). changed in response to the growth factor in the control cells Several serine-phosphorylated peptides derived from CYLD

Molecular & Cellular Proteomics 16.8 1437 CYLD Contributes to Ubiquitination and Degradation of EGFR

A Lys0, Arg0 Lys4, Arg6 Lys8, Arg10 B 4 2 Log2 (M/L_1) REPLICA 1 0

Exp 1 Exp shCYLD shControl shCYLD -8 -6 -4 -2 0 2 4 -2 Unstimulated 6’ EGF 6’ EGF Qed -4 1812 Pearson Corr = 0.93 -6 (M/L_3) Log2 shCYLD shControl shControl Exp 2 Exp Unstimulated 6’ EGF Unstimulated 4 REPLICA 2 2 Log2 (M/L_3) 0 Combine Lysates 1:1:1 from each experiment -8 -6 -4 -2 0 2 4 -2 -4 Pearson

Enrichment of Corr = 0.96 -6 (M/L_4) Log2 ubiquitinated Bead Ni2+ proteins using Ubi K 6xHistidines tag C Log2 Ratio 6’EGF / Unstimulated Enrichment of Bead shCYLD shControl ubiquitinated Ubi K EGFR proteins using Cbl-b FLAG tag SHC1 INPPL1 CALM2; CALM1 LysC + Trypsin Digestion EPS15L1 Peptide Fractionation PEAK1 LC-MS/MS EPS15 GRB2 UBASH3B Intensity m/z ASAP1

Data Analysis 0 0.5 1 1.5 2 2.5 3 3.5 4

Log2 Ratio value

D 16

shControl Unstimulated shControl 6’EGF 12 Units Units shCYLD Unstimulated 8 shCYLD 6’EGF Enrichment 4 Relavive

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.

1438 Molecular & Cellular Proteomics 16.8 CYLD Contributes to Ubiquitination and Degradation of EGFR

y y y y y y y y A C 8 7 6 5 4 3 2 1 y series - V T S P Y W E E R -

b series Pi b2 b5 Lys0, Arg0 Lys4, Arg6 Lys8, Arg10 y6 Intensity [10e5] y y 4 y shCYLD Wild Type Wild Type 1 7 b b5 6’ EGF Unstimulated 6’EGF 2 y2 y3 pY y5 Relative Abundance Abundance Relative y8 CYLD IMMUNOPRECIPITATION m/z

D SDS-PAGE Protein Separation WT 6’ WT 6’ EGF EGF

LC-MS/MS WT WT

Abundance 0’ EGF shCYLD 0’ EGF shCYLD Abundance Intensity Intensity

m/z m/z Data Analysis E Ratio WT 6’EGF / Unstimulated B phosphorylated non-phosphorylated CYLD CYLD protein Protein SILAC ratios Tyr15 peptide Tyr15 peptide Unique Sequence M/L H/L H/M >10 0.99 1.03 peptides coverage Unstimulated - stoichiometry of pTyr15 ~ 0.45% 47 55.8 23.71 24.18 1.03 6'EGF - stoichiometry of pTyr15 ~ 4.44%

