The Deubiquitinating Enzyme USP8 Promotes Trafficking and Degradation of the Chemokine Receptor 4 at the Sorting Endosome

Abstract

Reversible ubiquitination orchestrated by the opposition of ubiquitin ligases and deubiquitinating enzymes mediates endocytic trafficking of cell surface receptors for lysosomal degradation. Ubiquitin-specific protease 8 (USP8) has previously been implicated in endocytosis of several receptors by virtue of their deubiquitination. The present study explores an indirect role for USP8 in cargo trafficking through its regulation of the chemokine receptor 4 (CXCR4). Contrary to the effects of USP8 loss on enhanced green fluorescent protein, we find that USP8 depletion stabilizes CXCR4 on the cell surface and attenuates receptor degradation without affecting its ubiquitination status. In the presence of ligand, diminished CXCR4 turnover is accompanied by receptor accumulation on enlarged early endosomes and leads to enhancement of phospho-ERK signaling. Perturbation in CXCR4 trafficking, resulting from USP8 inactivation, occurs at the ESCRT-0 checkpoint, and catalytic mutation of USP8 specifically targeted to the ESCRT-0 complex impairs the spatial and temporal organization of the sorting endosome. USP8 functionally opposes the ubiquitin ligase AIP4 with respect to ESCRT-0 ubiquitination, thereby promoting trafficking of CXCR4. Collectively, our findings demonstrate a functional cooperation between USP8, AIP4, and the ESCRT-0 machinery at the early sorting phase of CXCR4 and underscore the versatility of USP8 in shaping trafficking events at the earlyto-late endosome transition.Endocytosis and trafficking of cell surface receptors is essential for organized signal transduction and maintenance of homeostasis. Malfunctions along the molecular pathways governing endocytosis can lead to a wide range of human pathologies including metabolic disorders, autoimmune diseases, and cancer (1, 2). Ubiquitination, a reversible post-translational modification of proteins (3), mediates spatial and temporal aspects of endocytosis by dictating macromolecular complex assembly and cargo fate (4). Although polyubiquitination linked through Lys 48 of ubiquitin targets substrates for proteasomal degradation (5), mono-and Lys 63 -linked ubiquitination signal cargo trafficking (6), and modulate function of endocytic sorting machinery (7). The reversible nature of ubiquitination is imparted by deubiquitinating enzymes (DUBs) 4 and enables dynamic regulation of these ubiquitin-dependent processes in the cell (8).Trafficking of endocytosed cargo for degradation by the lysosome occurs in a series of discrete selection stages that utilize the endosomal sorting complexes required for transport (ESCRTs) -0, -I, -II, and -III. The ESCRT-0 complex, consisting of the adaptor proteins hepatocyte growth factor-regulated substrate (Hrs) and signal transducing adaptor molecule (STAM), resides at the sorting endosome and functions in the first step of cargo selection. ESCRT-0 partitions the endocytosed material arriving on the early endosomes between recycling and the multivesicular body (9 -11) and interacts with downstream ESCRT machinery (12, 13) respon...

Differential phosphorylation of the phosphoinositide 3-phosphatase MTMR2 regulates its association with early endosomal subtypes

“…The significant increase in ERK1/2 activation upon expression of MTMR2 S58A/S631A and the observed enlarged MTMR2/ APPL1 endosomes supports this hypothesis. One possibility is that this effect is due to MTMR2-mediated PI(3)P depletion that leads to halting or retardation of endosomal maturation, a phenotype which others have shown to result in endosome enlargement and continued receptor signaling (Akbergenova and Bykhovskaia, 2009;Berlin et al, 2010;Kim et al, 2010). However, further testing will be needed, as our current results don't directly demonstrate PI(3)P depletion on APPL1-positive endosomes.…”

Cellular functions of the DUBs

“…However, they also find that lysosomal degradation of the activated chemokine receptor 4 (CXCR4) is blocked under similar conditions (Berlin et al, 2010a).…”

TRE17/USP6 regulates ubiquitination and trafficking of cargo proteins that enter cells by clathrin-independent endocytosis

“…USP8 is an endosomal DUB that has been shown to deubiquitylate and regulate the trafficking of several plasma membrane proteins, including EGF receptor, the chemokine receptor CXCR4, the Ca 2+ -activated K + channel and the epithelial Na + channel ENaC (Berlin et al, 2010a;Berlin et al, 2010b;Balut et al, 2011;Zhou et al, 2013). Given that USP8 is suggested to function at the plasma membrane and also at endosomal compartments, it was possible that USP8 might also regulate CIE cargo trafficking as TRE17 does.…”