AMSH is an endosome-associated ubiquitin isopeptidase

Abstract

The JAMM (JAB1/MPN/Mov34 metalloenzyme) motif has been proposed to provide the active site for isopeptidase activity associated with the Rpn11/POH1 subunit of the 19S-proteasome and the Csn5-subunit of the signalosome. We have looked for similar activity in associated molecule with the SH3 domain of STAM (AMSH), a JAMM domain–containing protein that associates with the SH3-domain of STAM, a protein, which regulates receptor sorting at the endosome. We demonstrate isopeptidase activity against K48-linked tetraubiquitin and K63-linked polyubiquitin chains to generate di-ubiquitin and free ubiquitin, respectively. An inactivating mutation (D348A) in AMSH leads to accumulation of ubiquitin on endosomes and the concomitant stabilization of a ubiquitinated form of STAM, which requires an intact ubiquitin interaction motif (UIM) within STAM. Short interfering RNA knockdown of AMSH enhances the degradation rate of EGF receptor (EGFR) following acute stimulation and ubiquitinated EGFR provides a substrate for AMSH in vitro. We propose that AMSH is a deubiquitinating enzyme with functions at the endosome, which oppose the ubiquitin-dependent sorting of receptors to lysosomes.

AMSH, an ESCRT-III Associated Enzyme, Deubiquitinates Cargo on MVB/Late Endosomes

“…To clarify the intracellular localization of AMSH, we transfected HeLa cells with a CHMP3 expression vector and analyzed the localization of the exogenous CHMP3 as well as the endogenous AMSH by confocal microscopy. In accordance with previous findings (Itoh et al, 2001;McCullough et al, 2004), we detected AMSH as scattered puncta throughout the cytoplasm with moderate expression in the nucleus ( Fig. 2A).…”

“…In studies aimed at finding molecules associated with STAM, we identified the AMSH deubiquitinating (DUB) enzyme (Tanaka et al, 1999;McCullough et al, 2004). AMSH possesses an isopeptidase domain characterized by the JAB1/MPN/Mov34 metalloenzyme (JAMM) domain.…”

“…AMSH possesses an isopeptidase domain characterized by the JAB1/MPN/Mov34 metalloenzyme (JAMM) domain. In an in vitro assay, recombinant AMSH removed ubiquitin moieties from K63-linked but not K48-linked ubiquitins (McCullough et al, 2004). Since K63 ubiquitin, like monoubiquitin, is associated with cargo sorting, the K63-specific DUB activity suggested that AMSH might function in MVB cargo sorting.…”

Deubiquitinases in cancer: new functions and therapeutic options

“…Similarly, some OTU family components such as OTUB1 and A20 hydrolyze Lys48-linked chains, whereas TRABID and OTUD5 have preference for Lys63 linkages (Virdee et al, 2010). Furthermore, Cezanne preferentially cleaves Lys11 over Lys48 and Lys63 linkages (Bremm et al, 2010), whereas JAMMs share the specificity for Lys63-linked polyubiquitin (McCullough et al, 2004;Sato et al, 2008;Cooper et al, 2009). Finally, the Josephin ATXN3-editing activity shows a restricted specificity for K63-linked chains (Winborn et al, 2008).…”

“…USP8 has an important role in the stabilization of RTKs through deubiquitylation, allowing their recycling to the plasma membrane (Niendorf et al, 2007), although other studies have suggested that USP8 might promote degradation of RTKs (Alwan and van Leeuwen, 2007). In addition, the endosomeassociated AMSH (also known as STAMBP) promotes EGFR recycling at the expense of lysosomal sorting (McCullough et al, 2004;Clague and Urbe, 2006). USP18 has been identified in a recent RNA interference screen as a new regulator of EGFR synthesis by modulating its translation (Duex and Sorkin, 2009).…”