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. were detected in this experiment, however their phosphoryl- no presence of this modified site, the EGF stimulation pro- ation status was unaffected by the EGF stimulation (supple- moted Tyr15 phosphorylation on ϳ4% or less of the total pool mental Table S6). On the other hand, one of the identified of CYLD in the cells (Fig. 3E). This data, together with our modified peptides contained a tyrosine phosphorylated site, previous results (Fig. 1A), indicated again that only a small corresponding to the residue 15 (Fig. 3C). Manual inspection fraction of the total cellular pool of this DUB is involved in the of the mass spectrometry signal showed a large increase of its EGF signaling pathway. Accordingly, CYLD appears to be ubiq- abundance at the EGF-stimulated condition (Fig. 3D). Al- uitously present in the cell and has been described in several though the intensities of the pY15-containing peptide in the subcellular locations (49, 50), implying that it can therefore play light and medium channels (corresponding to shCYLD cells distinct roles dependent on its molecular environment. and WT unstimulated cells, respectively) were not sufficiently Cbl-b is Recruited to the pY15 of CYLD—Taking into ac- above the signal-to-noise level to allow calculation of SILAC count that phosphorylated tyrosine residues often act as ratios, the profile of the corresponding signals evidenced a docking sites for the interaction with SH2- or PTB domain- strong increment of at least 10-fold of the phosphorylated containing proteins (47), we next investigated a potential abil- form in the EGF stimulated WT cells. To estimate the fraction ity of CYLD pY15 to recruit molecular effectors belonging to of CYLD that responded to the growth factor treatment, we the EGF pathway. We implemented a quantitative proteomics calculated the stoichiometry of pY15 according to the SILAC approach based on an affinity pull-down strategy using as ratios obtained from the corresponding unmodified peptide bait the Tyr15-containing peptide in phosphorylated or un- and the protein, as described elsewhere (48). Assuming a modified form (Fig. 4A). Two replicates were performed, pY15 6ЈEGF/unstimulated ratio of at least 10, our results swapping the medium and heavy SILAC conditions in order to suggested that, although at the basal state there is practically avoid any technical bias of the method, and we quantified a

Molecular & Cellular Proteomics 16.8 1439 CYLD Contributes to Ubiquitination and Degradation of EGFR

A B C 6 Lys0 Arg0 Lys4 Arg6 Lys8 Arg10 REPLICA 1 4 CHST3 UBASH3B CBL-B Qed 2 pY15/Y15 Rep2 WT WT WT 1451 0 & 6min EGF 0 & 6min EGF 0 & 6min EGF 0 pY15/Y15 Rep1 REPLICA 2 -2

PEPTIDE PULL-DOWN -4

-6 -6 -4 -2 0 2 4

D 10

8 Empty Tyr15 pTyr15 CBL-B UBASH3B

6 CHST3 Combination of beads + Log10 Intensity SDS-PAGE Protein Separation + 4 In Gel Tryptic Digestion -4 -2 0 2 4 Log2 pTyr15 / Tyr15 E LC-MS/MS

Unique Unique sequence Log2 pTyr15/Tyr15 Log2 Log10

Proteins peptides coverage (%) p value pTyr15/Tyr15 Intensity

Intensity CHST3 11 26.3 1.32E-19 3.09 6.54 UBASH3B 14 27.9 6.66E-17 2.87 6.67 m/z Data Analysis CBLB 16 19.2 5.14E-15 2.67 6.56

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)ofthe 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. total of 1451 protein groups in both replicas (Fig. 4B and factor stimulation and that this DUB appears to recruit Cbl-b, we supplemental Table S7). CHST3, UBASH3B and Cbl-b dis- hypothesized that CYLD could be involved in the regulation of played the most selective binding to the phosphorylated the EGFR ubiquitination through its binding with Cbl-b. Notably, Tyr15-containing peptide with more than 5-fold enrichment Cbl ligases have already been described not only to interact over the non-phosphorylated version of the peptide (Signifi- directly with the receptor (52, 53) but also to be recruited by cance B, p Ͻ 0.001; Fig. 4C,4D and supplemental Table S7). accessory adaptor proteins to the activated complexes follow- Notably, UBASH3B and Cbl-b are well known molecular char- ing EGF stimulation (54). Here, our data provided cues suggest- acters involved in the ubiquitination-dependent EGFR signaling such a role for the CYLD deubiquitinase as well. ing and both were already identified as proteins with a de- Downregulation of CYLD Impairs EGFR Ubiquitination creased EGF-dependent ubiquitination upon CYLD deficiency Through a Deficient Recruitment of Cbl-b to the Receptor—To (Fig. 2). The presence of Cbl-b was of interest, as it contains corroborate the involvement of CYLD in the events described a phospho-tyrosine binding domain and also represents a E3 above, we first immunoprecipitated EGFR from control and ligase responsible for the ubiquitination of several effectors of CYLD silenced cells, followed by immunoblotting. In agree- the EGF-dependent signaling cascades, including the EGFR ment with our quantitative proteomics analysis (Fig. 2), we (51). Considering that the silencing of CYLD showed an unex- observed a decrease of growth factor-dependent ubiquitina- pected impairment of the ubiquitination downstream the growth tion of the receptor in CYLD deficient HeLa cells (Fig. 5A). As

