Deubiquitination of CXCR4 by USP14 Is Critical for Both CXCL12-induced CXCR4 Degradation and Chemotaxis but Not ERK Activation

Abstract

The chemokine receptor CXCR4 plays important roles in the immune and nervous systems. Abnormal expression of CXCR4 contributes to cancer and inflammatory and neurodegenerative disorders. Although ligand-dependent CXCR4 ubiquitination is known to accelerate CXCR4 degradation, little is known about counter mechanisms for receptor deubiquitination. CXCL12, a CXCR4 agonist, induces a time-dependent association of USP14 with CXCR4, or its C terminus, that is not mimicked by USP2A, USP4, or USP7, other members of the deubiquitination catalytic family. Co-localization of CXCR4 and USP14 also is time-dependent following CXCL12 stimulation. The physical interaction of CXCR4 and USP14 is paralleled by USP14-catalyzed deubiquitination of the receptor; knockdown of endogenous USP14 by RNA interference (RNAi) blocks CXCR4 deubiquitination, whereas overexpression of USP14 promotes CXCR4 deubiquitination. We also observed that ubiquitination of CXCR4 facilitated receptor degradation, whereas overexpression of USP14 or RNAi-induced knockdown of USP14 blocked CXCL12-mediated CXCR4 degradation. Most interestingly, CXCR4-mediated chemotactic cell migration was blocked by either overexpression or RNAi-mediated knockdown of USP14, implying that a CXCR4-ubiquitin cycle on the receptor, rather than a particular ubiquitinated state of the receptor, is critical for the ligand gradient sensing and directed motility required for chemokine-mediated chemotaxis. Our observation that a mutant of CXCR4, HA-3K/R CXCR4, which cannot be ubiquitinated and does not mediate a chemotactic response to CXCL12, indicates the importance of this covalent modification not only in marking receptors for degradation but also for permitting CXCR4-mediated signaling. Finally, the indistinguishable activation of ERK by wild type-or 3K/R-CXCR4 suggests that chemotaxis in response to CXCL12 may be independent of the ERK cascade.

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

“…USP14 has been shown to regulate CXCR4 ubiquitination at the plasma membrane during the early phase of CXCR4 endocytosis without affecting ERK signaling (31). Unlike USP14, USP8 is not required for deubiquitination of CXCR4 (Fig.…”

“…CXCR4 mediated chemotaxis along a gradient of its cognate ligand, stromal-derived factor 1␣ (SDF-1␣/CXCL12), and supports metastasis of cancer to the bone marrow (25,28). Ligand-induced lysosomal degradation of CXCR4 is critical for downstream signal attenuation and is mediated by the HECT family E3 ligase atrophininteracting protein 4 (AIP4/Itch) (29,30) and deubiquitinase ubiquitin-specific protease 14 (USP14) (31). CXCR4 activation has also been linked to ubiquitination of Hrs by AIP4, and its trafficking proceeds along an Hrs-dependent pathway (32), suggesting that alteration in the Hrs ubiquitination status may modulate CXCR4 turnover.…”

A novel small molecule inhibitor of deubiquitylating enzyme USP14 and UCHL5 induces apoptosis in multiple myeloma and overcomes bortezomib resistance

“…SDF-1 binds to G-protein-coupled CXCR4, the known substrate of USP14, 15 and most important, CXCR4 is implicated in MM metastasis. 26 We therefore examined serum or SDF-1-induced MM cell migration.…”

“…15 UCLH5, like USP14, plays an important role in regulating oncogenic signaling. 16 For example, transforming growth factor-b (TGF-b) is a critical regulator of cell proliferation, differentiation, and tumor pathogenesis; UCHL5 regulates TGF-b/Smad signaling by binding to intracellular Smad transcription factors, thereby allowing for deubiquitylation and stabilization of the TGF receptor.…”

Ubiquitylation of the chemokine receptor CCR7 enables efficient receptor recycling and cell migration

“…Upon CXCL12 binding, CXCR4 was ubiquitylated by the E3 ubiquitin ligase AIP4, resulting in lysosomal sorting and subsequent degradation of CXCR4 (Marchese and Benovic, 2001;Marchese et al, 2003). Interestingly, CXCL12 was shown to recruit the de-ubiquitylation enzyme USP14 to CXCR4, thereby regulating CXCR4 degradation and cell migration (Mines et al, 2009). In addition, depletion of the de-ubiquitylation enzyme USP8 was found to stabilize CXCR4 surface expression without affecting receptor ubiquitylation, indicating a role of USP8 in CXCR4 trafficking and degradation (Berlin et al, 2010).…”