USP25 Regulates EGFR Fate by Modulating EGF-Induced Ubiquitylation Dynamics.

Deregulated epidermal growth factor receptor (EGFR) signaling is a key feature in different stages of oncogenesis. One important mechanism whereby cancer cells achieve increased and uncontrolled EGFR signaling is escaping down-modulation of the receptor. Ubiquitylation of the EGFR plays a decisive role in this process, as it regulates receptor internalization, trafficking and degradation. Deubiquitinating enzymes (DUBs) may oppose the ubiquitylation process, antagonizing or even promoting receptor degradation. Here, we use qualitative and quantitative assays to measure EGFR internalization and degradation after Ubiquitin Specific Peptidase 25 (USP25) depletion. We show that, by acting at the early steps of EGFR internalization, USP25 restrains the degradation of the EGFR by assisting in the association of the E3 ubiquitin ligase c-Cbl with EGFR, thereby modulating the amplitude of ubiquitylation on the receptor. This study establishes USP25 as a negative regulator of the EGFR down-modulation process and suggests that it is a promising target for pharmacological intervention to hamper oncogenic growth signals in tumors that depend on the EGFR.

USP9X Controls EGFR Fate by Deubiquitinating the Endocytic Adaptor Eps15.

Following activation by its cognate ligand(s), the epidermal growth factor receptor (EGFR) is rapidly routed to the lysosome for degradation in a ubiquitination-dependent fashion. This pathway represents the major mechanism of long-term attenuation of EGFR signaling, and its deregulation is a significant feature in different types of cancers. Here we demonstrate, through a systematic RNAi-based approach, that several deubiquitinating (DUB) enzymes extend or decrease EGFR half-life upon EGF stimulation. We focus on USP9X, whose depletion severely affects EGFR turnover, interfering with its internalization and trafficking. We identify the endocytic protein Eps15 as one of the critical substrates of USP9X, and we map the Eps15 ubiquitination sites. We found that Eps15 monoubiquitination occurs already at minimal dose of EGF stimulation and is essential for EGFR internalization. Overall, our findings identify USP9X as a novel regulator of EGFR endocytosis and suggest a model whereby cycles of ubiquitination and deubiquitination events on endocytic accessory proteins may regulate the internalization and trafficking of the EGFR toward the lysosomes.

ESCRT-0 is not required for ectopic Notch activation and tumor suppression in Drosophila.

Multivesicular endosome (MVE) sorting depends on proteins of the Endosomal Sorting Complex Required for Transport (ESCRT) family. These are organized in four complexes (ESCRT-0, -I, -II, -III) that act in a sequential fashion to deliver ubiquitylated cargoes into the internal luminal vesicles (ILVs) of the MVE. Drosophila genes encoding ESCRT-I, -II, -III components function in sorting signaling receptors, including Notch and the JAK/STAT signaling receptor Domeless. Loss of ESCRT-I, -II, -III in Drosophila epithelia causes altered signaling and cell polarity, suggesting that ESCRTs genes are tumor suppressors. However, the nature of the tumor suppressive function of ESCRTs, and whether tumor suppression is linked to receptor sorting is unclear. Unexpectedly, a null mutant in Hrs, encoding one of the components of the ESCRT-0 complex, which acts upstream of ESCRT-I, -II, -III in MVE sorting is dispensable for tumor suppression. Here, we report that two Drosophila epithelia lacking activity of Stam, the other known components of the ESCRT-0 complex, or of both Hrs and Stam, accumulate the signaling receptors Notch and Dome in endosomes. However, mutant tissue surprisingly maintains normal apico-basal polarity and proliferation control and does not display ectopic Notch signaling activation, unlike cells that lack ESCRT-I, -II, -III activity. Overall, our in vivo data confirm previous evidence indicating that the ESCRT-0 complex plays no crucial role in regulation of tumor suppression, and suggest re-evaluation of the relationship of signaling modulation in endosomes and tumorigenesis.