1440 Molecular & Cellular Proteomics 16.8 CYLD Contributes to Ubiquitination and Degradation of EGFR

A B D

Ubi Cbl-b IP IP EGFR IP EGFR pTyr EGFR EGFR c-Cbl CYLD Cbl-b Cbl-b CYLD WCL c-Cbl α-Tubulin EGFR WCL CYLD EGF 150 ng/ml 0’ 6’ 0’ 6’ Cbl-b α-Tubulin WCL CYLD EGF 150 ng/ml 0’ 6’ 0’ 6’ α-Tubulin

EGF 150 ng/ml 0’ 6’ 0’ 6’

E SKOV3 HEP2

C IP Ubi EGFR EGFR IP Cbl-b IP GFP WCL Input IP Cbl-b CYLD EGFR Cbl-b CYLD αTubulin Cbl-b 0’ 6’ 0’ 6’ EGFR CYLD WCL CYLD EGF 150 ng/ml 0’ 6’ 0’ 6’ 0’ 6’ 0’ 6’ α-Tubulin EGF 150 ng/ml 0’ 6’ 0’ 6’ 0’ 6’ 0’ 6’

FIG.5.CYLD facilitates ligand-dependent recruitment of Cbl-b to EGFR and subsequent receptor ubiquitination. A, Immunoprecipi- tation (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. further seen on Fig. 5A, the interaction of EGFR and Cbl-b 5A). Furthermore, reduced amounts of EGFR were seen in the was clearly impaired in CYLD-silenced cells, consolidating the Cbl-b immunoprecipitated complexes from the CYLD-si- notion that CYLD is in part responsible for the recruitment of lenced cells (Fig. 5C), in correlation with the results from the this E3 ligase to the receptor. Accordingly, this also resulted in EGFR co-immunoprecipitation experiments shown in Fig. 5A. decreased levels of Cbl-b in the tyrosine-phosphorylated Consistent with the proposed role of CYLD in assisting the complexes upon EGF stimulation (Fig. 5B). In contrast, the interaction between the receptor and Cbl-b, we also observed engagement of the closely related E3 ligase c-Cbl, which is enrichment of CYLD in the EGFR-immunoprecipitated com- also strongly involved in EGFR signaling (53), was unaffected plexes upon ligand stimulation (Fig. 5D). To further confirm by the silencing of CYLD (Fig. 5B). Therefore, the impact of the these results and exclude potential cell type- specific effects, DUB on the EGF-dependent ubiquitination events appears to we performed equivalent experiments in two additional cell be driven specifically through Cbl-b and not c-Cbl. lines. We corroborated the interaction of CYLD with Cbl-b and To further explore a potential interaction of CYLD, Cbl-b its contribution to the ubiquitination of EGFR in ovarian and and EGFR we used series of co-immunoprecipitation experi- laryngeal carcinoma cells (Skov3 and Hep2, respectively), ments, which indeed demonstrate the existence of an endog- hence demonstrating the generic nature of this unexpected enous complex among these proteins. The interactions of role of CYLD (Fig. 5E). CYLD with Cbl-b and CYLD with EGFR appeared to occur CYLD Deficiency Results in Deregulated Trafficking and already at basal levels in unstimulated cells and was further Degradation of EGFR—The ubiquitination-dependent signal- strengthened by the addition of EGF (Fig. 5C,5D), whereas ing events are crucial for proper endocytosis and intracel- EGFR and Cbl-b did not interact in the absence of ligand (Fig. lular trafficking of EGFR (42) and its subsequent lysosomal

Molecular & Cellular Proteomics 16.8 1441 CYLD Contributes to Ubiquitination and Degradation of EGFR

A shControl shCYLD B shControl shCYLD Rab7-GFP EGFR EGFR DAPI DAPI 0’

30’ 2h Post-stimulation

60’

