The epidermal growth factor receptor (EGFR) is involved in a broad range of cellular responses. Deregulated EGFR signalling is a significant feature in different stages of oncogenesis and it contributes to several cancer types. One important mechanism whereby cancer cells can obtain increased and uncontrolled EGFR signalling is to escape down-modulation of the receptor. Ubiquitination of EGFR and of members of the endocytic machinery has a key role in this process, regulating receptor internalization, trafficking and degradation. Deubiquitinating enzymes (DUBs) can reverse the ubiquitination process, antagonizing or even promoting receptor degradation. To identify DUBs altering EGFR degradation we undertook a genome-wide small interfering RNA screen targeting all known active DUBs. In addition to previously described AMSH, USP8, USP2 and OTUD7B enzymes, we identified twelve novel DUBs affecting EGFR degradation by using immunoblot-based approaches complemented by an ELISA-based assay. Among them USP25, a member of the ubiquitin-specific protease (USP) family, displayed one of the strongest effects. We found that the degradation rate of EGFR is enhanced upon USP25 knock-down. Quantitative internalization assays revealed that depletion of USP25 leads to a faster internalization rate of EGFR. Consistently, overexpression of wild-type USP25, but not its catalytic inactive mutant, partially blocked EGFR internalization. Pathway analyses suggest that upon knock-down of USP25 a dynamin-independent endocytic route is activated, accounting for the increased EGFR internalization. Furthermore, we scored an increase in the EGFR ubiquitination upon USP25 knock-down, in particular at early time points post EGF stimulation, suggesting that EGFR is a direct target of USP25. Details regarding the kind of ubiquitin chains attached to the EGFR in the absence of USP25 are still lacking and are currently under investigation. We also validated the E3 ligase component Cullin 3 (CUL3) as USP25 interactor and we found that USP25 preferentially binds the neddylated active form of CUL3. Initial analyses with CUL3 suggest that ablation of USP25 and CUL3 have opposite effects on EGFR internalization and that the observed phenotypes compensate each other in double knock-down. Our data suggest that USP25 and CUL3 may form a stable complex with opposing activity on EGFR internalization, and led us to hypothesize that they are involved in a novel “quality control” mechanism working at the plasma membrane. Taken together our study identifies USP25 as a novel negative regulator of EGFR ubiquitination and endocytosis, involved in early internalization events. USP25 may represent a suitable “druggable” target for pathological conditions where EGFR is deregulated and opens up a promising direction for future investigations.
|Titolo:||CHARACTERIZATION OF THE ROLE OF USP25 IN EGFR ENDOCYTOSIS|
|Data di pubblicazione:||18-mar-2015|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||CHARACTERIZATION OF THE ROLE OF USP25 IN EGFR ENDOCYTOSIS / N.c. Woessner ; internal advisor: PP Di Fiore ; external advisor: M. Clague. - Milano : Università degli studi di Milano. DIPARTIMENTO DI SCIENZE DELLA SALUTE, 2015 Mar 18. ((25. ciclo, Anno Accademico 2013.|
|Digital Object Identifier (DOI):||10.13130/n-c-woessner_phd2015-03-18|
|Appare nelle tipologie:||Tesi di dottorato|