Abstract
Reversible ubiquitination of activated receptor complexes signals their sorting between recycling and degradation and thereby dictates receptor fate. The deubiquitinating enzyme ubiquitin-specific protease 8 (USP8/UBPy) has been previously implicated in the regulation of the epidermal growth factor receptor (EGFR); however, the molecular mechanisms governing its recruitment and activity in this context remain unclear. Herein, we investigate the role of USP8 in countering ligand-induced ubiquitination and down-regulation of EGFR and characterize a subset of protein-protein interaction determinants critical for this function. USP8 depletion accelerates receptor turnover, whereas loss of hepatocyte growth factor-regulated substrate (Hrs) rescues this phenotype, indicating that USP8 protects EGFR from degradation via an Hrs-dependent pathway. Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content. These phenotypes require the central region of USP8, containing three extended Arg-X-X-Lys (RXXK) motifs that specify direct low affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2. The USP8·STAM complex critically impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes. Consequently, USP8-mediated deubiquitination slows progression of EGFR past the early-to-recycling endosome circuit in a manner dependent upon the RXXK motifs. Collectively, these findings demonstrate a role for the USP8·STAM complex as a protective mechanism regulating early endosomal sorting of EGFR between pathways destined for lysosomal degradation and recycling.
Highlights
Research Programs) Breast Cancer pre-doctoral fellowship. 2 To whom correspondence should be addressed: 929 East 57th St
We find that USP8 depletion accelerates ligand-induced degradation of epidermal growth factor receptor (EGFR) through a pathway regulated by hepatocyte growth factor-regulated substrate (Hrs) and describe three low affinity RXXK motifs responsible for the interaction with the Hrs partner, signal transducing adaptor molecule (STAM)
To address the mechanisms of USP8 action in this context, we examined the consequences of USP8 depletion on EGFR turnover in response to low concentrations (2.5 ng/ml) of stimulating ligand, EGF
Summary
Research Programs) Breast Cancer pre-doctoral fellowship. 2 To whom correspondence should be addressed: 929 East 57th St. 2 To whom correspondence should be addressed: 929 East 57th St. W432, receptor; MVB, multivesicular body; ESCRT, endosomal sorting complexes required for transport; Hrs, hepatocyte growth factor-regulated substrate; STAM, signal transducing adaptor molecule; AMSH, associated molecule. A high degree of structural conservation between the yeast and human ESCRT-0 complexes [18] implies functional and regulatory similarities In support of this notion, the mammalian STAM1/2 proteins have been shown to interact in vitro with two DUBs: USP8 and the associated molecule with the SH3 domain of STAM (AMSH) (19 –22). Analysis of a conditional USP8 mouse knock-out has revealed a drastic loss of growth factor receptors, including the EGFR, its family member, Erb-B3, and c-Met [24] These phenotypes are further accompanied by accumulation of ubiquitinated species at the early-to-late endosome transition as well as endosomal swelling akin to inhibition of Hrs [25]. With the SH3 domain of STAM; USP8, ubiquitin-specific protease 8; MIT, microtubule interacting and transport; Rhod, rhodanese-like domain; IF, immunofluorescence; BiFC, bimolecular fluorescence complementation; VFP, venus fluorescent protein; USP, ubiquitin-specific peptidase; WCL, whole cell lysate; IP, immunoprecipitate; Fl, fluorescein
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