120’

Exposure shControl C D 1.2 shCYLD EGFR 1.0 0.8 0.6 CYLD 0.4 αTubulin 0.2 EGF 150 ng/ml 0’ 15’ 30’ 60’ 120’ 0’ 15’ 30’ 60’ 120’ Relative Expression 0.0 shControl shCYLD EGFR CYLD

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. degradation or recycling. Ubiquitin moieties are recognized Rab7 both in the control and in the CYLD-silenced cells. by adaptor proteins containing ubiquitin-binding domains Nevertheless, the trafficking pattern of the receptor through- (UBDs) that sort the modified receptors through the endocytic out the cells differed noticeably. Although the control cells pathway (55). Consequently, an altered ubiquitination of the exhibited EGFR distribution in fewer discrete foci, the shCYLD EGFR can promote a de-regulated intracellular trafficking of cells presented a more scattered image of the receptor that activated receptors. The activities of the Cbl proteins consti- spread over the cells (Fig. 6A; 30 and 60 min after EGF tute an essential pillar for the negative regulation of RTKs addition). We also noticed much stronger EGFR signal in pathways and defective binding of these E3 ligases with the CYLD-silenced cells at 2 h poststimulation, which could indi- EGFR promotes a compromised ubiquitination and degrada- cate a decreased degradation of the receptor (Fig. 6B). tion of the receptor (56). Considering the effects of CYLD We next monitored EGFR cellular levels by immunoblotting silencing on the EGFR ubiquitination, we reasoned that this at different times after ligand stimulation, in combination with DUB could accordingly impact the trafficking of the receptor cycloheximide treatment to prevent contribution from the de following ligand stimulation. novo protein synthesis. We observed that the amounts of the To explore this option, we transfected HeLa cells with GFP- receptor after 60 and 120 min of EGF stimulation were indeed Rab7, a marker for late endosomes, prior to their stimulation higher in the CYLD deficient cells (Fig. 6C). In addition, this with EGF at different time points. As seen in Fig. 6A, the EGFR effect was not because of an overall increased expression of was internalized and displayed partial co-localization with the EGFR in the shCYLD cells. As seen by quantitative PCR

1442 Molecular & Cellular Proteomics 16.8 CYLD Contributes to Ubiquitination and Degradation of EGFR

A C D 1.0 IP: Cbl-b IP: Cbl-b PRM: EGFR PRM: CYLD EGFR Ubi IP 0.8 CYLD EGFR EGFR FLAG Cbl -b 0.6 αTubulin EGFR 0.4 Cbl -b WCL shControlshCYLD CYLD 0.2 α Quantification MS Relative Flag-CYLD WT Tubulin Flag-CYLD Y15F 0.0 EGF 150 ng/ml 0 6 0 6 060606 EGF 150 ng/ml 060606 (minutes) B

pTyr CYLD WT shCYLD shCYLD IP CYLD Y15F CYLD WT CYLD WT FLAG CYLD CYLD Y15F CYLD Y15F

02 02EGF 150 ng/ml (minutes) F 0.025 CYLD WT shCYLD CYLD WT CYLD Y15F CYLD Y15F 0.020 * p-val <0.01 E 0.015 EGFR * CYLD 0.010 * αActin 0.005 012012012EGF 150 ng/ml (hours) Quantification MS Relative shCYLD CYLD WT CYLD Y15F 0 01h2hEGF 150 ng/ml (hours)

