Biotechnol. strategies, we demonstrate the involvement of CYLD in the regulation of the ubiquitination events brought on 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 conversation 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. A-841720 This represents a previously uncharacterized strategy exerted by this deubiquitinase and tumors suppressor for the unfavorable regulation of a tumorigenic signaling pathway. Epidermal Growth Factor Receptor (EGFR)1 belongs to the family of receptor tyrosine kinases (RTKs) and plays a crucial role in the maintenance of a correct cellular homeostasis, controlling central processes such as cell proliferation, migration, differentiation or survival (reviewed in (1)). EGFR A-841720 consists of an extracellular domain name for the recognition of the ligands, a single pass transmembrane region and an intracellular tyrosine kinase- made up of domain name. The binding of a ligand, such as Epidermal Growth Factor (EGF), promotes the dimerization of the receptor and the subsequent activation of its kinase activity, which further leads to the autophosphorylation of A-841720 tyrosine residues around the intracellular region of the EGFR (1, 2). These altered residues act as docking sites for recruiting SH2 or PTB domain name- made up of signaling proteins (3, 4), hence the stimulation triggers the association of large intracellular complexes that support rapid spread and amplification of the signal, eventually resulting in a specific cellular output (5). To facilitate an adequate response in intensity and duration, the downstream events following the activation of the receptor necessitate tight negative regulation that counteracts the positive signals. In this regard, the attachment of ubiquitin moieties to the EGFR plays a key role in directing its internalization and further endocytic trafficking that eventually leads to its lysosomal degradation or recycling (6, 7). Hence, the regulated A-841720 turnover of EGFR is usually pivotal Arnt for a correct cellular output. E3 ubiquitin ligases of the casitas B-lineage lymphoma (Cbl) family play a crucial part in this chain of events. Cbl proteins are recruited to activated receptors, either binding directly to their phosphorylated tyrosine residues or through the assistance of other adaptor proteins (8), thereby ubiquitinating the EGFR, which can in turn be acknowledged and sorted by the endocytic machinery (9, 10). In this context, further level of signaling modulation can be reached by ubiquitin hydrolases (deubiquitinases, DUBs), which may oppose the activity of Cbl ligases by removing ubiquitin moieties from the receptor (11, 12). These latter enzymes can thereby have a decisive role for the final cellular response. Cylindromatosis protein (CYLD) is usually a tumor suppressor that displays a specific ubiquitin hydrolase activity for K63-linked polyubiquitin chains (13). Unfavorable regulatory actions of CYLD have been reported for several signaling paradigms, the most explored to date being its role in the NF-kB pathway (14C16). It has also been shown an involvement of CYLD in the control of essential cellular processes as apoptosis (14C16), inflammation (17), proliferation (18) and tumorigenesis (19). The downregulation of this DUB has been linked with oncogenesis in different cellular contexts including melanoma (20), myeloma (21), uterine cervix carcinoma (22), hepatocellular and A-841720 colon carcinoma (23). To our knowledge, the investigation of CYLD and its spectrum of actions have mostly been carried out through targeted approaches that explored, one at a time, the different regulatory strategies of this DUB on specific, discrete targets. However, advances in quantitative mass spectrometry (MS)-based proteomics allow studies of signaling cascades in a system-wide, unbiased manner for uncovering novel molecular character types with a key part in the transduction mechanisms. Currently, MS-based quantitative proteomics represents a powerful tool for the comprehensive characterization of signaling networks (24, 25), including their dynamics changes (26C28), protein interactions (29, 30) and numerous post-translational modifications (31). Indeed, the first evidences pointing to an involvement of CYLD in the molecular events downstream the EGFR were obtained by MS-based proteomics (32). Following addition of EGF, the presence of CYLD among the tyrosine-phosphorylated complexes showed a very rapid and strong increase, which could indicate a role for this deubiquitinase in the RTK signaling and make CYLD-related ubiquitination events in EGFR pathway worth investigating. Here, we conducted a global analysis of the cellular ubiquitinome by employing a recently described approach termed StUbEx (Stable Tagged Ubiquitin Exchange) followed.