First name
Nadine
Last name
Wossner
Year of Study
Thesis Title
Characterization of the role of USP25 in EGFR endocytosis
Thesis Abstract
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. 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. 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.
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.
To identify DUBs altering EGFR degradation we undertook a genome-wide small interfering RNA screen targeting all known active DUBs. 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. 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.
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.
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