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. analyses, the EGFR mRNA levels were lowered when the DUB immunoprecipitation of the reconstituted CYLD proteins un- is silenced (Fig. 6D). Altogether, these data indicate a specific der stringent conditions demonstrated that the Y15F substi- stabilization of EGFR through a post-translational mechanism tution results in a dramatic decrease of the tyrosine-phos- in CYLD-silenced cells, thus in agreement with our proposed phorylation of the DUB upon EGFR stimulation (Fig. 7B), role of CYLD in the regulation of receptor ubiquitination, im- indicating Tyr15 as the major site of CYLD phosphorylation in portant for its subsequent trafficking and degradation. response to EGF. Like the effects seen in the CYLD-silenced CYLD Phosphotyr15-deficient Mutant Mimics the Effects cells, we observed decreased ligand dependent ubiquitination of CYLD Silencing—To corroborate that the observed EGF- of the EGFR in the cells reconstituted with the Y15F mutant, triggered effects upon CYLD silencing are indeed driven together with a reduced interaction between the activated through the Tyr15 phosphorylation of the DUB, the following RTK and the ubiquitin ligase Cbl-b (Fig. 7C). To simultane- experiments were performed. Using RNAi-resistant con- ously measure to what extent the interaction of CYLD with structs, we reconstituted in the CYLD-silenced HeLa cells Cbl-b is affected by the Y15F mutation and its consequence either a wild type CYLD or a Y15F-CYLD mutant, in which for the Cbl-b association with EGFR, we implemented MS tyrosine 15 was exchanged with phenylalanine, hence inca- procedure based on Parallel Reaction Monitoring (PRM) anal- pable of being phosphorylated at this position (Fig. 7A). The ysis coupled to immunoprecipitation of Cbl-b (supplemental

Molecular & Cellular Proteomics 16.8 1443 CYLD Contributes to Ubiquitination and Degradation of EGFR

