Essential Role of Ubiquitin-Specific Protease 8 for Receptor Tyrosine Kinase Stability and Endocytic Trafficking In Vivo

Abstract

Posttranslational modification by ubiquitin controls multiple cellular functions and is counteracted by the activities of deubiquitinating enzymes. UBPy (USP8) is a growth-regulated ubiquitin isopeptidase that interacts with the HRS-STAM complex. Using Cre-loxP-mediated gene targeting in mice, we show that lack of UBPy results in embryonic lethality, whereas its conditional inactivation in adults causes fatal liver failure. The defect is accompanied by a strong reduction or absence of several growth factor receptor tyrosine kinases (RTKs), like epidermal growth factor receptor, hepatocyte growth factor receptor (c-met), and ERBB3. UBPydeficient cells exhibit aberrantly enlarged early endosomes colocalizing with enhanced ubiquitination and have reduced levels of HRS and STAM2. Congruently immortalized cells gradually stop proliferation upon induced deletion of UBPy. These results unveil a central and nonredundant role of UBPy in growth regulation, endosomal sorting, and the control of RTKs in vivo.Posttranslational modification of proteins by mono-or polyubiquitination represents a central mechanism for modulating a wide range of cellular functions, like protein stability, intracellular transport, protein interactions, and transcriptional activity. Ubiquitin is covalently bound to substrates by the activity of E1, E2, and E3 conjugating enzymes (17,44,53). Analogous to other posttranslational modifications, ubiquitination is a reversible process counteracted by deubiquitinating enzymes (DUBs), which cleave the isopeptide linkage between the protein substrate and the ubiquitin residue (14). While the processes and biological consequences of ubiquitin conjugation have been intensively studied, the role of DUBs is just beginning to emerge. Based on structural predictions, the human genome contains more than 90 putative DUBs. These fall into the subclasses of ubiquitin C-terminal hydrolases, ubiquitin-specific proteases (USPs), Machado Joseph disease protein domain proteases, ovarian tumor proteases, and JAMM motif proteases (1, 37, 42). The USPs, with more than 50 members, comprise the largest class of DUBs. Members of the USP family have been associated with the regulation of different cellular pathways. USP7 (HAUSP) regulates p53 stability by deubiquitination of p53 and Mdm2 (32, 33), USP2a was reported to regulate the stability of fatty acid synthase (12), and USP1 has been shown to deubiquitinate the monoubiquitinated proteins Fanconia anemia protein FANCD2 (41) and DNA replication processivity factor PCNA (19).USP8 (UBPy/HUMORF8) was first described as a growthregulated ubiquitin isopeptidase that accumulates upon growth stimulation. Protein levels of UBPy decrease, when cells undergo growth arrest by contact inhibition, suggesting a possible role in the control of mammalian-cell proliferation (40). An oncogenic fusion product of the 5Ј end of phosphatidylinositol 3-kinase p85␤ fused to the 3Ј end of UBPy, which contains the catalytic domain, was isolated from a patient with chronic myelogenous leukemia (21). B...

Deubiquitylases From Genes to Organism

“…Its depletion has more severe effects on the organization of the endocytic pathway that combine to inhibit EGFR downregulation (25,177,218,219). One contributing factor to such defective EGFR trafficking is that USP8 controls the stability of ESCRT-0 components, Hrs and STAM in cells and in conditional knockout mice (185,219). However, for Frizzled receptor (Fz) and Smoothened (Smo), key components of Wingless (Wnt) and Hedgehog signaling pathways, respectively, USP8 plays a negative regulatory role with regard to receptor degradation more akin to that originally proposed for AMSH (153,180,281).…”

Deubiquitinases in cancer: new functions and therapeutic options

“…There are at least four DUBs-USP8, USP18, AMSH and POH1-that affect the trafficking of RTKs like epidermal growth factor receptor (EGFR), Met and ErbB2. 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).…”

The Endosome-associated Deubiquitinating Enzyme USP8 Regulates BACE1 Enzyme Ubiquitination and Degradation

“…and Presenilin 1 Protein Levels-Depletion of USP8 has been shown to decrease protein levels of USP8 substrates, as a result of increased substrate ubiquitination and degradation (23,36). To determine whether USP8 depletion resulted in a decrease in BACE1, we performed siRNA-mediated knockdown of USP8, using two different siRNAs targeting USP8 in an H4 neuroglioma cell line overexpressing BACE1-GFP (H4-BACE1-GFP).…”

“…Although some investigators have noted an increase in total cellular EGFR when USP8 is depleted (24,28), others have determined that USP8 depletion leads to an increase in EGFR lysosomal degradation, accompanied by a decrease in EGFR protein levels in both cellular and mouse conditional knock-out models (23,36,37). USP8 has a role not only in protein degradation but also in protein trafficking.…”

“…As USP8 stabilizes STAM, USP8 may play a role in facilitating the passage of molecules destined for degradation along the endosome-lysosome degradation pathway. Notably, USP8 depletion or overexpression of catalytically inactive USP8 leads to the accumulation of ubiquitinated proteins in the endosomes (24,34,36,41).…”