Regulation of cell surface receptor trafficking through the ESCRT-0 complex by the deubiquitinating enzyme ESP8

Regulation of cell surface receptor abundance and the spatial and temporal control of ligand-induced signaling is essential for proper cellular growth and homeostasis. Malfunctions along such regulatory pathways can lead to a wide variety of pathologies, including various cancers. Ubiquitination, the process of covalent modification of a target protein with one or more ubiquitin moieties, plays a key role in ligand-mediated downregulation of receptor tyrosine kinases and G protein-coupled receptors. Orchestrated by a cascade of ubiquitin activating, conjugating and ligating enzymes, ubiquitination serves as a signal for endosomal trafficking of cell surface receptors for lysosomal degradation. Moreover, ubiquitination of endosomal sorting machinery is critical for their function. The reversible nature of ubiquitination, imparted by deubiquitinating enzymes specific for cleavage of ubiquitin from modified substrate proteins enables coordinated and dynamic regulation of ubiquitin-dependent events in endocytosis.;The work presented herein investigates the role of the deubiquitinating enzyme Ubiquitin-Specific Protease 8 (USP8) in endocytosis and ligand-mediated downregulation of two receptors: Epidermal Growth Factor Receptor (EGFR) and Chemokine Receptor 4 (CXCR4). The central hypothesis of this study is based on the premise that USP8-mediated deubiquitination exhibits two distinct consequences with respect to receptor endocytosis---protection of direct receptor substrates from degradation and modulation of endosomal sorting machinery in support of general cargo trafficking. With particular emphasis on the functional relationship between USP8 and the early endosomal sorting complex required for transport (ESCRT-0), EGFR and CXCR4 illustrate the different modes of receptor regulation by USP8. To study USP8 function in this context, the following experimental aims were proposed: (i) to compare and contrast the implications of USP8 depletion on cellular abundance, signaling and ligand-mediated degradation of EGFR and CXCR4, (ii) to probe the effects of USP8 catalytic activity on ubiquitination and stability of endosomal adaptor proteins HRS and STAM, and (iii) to characterize the direct interactions of the three Arg-X-X-Lys (RXXK) motifs of USP8 with the SH3 domain of STAM and examine the contribution of these interactions to cellular activity of USP8. Collectively, the present body of work explores the role of USP8 as a global regulator of ubiquitin-mediated events governing receptor trafficking and degradation and seeks to contribute mechanistic insights into the biology of deubiquitination in endocytosis.;The investigation described herein reveals opposing consequences of USP8 activity for the regulation of EGFR and CXCR4 cellular abundance. USP8 loss-of-function studies, employing siRNA interference and over-expression of catalytically inactive mutants of USP8, demonstrate that USP8 opposes the early phase of EGF-induced EGFR ubiquitination and protects EGFR from ligand-mediated degradation. Importantly, the rate of EGFR turnover is most susceptible to USP8 activity in the presence of low, physiological concentrations of EGF. In addition to EGFR, USP8 catalytic activity impacts the ubiquitination and stability of early endocytic sorting adaptors, hepatocyte growth factor-regulated substrate (HRS) and signal transducing adaptor molecule (STAM). Three RXXK motifs within USP8 support a direct low-affinity polyvalent interaction with the SH3 domain(s) of STAM1/2 proteins, specifically localizing the USP8/STAM complex to the ESCRT-0 machinery. Importantly, the ability of USP8 to form a complex with STAM is required for USP8-mediated deubiquitination of activated EGFR. These findings suggest that USP8 cooperates with the ESCRT-0 sorting machinery to support recycling of signaling growth factor receptors. While USP8 also regulates the ubiquitination status of ESCRT-0 in a manner dependent upon the RXXK/SH3 interaction, stability of HRS and STAM is only marginally affected by its ability to directly interact with the SH3 domain of STAM, indicate that USP8 must possess additional means of localizing to the ESCRT-0 complex.;In contrast to EGFR, CXCR4 stability is enhanced in cells compromised for USP8 function. CXCR4 accumulates on enlarged early endosomes and co-localizes with abnormally distributed ESCRT-0 complex as a result of USP8 inactivation. However, regulation of CXCR4 trafficking and downregulation by USP8 is not susceptible to mutations in the RXXK motifs. Such evidence demonstrates that USP8 is not functionally beholden to a single mode of recruitment to the endosomal compartment and implicates USP8 as a multifaceted regulator of cell surface receptor endocytosis. Collectively, the findings described herein portray a complex cooperation between USP8 and the ESCRT-0 proteins that shapes ubiquitin-mediated events at the early-to-late endosomal transition.