Table S8). Compared with the CYLD WT cells, the association ligase to the receptor upon EGF stimulation. Accordingly, we of the DUB with Cbl-b was strongly impaired in the CYLD- observed compromised recruitment of Cbl-b to the activated silenced cells as well as in the Y15F-CYLD expressing cells EGFR in CYLD-silenced or CYLD Y15F-expressing cells, (Fig. 7D). Likewise, the recruitment of EGFR to Cbl-b was also which resulted in deficient ubiquitination and subsequent diminished to more than 50% when the DUB was either degradation of the receptor. It should be noted that previous downregulated or Y15F mutated (Fig. 7D). We next examined research has shown that RNAi-based silencing of Cbl-b re- the levels of EGFR in the cells following 1 and 2 h treatment sults in similar EGFR stabilization (59), and that the interaction with the ligand and observed that the receptor is slightly of Cbl-b with EGFR as an important factor for the degradation stabilized in the Y15F-CYLD cells, like the CYLD-silenced of the activated receptor complexes (60). cells (Fig. 7E). To obtain quantitative estimate of this obser- Interestingly, the contribution of CYLD to the ubiquitination vation we utilized PRM measurements, which also indicated a signaling downstream of this RTK appears to be driven spe- significant decrease in the degradation of EGFR when CYLD cifically through the Cbl-b and not c-Cbl, as the later was not is either silenced in the cells or incapable of being phosphor- recruited to the CYLD pTyr15 and its engagement to the ylated on Tyr15 (Fig. 7F). activated complexes was unaffected by the silencing of the DUB. These two closely-related proteins, despite exerting DISCUSSION redundant functions, seem to be subjected to different regu- This study aimed to understand the engagement of the latory mechanisms (51, 61, 62) and CYLD could indeed tumor suppressor CYLD deubiquitinase within the EGF-acti- contribute to their differential modulation. In the context of vated complexes and its impact on the ubiquitination events EGF-dependent pathway, both Cbl-b and c-Cbl have been in the pathway. Here, we demonstrate that CYLD is necessary reported to directly interact with the activated EGFR through for proper EGF-dependent ubiquitination of several well- its phosphorylated tyrosine residues (51, 52). However, an known effectors of the EGFR pathway, including the receptor additional association of the E3 ligases to the receptor could itself, UBASH3B and Cbl-b. We identified an EGF-dependent be facilitated by the Grb2 adaptor protein as well (54). Cer- phosphorylation of Tyr15 on CYLD, a modification that cre- tainly, Grb2 deficiency promotes a reduced recruitment of ated a docking site for the E3 ligase Cbl-b, thereby allowing c-Cbl to the EGFR with a subsequent impairment of its ubiq- the assembly of additional Cbl-b to the activated EGFR. Ac- uitination and trafficking (63). Grb2-mediated association of cordingly, the silencing of CYLD or the substitution of its Cbl-b to EGFR is not explored, to our knowledge, being the Tyr15 with a phosphorylation-incapable residue promoted a interaction between Cbl-b and Grb2 only described in the decrease in the recruitment of the ligase to the receptor, context of TCR stimulation (64). It is possible that the recruit- triggering an impaired ubiquitination of EGFR, its posterior ment of additional Cbl-b to activated EGFR is controlled by trafficking and degradation. CYLD instead, similarly as Grb2 does for the related ligase Although the function of CYLD in NFkB signaling is well c-Cbl. Future investigations on the importance of CYLD’s explored (15, 16), its role in the RTKs signaling, particularly deubiquitinating activity in the process, the role of the basal downstream of the EGFR, is less understood. Earlier work association of CYLD with Cbl-b in the absence of EGF stim- from our group initially pointed to a potential involvement of ulation or the underlying mechanisms of differential recruit- CYLD in the EGFR pathway (32). More recently, CYLD was ment of the two closely related ubiquitin ligases could provide revealed as a key character for integrating EGF and integrin- further insights into the complex interplay that control the dependent signals for the formation of dorsal ruffles in fibro- fine-tuned regulation of EGFR trafficking and degradation. blasts (57). Interestingly, the crosstalk of these molecular EGFR pathway represents a central molecular network in networks also resulted in the tyrosine phosphorylation of cellular physiology, controlling key processes such as prolif- CYLD, albeit trough distinct molecular mechanisms com- eration and migration. To orchestrate adequate cellular re- pared with our current study. EGF stimulation and fibronectin- sponses, the EGF-dependent signaling cascades are sub- dependent activation of ␤1 integrin signaling were both re- jected to a tight regulation that is frequently found altered in quired for the observed tyrosine phosphorylation of CYLD and various human cancers. Understanding the molecular events although the exact sites of modification were not elucidated, controlling EGFR signaling constitutes the key for its manage- it appeared not to involve Tyr15 (57). ment in case of alteration. Being best described as a tumor In contrast, our study identified a specific role of CYLD, suppressor in the NFkB pathway, the role of CYLD in the through its Tyr15 phosphorylation, in the molecular cascade EGFR axis remained however elusive. In the NFkB pathway, downstream of EGFR. Of note, this site has previously been CYLD directly removes K63-polyubiquitin chains from key identified in a large-scale proteomic screen; nonetheless, the molecular effectors, such as TRAF2, TRAF6, and NEMO (15, investigation of CYLD was out of the scope of the study and 16). These K63-polyubiquitin chains facilitate protein-protein thus not explored (58). Here, we demonstrated that Cbl-b is interactions that, hence, are inhibited by the action of CYLD, recruited to the phosphorylated Tyr15 of CYLD and this inter- resulting in decreased signal transduction and diminished action is necessary for the proper engagement of the ubiquitin translocation of NFkB to the nucleus. We uncover herein an

