USP9X Controls EGFR Fate by Deubiquitinating the Endocytic Adaptor Eps15

Abstract

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.

Deubiquitylating enzymes and drug discovery: emerging opportunities

“…Third, through their deubiquitylating activities, various DUBs, such as USP7 and USP28, have been reported to regulate the levels and/or activities of various oncogene or tumour suppressor proteins 30,31 . Fourth, DUBs modulate other therapeutically relevant cellular components and processes, such as the UPS (for example, USP14 and UCHL5 (also known as UCH37)) 32 , stem cell renewal (for example, USP16 or USP22) 29,33 , DNA damage response (DDR) and DNA repair (for example, USP1 or USP11) 9 , immunooncology (for example, USP7) 34 or receptor tyrosine kinases (for example, USP8 or USP9X) 35,36 . Consequently, and as described in more detail below, various DUBs are emerging as attractive targets for the development of novel cancer therapies.…”

Ubiquitin-specific Protease 36 (USP36) Controls Neuronal Precursor Cell-expressed Developmentally Down-regulated 4-2 (Nedd4-2) Actions over the Neurotrophin Receptor TrkA and Potassium Voltage-gated Channels 7.2/3 (Kv7.2/3)

“…1D). Therefore, it seems that the ubiquitination/deubiquitination machinery regulating TrkA may appear as complex as in the case of other receptor tyrosine kinases, such as EGFR and Met, for which many identified DUBs regulate in a direct or indirect way the response to their activation by their cognate ligands (34,41). Additional studies need to be carried out to address the role of other DUBs identified in this work within the NGF-TrkA system.…”

“…We looked for DUBs that could alter TrkA activation kinetics in response to NGF performing a siRNA screen, an approach that has been successfully used by several groups trying to identify DUBs involved in the modulation of signaling cascades triggered by different receptors (33,34). We identified USP36 as a DUB that affected not only TrkA signaling but also ubiquitination.…”

“…To identify DUBs modulating TrkA activation kinetics, we performed a screen using a library of siRNAs targeting dub genes that has been previously used (33,34). The DUB library consisted of pools of four nonoverlapping siRNAs, which targeted all known or putative DUBs (see "Experimental Procedures").…”

The deubiquitylase USP9X controls ribosomal stalling

“…Recent work, focused on this enzyme, has established proof of principle that selective small molecule inhibition amongst the USP family can be achieved [15][16][17][18]. USP9X is one of the most abundant members of the USP family and has been linked with many processes, including centrosome function, chromosome alignment during mitosis, EGF receptor degradation, chemo-sensitisation and circadian rhythms [19][20][21][22][23]. Loss of function mutations in females lead to congenital malformations and intellectual disability [24].…”