1444 Molecular & Cellular Proteomics 16.8 CYLD Contributes to Ubiquitination and Degradation of EGFR

additional molecular strategy utilized by CYLD for the nega- 11. Pareja, F., Ferraro, D. A., Rubin, C., Cohen-Dvashi, H., Zhang, F., Aulmann, tive regulation of another tumorigenic signaling network, S., Ben-Chetrit, N., Pines, G., Navon, R., Crosetto, N., Kostler, W., Carvalho, S., Lavi, S., Schmitt, F., Dikic, I., Yakhini, Z., Sinn, P., Mills, namely the one controlled by EGFR. Here, CYLD appears to G. B., and Yarden, Y. (2012) Deubiquitination of EGFR by Cezanne-1 control the fine-tuned regulation of EGFR trafficking and deg- contributes to cancer progression. Oncogene 31, 4599–4608 radation by assisting in the association of the E3 ubiquitin 12. Liu, Z., Zanata, S. M., Kim, J., Peterson, M. A., Di Vizio, D., Chirieac, L. R., Pyne, S., Agostini, M., Freeman, M. R., and Loda, M. (2013) The ubiq- ligase Cbl-b with EGFR, thereby modulating the amplitude of uitin-specific protease USP2a prevents endocytosis-mediated EGFR ubiquitination on the receptor and several other major players degradation. Oncogene 32, 1660–1669 in the EGFR network. 13. Komander, D., Lord, C. J., Scheel, H., Swift, S., Hofmann, K., Ashworth, A., and Barford, D. (2008) The structure of the CYLD USP domain explains The unbiased proteomic surveys used in our work prove to its specificity for Lys63-linked polyubiquitin and reveals a B box module. be efficient for addressing complex regulatory mechanisms in Mol. Cell 29, 451–464 a systematic manner, opening the line to a new understanding 14. Brummelkamp, T. R., Nijman, S. M., Dirac, A. M., and Bernards, R. (2003) Loss of the cylindromatosis tumour suppressor inhibits apoptosis by of the captivating and sometimes, like the case with CYLD, activating NF-kappaB. Nature 424, 797–801 counterintuitive nature of the fine-tuned modulation of cellular 15. Kovalenko, A., Chable-Bessia, C., Cantarella, G., Israel, A., Wallach, D., and signaling. Courtois, G. (2003) The tumour suppressor CYLD negatively regulates NF-kappaB signalling by deubiquitination. Nature 424, 801–805 16. Trompouki, E., Hatzivassiliou, E., Tsichritzis, T., Farmer, H., Ashworth, A., DATA AVAILABILITY and Mosialos, G. (2003) CYLD is a deubiquitinating enzyme that nega- The mass spectrometry proteomics data have been depos- tively regulates NF-kappaB activation by TNFR family members. Nature ited to the ProteomeXchange Consortium (http://proteome- 424, 793–796 17. Yoshida, H., Jono, H., Kai, H., and Li, J. D. (2005) The tumor suppressor central.proteomexchange.org) via the PRIDE partner reposi- cylindromatosis (CYLD) acts as a negative regulator for toll-like receptor tory with the dataset identifiers PXD003423 and PXD006390. 2 signaling via negative cross-talk with TRAF6 AND TRAF7. J. Biol. Chem. 280, 41111–41121 * This work was supported by the Danish Natural Science Research 18. Massoumi, R., Chmielarska, K., Hennecke, K., Pfeifer, A., and Fassler, R. Council (DFF - 1323–00191), the Novo Nordisk Foundation, the Lund- (2006) Cyld inhibits tumor cell proliferation by blocking Bcl-3-dependent NF-kappaB signaling. Cell 125, 665–677 beck Foundation, the Danish Strategic Research Council and the Villum 19. Zhang, J., Stirling, B., Temmerman, S. T., Ma, C. A., Fuss, I. J., Derry, J. M., Foundation through the Villum Center for Bioanalytical Sciences. □ and Jain, A. (2006) Impaired regulation of NF-kappaB and increased S This article contains supplemental material. susceptibility to colitis-associated tumorigenesis in CYLD-deficient ‡‡ To whom correspondence should be addressed: Department of mice. J. Clin. Invest. 116, 3042–3049 Biochemistry and Molecular Biology, University of Southern Denmark, 20. Massoumi, R., Kuphal, S., Hellerbrand, C., Haas, B., Wild, P., Spruss, T., Campusvej 55, DK-5230 Odense, Denmark. Tel.: ϩ45 6550 2366; Pfeifer, A., Fassler, R., and Bosserhoff, A. K. (2009) Downregulation of E-mail: [email protected]. CYLD expression by Snail promotes tumor progression in malignant ¶ Present address: Molecular Oncology, UMR 144 CNRS, Curie melanoma. J. Exp. Med. 206, 221–232 Institute. 26, rue d’Ulm. 75248 Paris, France. 21. Annunziata, C. M., Davis, R. E., Demchenko, Y., Bellamy, W., Gabrea, A., ** Present address: Department of Biochemistry and Molecular Bi- Zhan, F., Lenz, G., Hanamura, I., Wright, G., Xiao, W., Dave, S., Hurt, E. 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